Enzyme

Caffeic acid phenethyl ester (CAPE) is the major pharmacologically-active component of some propolis types, rich in polyphenols, such as poplar propolis types. CAPE has the potential to be applied as a pharmaceutical as it possesses most of the pharmacological activities of propolis, such as anti-proliferative, antioxidant, immunomodulatory, antidiabetic, anti-inflammatory and antimicrobial. Its advantage is that it lacks some of the downsides of total propolis extracts, such as inability for unified standardization, which is cornerstone for implementing its therapeutic potential as a drug. The current paper provides an overview on the pharmacodynamic principles of CAPE. We present literature search outcomes form ClinicalTrials.gov database and from scientific publications, available on Scopus and Crossref databases. We take a round view of CAPE’s potential therapeutic implications in light of approved drugs with related modes of action.

Phenylpropenes such as chavicol, t-anol, eugenol, and isoeugenol are produced by plants as defense compounds against animals and microorganisms and as floral attractants of pollinators. Moreover, humans have used phenylpropenes since antiquity for food preservation and flavoring and as medicinal agents. Previous research suggested that the phenylpropenes are synthesized in plants from substituted phenylpropenols, although the identity of the enzymes and the nature of the reaction mechanism involved in this transformation have remained obscure. We show here that glandular trichomes of sweet basil (Ocimum basilicum), which synthesize and accumulate phenylpropenes, possess an enzyme that can use coniferyl acetate and NADPH to form eugenol. Petunia (Petunia hybrida cv. Mitchell) flowers, which emit large amounts of isoeugenol, possess an enzyme homologous to the basil eugenol-forming enzyme that also uses coniferyl acetate and NADPH as substrates but catalyzes the formation of isoeugenol. The basil and petunia phenylpropene-forming enzymes belong to a structural family of NADPH-dependent reductases that also includes pinoresinol-lariciresinol reductase, isoflavone reductase, and phenylcoumaran benzylic ether reductase.

The oxidative phosphorylation (OXPHOS) system consists of five multiprotein complexes and two mobile electron carriers embedded in the lipid bilayer of the mitochondrial inner membrane. With the exception of complex II and the mobile carriers, the other parts of the OXPHOS system are under dual genetic control. Due to this bigenomic control, the inheritance of OXPHOS system defects is either maternal, in the case of mitochondrial DNA mutations, autosomal or X-linked, in the case of nuclear gene defects. In this review, our current genetic understanding of OXPHOS system enzyme deficiencies will be summarized, and future directions that the field might take to unravel so-far genetically unresolved OXPHOS system enzyme deficiencies will be described, with special emphasis on complex I biogenesis.

A biosensor is an analytical device that consists of an immobilized biocomponent in conjunction with a transducer, and represents a synergistic combination of biotechnology and microelectronics. This review summarizes the use of biosensors for detecting and quantifying heavy metal ions. Heavy metal contamination is of serious concern to human health since these substances are non-biodegradable and retained by the ecological system. Conventional analytical techniques for heavy metals (such as cold vapour atomic absorption spectrometry, and inductively coupled plasma mass spectrometry) are precise but suffer from the disadvantages of high cost, the need for trained personnel and the fact that they are mostly laboratory bound. Biosensors have the advantages of specificity, low cost, ease of use, portability and the ability to furnish continuous real time signals. The analysis of heavy metal ions can be carried out with biosensors by using both protein (enzyme, metal-binding protein and antibody)-based and whole-cell (natural and genetically engineered microorganism)-based approaches.

The principles and recent progress in the research and development of photobiological hydrogen production are reviewed. Cyanobacteria produce hydrogen gas using nitrogenase and/or hydrogenase. Hydrogen production mediated by native hydrogenases in cyanobacteria occurs under in the dark under anaerobic conditions by degradation of intracellular glycogen. In vitro and in vivo coupling of the cyanobacterial photosynthetic system with a clostridial hydrogenase

In this study the applicability of membrane distillation (MD) was investigated to produce black-currant juice concentrate. For the experiments enzyme treated, pre-filtered by microfiltration and pre-concentrated by reverse osmosis fruit juice was applied. The measurements were carried out in a hollow fibre membrane contactor. Two different temperature differences were investigated as driving force: 15 and 198C. The shape of the curves of the fluxes appeared very similar at both temperature differences, however in case of the higher driving force the observed flux was 80% higher than in case of the lower driving force. By the MD process the pre-treated initial juice with 228Brix total soluble solid (TSS) content was concentrated up to 58.28Brix.

The genes encoding enzymes of the tyrosinase family are strong candidates for coat color variation in mammals. To investigate their influence in domestic cat coat color, we determined the complete nucleotide coding sequence of the domestic cat genes tyrosinase (TYR)-a plausible candidate gene for the albino (C) locus, and tyrosinase related protein 1 (TYRP1)-a candidate gene for the brown (B) locus. Sequence variants between individuals exhibiting variation in pigmentation were submitted to association studies. In TYR, two nonsynonymous substitutions encoding TYR-G301R and TYR-G227W were associated with the siamese and burmese phenotypes of the albino locus, respectively. TYRP1 was mapped on chromosome D4 within 5 cM of a highly polymorphic microsatellite, previously found to be fixed in a cat breed selected for the chocolate (b) allele of the B locus, which reinforced TYRP1 as a candidate gene for the B locus in the domestic cat. Two DNA polymorphisms, one leading to a TYRP1-A3G substitution in the signal peptide and another to an in-frame insertion TYRP1-421ins17/18 caused by a donor splice site mutation in intron 6, were associated with the chocolate (b) allele. A premature UAG stop codon at position 100 of TYRP1 was associated with a second allele of the B locus, cinnamon (b l ). The results provide very strong evidence that the specific nucleotide variants of feline TYR (chromosome D1) are causative of the siamese (c s ) and burmese (c b ) alleles of the albino locus, as well as nucleotide variants of TYRP1 (chromosome D4) as specifying the chocolate (b) and cinnamon (b l ) alleles of the B locus.

Peroxisomes play an essential role in cellular lipid metabolism as exemplified by the existence of a number of genetic diseases in humans caused by the impaired function of one of the peroxisomal enzymes involved in lipid metabolism. Key pathways in which peroxisomes are involved include: (1.) fatty acid beta-oxidation; (2.) etherphospholipid biosynthesis, and (3.) fatty acid alpha-oxidation. In this paper we will describe these different pathways in some detail and will provide an overview of peroxisomal disorders of metabolism and in addition discuss the toxicity of the intermediates of peroxisomal metabolism as they accumulate in the different peroxisomal deficiencies.

Molybdenum cofactor deficiency (MoCD) is a rare metabolic disorder characterized by severe and rapidly progressive neurologic damage caused by the functional loss of sulfite oxidase, 1 of 4 molybdenum-dependent enzymes. To date, no effective therapy is available for MoCD, and death in early infancy has been the usual outcome. We report here the case of a patient who was diagnosed with MoCD at the age of 6 days. Substitution therapy with purified cyclic pyranopterin monophosphate (cPMP) was started on day 36 by daily intravenous administration of 80 to 160 μg of cPMP/kg of body weight. Within 1 to 2 weeks, all urinary markers of sulfite oxidase (sulfite, S-sulfocysteine, thiosulfate) and xanthine oxidase deficiency (xanthine, uric acid) returned to almost normal readings and stayed constant (>450 days of treatment). Clinically, the infant became more alert, convulsions and twitching disappeared within the first 2 weeks, and an electroencephalogram showed the return of rhythmic ele...

Solid tumours account for 90% of all cancers. Gene therapy represents a potential new modality for their treatment. Up to now, several approaches have been developed, but the most efficient ones are the viral vector based gene therapy systems. However, viral vectors suffer from several deficiencies: firstly most vectors currently in use require intratumoural injection to elicit an effect. This is far from ideal as many tumours are inaccessible and many may have already spread to other parts of the body, making them difficult to locate and inject gene therapy vectors into. Second, because of cell heterogeneity within a given cancer, the vectors do not efficiently enter and kill every cancer cell. Third, hypoxia, a prevalent characteristic feature of most solid tumours, reduces the ability of the viral vectors to function and decreases viral gene expression and production. Consequently, a proportion of the tumour is left unaffected, from which tumour regrowth occurs. Thus, cancer gene therapy has yet to realise its full potential. The facultative or obligate anaerobic bacteria have been shown to selectively colonise and regerminate in solid tumours when delivered systemically. Among them, the clostridial spores were easy to produce, stable to store and safe to use as well as having extensive oncolytic ability. However, research in animals and humans has shown that oncolysis was almost always interrupted sharply at the outer rim of the viable tumour tissue where the blood supply was sufficient. These clostridial spores, though, could serve as "Trojan horse" for cancer gene therapy. Indeed, various spores harbouring genes for cancerstatic factors, prodrug enzymes, or proteins or cytokines had endowed with additional tumour-killing capability. Furthermore, combination of these "Trojan horses" with conventional chemotherapy or radiation therapies often significantly perform better, resulting in the "cure" of solid tumours in a high percentage of animals. It is, thus, not too difficult to predict the potential outcomes for the use of clostridial spores as "Trojan horse" vectors for oncolytic therapy when compared with viral vector-mediated cancer therapy for it be replication-deficient or competent. However, to move the "Trojan horse" to a clinic, though, additional requirements need to be satisfied (i) target tumours only and not anywhere else, and (ii) be able to completely kill primary tumours as well as metastases. Current technologies are in place to achieve these goals.

Desulfovibrio vulgaris Hildenborough cytochrome c 3 contains four hemes in a low-spin state with bis-histidinyl coordination. Highspin forms of cytochrome c 3 can be generated by protonation of the axial ligands in order to probe spin equilibrium (low-spin/high-spin). The spin alterations occurring at acid pH, the associated changes in redox potentials, as well as the reactivity towards external ligands were followed by the conjunction of square wave voltammetry and UV-visible, CD, NMR and EPR spectroscopies. These processes may be used for modelling the action of enzymes that use spin equilibrium to promote enzyme activity and reactivity towards small molecules.

and total counts) correlated negatively. The overall assessment of soil activity, the soil respiration, did not correlate with either. Significant correlations were found between the principal components of pH, CLPP, EAA, and culturable and total number of bacteria, while no correlations were found to soil respiration, loss on ignition and soil moisture content. The pH in the test system might make differences between bacterial communities more detectable. The functional characterization of the bacterial communities by the growth based CLPP correlated positively to the bacterial community function assayed by the EAA, which is independent of growth and, hence, more closely reflects the in situ activities.

Infection of red blood cells (RBC) subjects the malaria parasite to oxidative stress. Therefore, efficient antioxidant and redox systems are required to prevent damage by reactive oxygen species. Plasmodium spp. have thioredoxin and glutathione (GSH) systems that are thought to play a major role as antioxidants during blood stage infection. In this report, we analyzed a critical component of the GSH biosynthesis pathway using reverse genetics. Plasmodium berghei parasites lacking expression of gamma-glutamylcysteine synthetase (c-GCS), the rate limiting enzyme in de novo synthesis of GSH, were generated through targeted gene disruption thus demonstrating, quite unexpectedly, that c-GCS is not essential for blood stage development. Despite a significant reduction in GSH levels, blood stage forms of pbggcs 2 parasites showed only a defect in growth as compared to wild type. In contrast, a dramatic effect on development of the parasites in the mosquito was observed. Infection of mosquitoes with pbggcs 2 parasites resulted in reduced numbers of stunted oocysts that did not produce sporozoites. These results have important implications for the design of drugs aiming at interfering with the GSH redox-system in blood stages and demonstrate that de novo synthesis of GSH is pivotal for development of Plasmodium in the mosquito.

Crude extracts were partially purified by organic solvents fractionation: best results (96% of proteins, 91% of total caseinolytic activity) were obtained by adding four volumes of cold acetone to the crude extract. This preparation (redissolved acetone precipitate, RAP) showed maximum activity (>80%) at pH 5-9, and exhibited high thermal stability (>90% of residual activity after heating for 60 min at 60 • C). The enzyme was completely inhibited by E-64 trans-epoxysuccinyl-leucyl-amido(4-guanidino)-butane and iodoacetic acid and activated by the addition of cysteine or ␤-mercaptoethanol; these results strongly suggest that the isolated protease should be included within the cysteine group, as all the other studied proteases belonging to the family Bromeliaceae. IEF-zymogram of RAP showed five bands (pI 7.3 to <9.3), most of them proteolytically actives, but only three of which (pI 7.6, 8.2 and 8.8) proved to be important. Ion exchange chromatography in DEAE-Sephadex, was selected to separate this active bands.

The role of the hexosamine biosynthetic pathway in diabetic decline in renal function in type 1 and type 2 diabetes nephropathy. The hexosamine biosynthetic pathway has been derived from an increase in mesangial matrix [2-5]. Imhypothesized to be involved in the development of insulin munohistochemical and biochemical studies indicated an resistance and diabetic vascular complications. In particular, it increased deposition of various collagen types, laminin, was demonstrated that hyperglycemia-induced production of and fibronectin, particularly in glomeruli of patients with transforming growth factor-␤ (TGF-␤1), a prosclerotic cytokine causally involved in the development of diabetic nephropdiabetes and in experimental animal models [6-11]. Nuathy. Several lines of evidence indicate that TGF-␤1 induction merous studies have shown that elevated glucose concenis mediated by the hexosamine pathway. In cultured mesangial trations can cause an increased expression of these comcells, high glucose levels induce TGF-␤1 production. This effect ponents of the extracellular matrix in cultured mesangial is eliminated by inhibition of glutamine : fructose-6-phosphatecells, which suggests that these cells are an useful model amidotransferase (GFAT), the rate-limiting enzyme of this pathway. Furthermore, stable overexpression of GFAT increased in studying the pathogenesis of diabetic nephropathy levels of TGF-␤1 protein, mRNA, and promoter activity. Inas-[10, 12, 13]. These in vitro studies provided evidence that much as stimulation or inhibition of GFAT increased or dethe high glucose-induced stimulation of mesangial and creased high glucose-stimulated activity of protein kinase C tubular matrix production is mediated by transforming (PKC), respectively, the observed effects appear to be transgrowth factor-␤1 (TGF-␤1) [14-17]. Early glomerular and duced by PKC. In similar experiments, involvement of the hexosamine pathway in hyperglycemia-induced production of tubular increase of TGF-␤1 expression has been shown cytokines (TGF-␣ and basic fibroblast growth factor [bFGF]) in experimental and human diabetic nephropathy [18-20].

A potentiometric biosensor based on urease was developed for the quantitative determination of urea concentration in aqueous solutions for biomedical applications. The urease was either physisorbed onto an electrodeposited polyaniline film (PANI), or immobilized on a layer-by-layer film (LbL) assembled over the PANI film, that was obtained by the alternate deposition of charged polysaccharides (carboxymethylpullulan (CMP) and chitosan (CHI)). In the latter case, the urease (Urs) enzyme was either physically adsorbed or covalently grafted to the LbL film using carbodiimide coupling reaction. Potentiometric responses of the enzymatic biosensors were measured as a function of the urea concentration in aqueous solutions (from 10 −6 to 10 −1 mol L −1 urea). Very high sensitivity and short response time were observed for the present biosensor. Moreover, a stability study showed a higher stability over time for the potentiometric response of the sensor with the enzyme-grafted LbL film, testifying for the protective nature of the polysaccharide coating and the interest of covalent grafting.

The use of microfluidic glass chips for continuous single-cell lysis and assay of internal-Galactosidase (-Gal) content is described. Cells were transported single file toward a Y-shaped mixing junction at which lytic agents were introduced by suction. Flow velocities of 100 and 40 m/s were used under protein denaturing [35 mM sodium dodecylsulfate (SDS)] and nondenaturing (0.1% Triton X-100) conditions, respectively. Complete and reproducible lysis of individual cells on-chip occurred within 30 s using Triton X-100 and 2 s when using SDS. Optimal concentrations of lysis and enzyme substrate reagents were determined using microtitre plate and chip-based procedures. Fluorescence peaks, due to the enzymatic product fluorescein mono-D-galactopyranoside (FMG), were detected downstream of the mixing and cell lysis point for the reaction of-Gal with 200 M of the fluorogenic substrate fluorescein-diD -galactopyranoside (FDG). FMG fluorescence was observed from cells preincubated with FDG off-chip then subsequently lysed on-chip with SDS. Unincubated cells were mixed on-chip with both FDG and Triton X-100, each individual cell generating FMG fluorescence downstream of the mixing point detected within 2 min of mixing. In contrast, viable cells incubated with FDG required 1 h or more in order to generate significant signal in a flow cytometer.

The native b-D-glucan exohydrolase isoenzyme ExoI from barley seedlings, designated HvExoI, was the first GH3 glycoside hydrolase, for which a crystal structure was determined. A precise understanding of relationships between structure and function in this enzyme has been gained by structural and enzymatic studies. To allow testing of hypotheses gained from these studies, an efficient system for expression of HvExoI in Pichia pastoris was developed using a codon-optimized cDNA. Protein expression at a temperature of 20°C yielded a recombinant enzyme, designated rHvExoI, which had molecular masses of 70-110 kDa due to heavy glycosylation at Asn221, Asn498 and Asn600, the three sites of N-glycosylation in native HvExoI. Most of the N-linked carbohydrate could be removed from rHvExoI, resulting in N-deglycosylated rHvExoI with a substantially decreased molecular mass of 67 kDa. rHvExoI was able to hydrolyse barley (1,3;1,4)-b-D-glucan, laminarin and lichenans. The catalytic efficiency value k cat /K M of rHvExoI with barley (1,3;1,4)-b-D-glucan was similar to that reported for native HvExoI. Further, laminaribiose, cellobiose and gentiobiose were formed through transglycosylation reactions with 4-nitrophenyl b-D-glucoside and barley (1,3;1,4)-b-D-glucan. Overall, the biochemical properties of rHvExoI were similar to those reported for native HvExoI, although differences were seen in thermostabilities and hydrolytic rates of certain blinked glucosides.

Lignocellulosic materials pretreated using liquid hot water (LHW) (220~ 5 MPa, 120 s) were fermented to ethanol by batch simultaneous saccharification and fermentation (SSF) using Saccharomyces cerevisiae in the presence of Trichoderma reesei cellulase. SSF of sugarcane bagasse (as received), aspen chips (smallest dimension 3 mm), and mixed hardwood flour (-60 +70 mesh) resulted in 90% conversion to ethanol in 2-5 d at enzyme loadings of 15-30 FPU/g. In most cases, 90% of the final conversion was achieved within 75 h of inoculation. Comminution of the pretreated substrates did not affect the conversion to ethanol. The hydrolysate produced from the LHW pretreatment showed slight inhibition of batch growth of S. cerevisiae. Solids pretreated at a concentration of 100 g/L were as reactive as those pretreated at a lower concentration, provided that the temperature was maintained at 220~

Previous works revealed the presence of the nPKC enzyme p105 in hemocytes of M. galloprovincialis Lmk. Specific mussel antibodies were obtained from mouse and used in confocal microscopy and Western blotting. These techniques allowed the observation of p105 cytosol-to-membrane translocation induced by TPA for the first time in hemocytes of molluscs. The incubation of mussel immune cells with TPA for longer than 30 min also triggered a down-regulation process. Mussel hemocytes are an excellent model to study the molecular processes of innate immunity.

The modeling of the severe acute respiratory syndrome coronavirus helicase ATPase catalytic domain was performed using the protein structure prediction Meta Server and the 3D Jury method for model selection, which resulted in the identification of 1JPR, 1UAA and 1W36 PDB structures as suitable templates for creating a full atom 3D model. This model was further utilized to design small molecules that are expected to block an ATPase catalytic pocket thus inhibit the enzymatic activity. Binding sites for various functional groups were identified in a series of molecular dynamics calculation. Their positions in the catalytic pocket were used as constraints in the Cam-bridge structural database search for molecules having the pharmacophores that interacted most strongly with the enzyme in a desired position. The subsequent MD simulations followed by calculations of binding energies of the designed molecules were compared to ATP identifying the most successful candidates, for likely inhibitors-molecules possessing two phosphonic acid moieties at distal ends of the molecule.

Fatty acid hydroperoxide lyase (HPO-lyase) was puri®ed 300-fold from tomatoes. The enzymatic activity appeared to be very unstable, but addition of Triton X 100 and b-mercaptoethanol to the buer yielded an active enzyme that could be stored for several months at À808C. The enzyme was inhibited by desferoxamine mesylate (desferal), 2-methyl-1,2-di-3-pyridyl-1-propanone (metyrapone), nordihydroguaiaretic acid (NDGA), n-propyl gallate and butylated hydroxyanisole, suggesting the involvement of free radicals in the reaction mechanism and the existence of a prosthetic group in the active center. However, no heme group could be demonstrated with the methods commonly used to identify heme groups in proteins. Only 13-hydroperoxides from linoleic acid (13-HPOD) and a-linolenic acid (a-13-HPOT) were cleaved by the tomato enzyme, with a clear preference for the latter substrate. The pH-optimum was 6.5, and for concentrations lower than 300 mM a typical Michaelis±Menten curve was found with a K m of 77 mM. At higher a-13-HPOT concentrations inhibition of the enzyme was observed, which could (at least in part) be attributed to 2E-hexenal. A curve of the substrate conversion as a function of the enzyme concentration revealed that 1 nkat of enzyme activity converts 0.7 mmol a-13-HPOT before inactivation. Headspace analysis showed that tomato HPO-lyase formed hexanal from 13-HPOD and 3Z-hexenal from a-13-HPOT. A trace of the latter compound was isomerized to 2Ehexenal. In addition to the aldehydes, 12-oxo-9Z-dodecenoic acid was found by GC/MS analysis. To a small extent, isomerization to 12-oxo-10E-dodecenoic acid occurred. #

The role of genes involved in sucrose catabolism was investigated with a view to designing effective prebiotic substrates to encourage the growth of Bifidobacterium in the gut. Two gene clusters coding for sucrose utilisation in Bifidobacterium longum NCC2705 were identified in the published genome. The genes encoding putative sucrose degrading enzymes, namely, the scrP (sucrose phosphorylase) and the cscA (β-fructofuranosidase), were cloned from B. longum NCIMB 702259 T and expressed in Escherichia coli DH5α. Both complemented the sucrase negative phenotype of untransformed cells and showed specific sucrase activity. Transcriptional analysis of the expression of the genes in B. longum grown in the presence of various carbohydrate substrates showed induction of scrP gene expression in the presence of sucrose and raffinose, but not in the presence of glucose. The cscA gene showed no increased transcription in B. longum grown in the presence of any of the carbohydrates tested. Phylogenetic analysis indicates that the B. longum CscA protein belongs to a distinct phylogenetic cluster of intracellular fructosidases, which specifically cleave the shorter fructose oligosaccharides.

A large number of micropropagated Euphorbia millii shoots from temporary immersion bioreactor showed thick broad leaves that were translucent, wrinkled and/or curled and brittle, symptoms of hyperhydricity. The environment inside bioreactor normally used in plant micropropagation is characterised by high relative humidity, poor gaseous exchange between the internal atmosphere of the bioreactor and its surrounding environment, and the accumulation of ethylene, conditions that may induce physiological disorders. A comparison of hyperhydric shoots (HS) with normal plants shows marked increase in malondialdehyde (MDA) content in HS plants. MDA, a decomposition product of polyunsaturated fatty acids hydroperoxides, has been utilized very often as a suitable biomarker for lipid peroxidation, which is an effect of oxidative damage. This hypothesis is also confirmed by the higher lipoxygenase (LOX) activity in HS plants. The potential role of antioxidant enzymes in protecting hyperhydric shoots from oxidative injury was examined by analyzing enzyme activities and isozyme profiles of hyperhydric and non-hyperhydric leaves of E. millii. Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity were significantly higher in hyperhydric tissue as compared to non-hyperhydric normal leaf tissue. After native polyacrylamide gel electrophoresis (PAGE) analysis, seven SOD isoenzymes were detected and the increase in SOD activity observed in hyperhydric tissue seemed to be mainly due to Mn-SOD and Cu/Zn-SOD. The activity of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) was proportionally increased in HS tissue compared to normal leaves indicating a crucial role in eliminating toxic H 2 O 2 from plant cells. The depletion of GSH and total glutathione in spite of higher GR activities observed in HS tissue indicates that mechanism of antioxidant defense was by enhanced oxidation of GSH to GSSG by DHAR yielding ascorbate (AA). The antioxidant metabolism has been shown to be important in determining the ability of plants to survive in hyperhydric stress and the up regulation of these enzymes would help to reduce the build up of ROS.

The cysteine-rich N-terminal domain of the micronemal adhesive protein MIC1 (MIC1-NT) from Toxoplasma gondii was cloned, expressed in Escherichia coli and purified. MIC1-NT is amenable to structural studies as shown by preliminary NMR and X-ray analysis. Positive results with two further micronemal proteins indicate that our strategy has wider application.

Fructose 2,6-bisphosphate is present at high concentrations in many established lines of transformed cells. It plays a key role in the maintenance of a high glycolytic rate by coupling hormonal and growth factor signals with metabolic demand. The concentration of fructose 2,6-bisphosphate is controlled by the activity of the homodimeric bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2). We report here the PFKFB-3 gene expression control by insulin in the human colon adenocarcinoma HT29 cell line. The incubation of these cells with 1 WM insulin resulted in an increase in the PFK-2 mRNA level after 6 h of treatment, this effect being blocked by actinomycin D. Furthermore, insulin induced ubiquitous PFK-2 protein levels, that were evident after a lag of 3 h and could be inhibited by incubation with cycloheximide. ß

IC 50 value obtained for the inhibition of COX-2 by phycocyanin is much lower (180 nM) as compared to those of celecoxib (255 nM) and rofecoxib (401 nM), the wellknown selective COX-2 inhibitors. In the human whole blood assay, phycocyanin very efficiently inhibited COX-2 with an IC 50 value of 80 nM. Reduced phycocyanin and phycocyanobilin, the chromophore of phycocyanin are poor inhibitors of COX-2 without COX-2 selectivity. This suggests that apoprotein in phycocyanin plays a key role in the selective inhibition of COX-2. The present study points out that the hepatoprotective, anti-inflammatory, and anti-arthritic properties of phycocyanin reported in the literature may be due, in part, to its selective COX-2 inhibitory property, although its ability to efficiently scavenge free radicals and effectively inhibit lipid peroxidation may also be involved.

The antimicrobial peptide human α-defensin 5 (HD5) is expressed in Paneth cells, secretory epithelial cells in the small intestine. Unlike other characterized defensins, HD5 is stored in secretory vesicles as a propeptide. The storage quantities of HD5 are ∼90-450 µg per cm 2 of mucosal surface area, which is sufficient to generate microbicidal concentrations in the intestinal lumen. HD5 peptides isolated from the intestinal lumen are proteolytically processed forms-HD5(56-94) and HD5(63-94)-that are cleaved at the Arg 55 -Ala 56 and Arg 62 -Thr 63 sites, respectively.We show here that a specific pattern of trypsin isozymes is expressed in Paneth cells, that trypsin colocalizes with HD5 and that this protease can efficiently cleave HD5 propeptide to forms identical to those isolated in vivo. By acting as a prodefensin convertase in human Paneth cells, trypsin is involved in the regulation of innate immunity in the small intestine.

Although the precise mechanisms explaining loss of, and failure to regain, function after spinal cord injury are unknown, there is increasing interest in the role of "secondary cell death." One prevalent theme in cell loss in other regions of the CNS involves apoptosis executed by the intracellular caspase proteases. A recent study demonstrated that spinal cord injury rapidly increased the activation of caspase-3. Our previous studies demonstrated peak apoptosis in three of four cellular compartments 3 days after controlled contusion in the rat. We have extended these analyses to include enzyme and substrate studies of caspase subfamilies both in rostral and in caudal adjacent segments compared to the lesion site. Although presumed activation of programmed proenzyme is considered the mechanism for enhanced caspases, our novel analyses were designed to detect upregulation of gene expression. We surveyed traumatically injured spinal cord for caspase family messages with a modified differential mRNA display approach and found that the caspase-3 (CASP3) message was present and upregulated severalfold after injury. Our results clearly demonstrate that cell death in the spinal cord occurs after posttranslational activation of caspases that follow, at least for caspase-3, initial upregulation of CASP3 mRNA levels.

The cacti family is a morphologically heterogeneous group comprising 100 genera and about 1500 species (Hernandez and Barcenas, 1996). With the exception of one genus, all members of this family are native to America (Hernandez and Barcenas, 1996). There are three subfamilies, Opuntioideae, Cactoideae, and Pereskioideae (Gibson and Nobel, 1986). DNA isolation from cacti is notoriously difficult because they contain high amounts of polysaccharides and secondary metabolites which form insoluble complexes with nucleic acids during extraction (Guillemaut and Marechal-Drouard, 1992). Like in other groups of plants, the secondary metabolites and polysaccharides in cacti inhibit enzyme action (Porebski et al., 1997). The polysaccharides are visually evident by their viscous, glue-like texture and they make the DNA unmanageable when pipeting and hard to amplify by the polymerase chain reaction (PCR) (Poresbski et al., 1997). We report an easy and inexpensive protocol to isolate DNA from cacti. We used this method to isolate DNA from 85 species (170 individuals) of 39 genera of the subfamilies Pereskioideae, Opuntioidea, and Cactoideae. This procedure is a modification of a protocol described by De la Cruz et al. (1995) for the Cacti family. It requires only a few grams of tissue and does not require destruction of the whole plant to produce high molecular weight genomic DNA. The DNA from this procedure can be amplified consistently by PCR and used for RAPD analysis.

Cellulose nanofibers with a size range of 5–100 nm have the potential to be a low cost renewable material that has application in a range of products. However, current chemical methods to produce crystalline nanofibers suffer from low yields and high chemical costs, while mechanical methods require high energy costs. Methods to lower the energy costs of the mechanical methods have not been well documented in the literature. A bleached softwood kraft pulp was processed using a mechanical dispersion mill and a homogenizer to produce cellulose nanofibers. Two different commercial enzymes were used to pretreat the wood fibers before the mechanical treatments. The resulting nanofibers were characterized by light microscopy, atomic force microscopy, and inverse gas chromatography. Results indicate that the dispersion mill does not affect the overall pulp fiber fibrillation, but does help prepare the sample for the homogenizer. Most fibrillation occurs after three passes through the homogenizer. The enzyme pretreatment has little effect on the size of the fibers, but does allow for higher solids to pass through the homogenizer without clogging. The dispersion component of surface energy of the resulting nanofibrils is impacted by the type of enzyme used. The measurement of acid–base properties proved to be challenging using current IGC experimental protocols.

EPR spectroscopic techniques have been developed for the measurement of oxygen and nitric oxide in vivo. Specifically, the methods for in vivo measurement of these molecules has been applied to the study of septic shock, utilising an experimental murine model developed in our laboratory. Oxygen was measured as pO 2 by the particlulate probes Gloxy and LiPc, which were surgically implanted at specific sites in tissues, and the soluble probe Trityl, which was administered intravenously. Nitric oxide was measured as the NO-Fe-(DETC) 2 complex after administration of Fe 2+ and DETC. LPS was seen to significantly decrease liver oxygen measured across the lobule and at the sinusoids by the Gloxy probe; there was a corresponding increase in nitric oxide both in the liver and systemically. The nitric oxide most likely originated from increased iNOS enzyme in the liver as demonstrated by Western blotting and the localisation of nitric oxide to the liver was confirmed with EPR imaging. LPS also caused a decrease in cerebral blood and tissue oxygenation, the rate of which was found to be dependent on the blood oxygenation. The development and applications of these in vivo EPR techniques for biomedical research and diagnostics is discussed.

Our objectives were to determine postnatal changes in the maximal enzyme activity (V ) and enzyme affinity (K ) max m of jejunal mucosal membrane-bound alkaline phosphatase, aminopeptidase N and sucrase using a porcine model which may more closely resemble the human intestine. Jejunal brush border membrane was prepared by Mg -precipitation 2q and differential centrifugation from pigs of suckling (8 days), weaning (28 days), post-weaning (35 days) and adult (70 days) stages. p-Nitrophenyl phosphate (0-8 mM), L-alanine-p-nitroanilide hydrochloride (0-28 mM) and sucrose (0-100 mM) were used in alkaline phosphatase, aminopeptidase N and sucrase kinetic measurements. V of alkaline max phosphatase was the lowest in the adult (4.27 mmolØmg proteinØmin ), intermediate in the suckling (9.75 y1 y1 mmolØmg proteinØmin ) and the highest in the weaning and post-weaning stage (12.83 and 10.40 mmolØmg yl yl yl proteinØmin ). K of alkaline phosphatase was high in the suckling and weaning stages (5.14 and 9.93 mM) and low yl m in the adult (0.66 mM). V of aminopeptidase N was low in the suckling (7.04 mmolØmg protein Ømin ) and high y1 y1 max in the post-weaning stage (13.36 mmolØmg proteinØmin ). K of aminopeptidase N was the highest in the two yl yl m weaning stages (2.96 and 3.39 mM), intermediate in the adult (2.33 mM) and the lowest in the suckling stage (1.66 mM). V of sucrase increased from the suckling to the adult (0.48-1.30 mmolØmg proteinØmin ). K of sucrase yl yl max m ranged from 11.19 to 16.57 mM. There are dramatic postnatal developmental changes in both the maximal enzyme activity and enzyme affinity of jejunal brush border membrane-bound alkaline phosphatase, aminopeptidase N and sucrase in the pig. ᮊ

A key characteristic of apoptosis is its regulated nuclear degradation. Apoptosis-like nuclear degradation also occurs in the ciliated unicellular organism, Tetrahymena thermophila. Chromatin of the macronucleus undergoes massive condensation, a process that can be blocked by caspase inhibitors. The nucleus becomes TUNEL-positive, and its DNA is cleaved into nucleosome-sized fragments. In a matter of hours the macronucleus is completely degraded, and disappears. The condensed nucleus sequesters acridine orange, which means that it might become an acidic compartment. We therefore asked whether lysosomal bodies fuse with the condensed macronucleus to form an autophagosome. We monitored acid phosphatase (AP) activity, which is associated with lysosomal bodies but is not found in normal nuclei. We find that after the macronucleus condenses AP activity is localized in cap-like structures at its cortex. Later, after the degrading macronucleus loses much of its DNA, acid phosphatase deposits appear deeper within the nucleus. We conclude that although macronuclear elimination is initiated by an apoptosis-like mechanism, its final degradation may be achieved through autophagosomy. Cell Death and Differentiation (2001) 8, 289 ± 297.

and aggression during the breeding season. However, the role of T in year-round territorial birds is still enigmatic, especially in tropical birds. Spotted antbirds (Hylophylax n. naevioides) defend territories in the Panamanian rainforest year-round but have low plasma T levels (0.1-0.2 ng/ml), except during brief periods of social challenges. We manipulated T action in captive male Spotted antbirds to test whether this hormone is involved in the regulation of song and aggression. Timplants increased plasma androgen levels (T and dihydrotestosterone) and enhanced song in nonbreeding males. During a staged male-male encounter, T-implanted males sang more and were more aggressive than controls. In a second experiment, we blocked the two known T actions: its binding to androgen receptors and its conversion into estradiol by the enzyme aromatase. For this, we administered the androgen receptor antagonist flutamide (Flut) in combination with the aromatase inhibitor 1-4-6 androstatrien-3,17-dione (ATD) to birds in breeding condition. ATD ؉ Flut treatment significantly elevated plasma levels of luteinizing hormone, presumably via the lack of T feedback from its receptors. ATD ؉ Flut-treated birds gave fewer spontaneous songs than control-implanted males. During staged male-male encounters, ATD ؉ Flut-treated males did not sing at all and showed reduced aggressive behavior. Our data indicate that T can regulate aggressive behavior in these tropical birds. Although plasma T levels can be low year-round, Spotted antbirds may use T either by secreting it briefly during social challenges, by having a high sensitivity to T action, or by enzymatically converting circulating T precursors directly at the site of action. We hypothesize that plasma T levels are kept low in these year-round territorial birds to avoid potentially detrimental effects of tonically elevated T. Future treatment of nonbreeding birds with ATD ؉ Flut will reveal whether T is indeed involved year-round in regulating aggressive behavior.

Background: Increasing evidence suggesting a link between cholesterol metabolism in the brain and Alzheimer's disease provides a rationale for studies of the enzymes involved in the maintenance of an accurate neuronal cholesterol balance. Cytochrome P450 46A 1 (CYP46A 1) catalyzes the 24Shydroxylation of cholesterol, the first step in the only pathway of cholesterol elimination from the brain in humans. CYP46A1 is a recently cloned enzyme, and it has not yet been purified and characterized at a biochemical level. Objectives: To develop an efficient Escherichia coli expression system for CYP46A1, partially purify the enzyme and characterize its kinetic and substrate binding properties. Methods. Molecular biology techniques were used to express CYP46A1 in E. coil; methods of protein chemistry to partially purify the enzyme; spectral assay to determine substrate binding properties of CYP46A1; the in vitro reconstituted system to measure Km and kcat; gas chromatography/mass-spectrometry and liquid chromatography/mass-spectrometry to identify CYP46A1 metabolites in the incubations with substrates other than cholesterol. Results: CYP46A1 was expressed in E. coli at 150 nmol/1 culture levels and partially purified. Novel, previously unsuspected activities were discovered. In addition to the known cholesterol 24S-hydroxylase activity, CYP46A1 was found to further hydroxylate the product, 24S-hydroxycholesterol, and to metabolize a number of naturally occurring cholesterol derivatives that differ in the length of the steroid side chain, position of the double bond, and substitutions in the steroid nucleus and the side chain (progesterone, testosterone, cortisol, 7a-hydroxycholesterol, and cholestauol). Several drugs that were tested were also metabolized (dextromethorphau, diclofenac, and phenacetin). Catalytic efficiencies (the kcat/Km ratio, min -1 .ram -1) of CYP46A1 for cholesterol and 24S-hydroxholesterol were determined (0.002 and 0.54, respectively). The data indicate that cholesterol is a worse substrate for CYP46A1 than 24Shydroxycholesterol. Low rate of cholesterol hydroxylation by CYP46A1 likely reflects physiological requirements for the slow cholesterol turnover in the brain. Conclusions: CYP46A1 has a broad substrate specificity and its biological role may not be limited to cholesterol 24S-hydroxylatlon: this enzyme may participate in the metabolism of neurosteroids and drugs that can cross the blood-brain barrier and play a regulatory role by producing and further metabolizing a biologically active product 24S-hydroxycholesterol.

The Ag-AS technique was used for staining the polytene chromosomes of D. melanogaster and D. lurnmei. Bands were stained dark reddish-brown, interbands light yellow. A toromere was heavily stained on the sixth chromosome of D. lummei. The staining intensity of nucleoli was lower than that of chromosomes. During a prolonged staining ectopic threads and the nonhomogeneous structure of nucleoli were revealed. Pretreatment with RNase caused slight changes in the silver staining pattern of chromosomes; pretreatment with DNase did not result in any visible changes, while after preincubation with proteolytic enzymes chromosome morphology was destroyed. Hyaluronidase and lysozyme removed the silver-reducing components from chromosomes without destroying the general chromosome structure. Each of these two enzymes acts specifically: hyaluronidase affects the morphology of chromosomes, but not nucleoli and bands at heat shock puffs, whereas the action of lysozyme is probably evenly distributed between chromosomes and nucleoli.

The present study investigated the dynamics of nutrient utilization and various growth and physiological parameters during in vitro proliferation of apple root stock 'M9 EMLA' in two different bioreactor systems, i.e. temporary and continuous immersions. Individual shoots obtained from temporary immersion system had higher dry mass and were of better quality than those obtained from continuous immersion. In continuous immersion bioreactor, apple shoots appeared to utilize more nutrients from liquid culture medium than that from temporary immersion. The shoot growth was limited by the availability of phosphate and nitrogen in continuous immersion system. The shoots produced in temporary immersion bioreactor showed higher photosynthetic rate, maximum quantum yield of photosystem-II and slow but steady rate of nutrient absorption, indicating the occurrence of higher photomixotrophic metabolism. The study also showed that high level of antioxidant scavenging enzymes in shoots grown in continuous immersion system induced physiological changes to foster adaptation to stresses.