PROTEIN ANALYSIS

We describe the use of a tri(ethylene glycol)-terminated alkyltrichlorosilane to create a very thin, protein-resistant "self-assembled monolayer" coating on the inner surface of a fused-silica capillary. The same compound has been demonstrated previously on flat silica substrates to resist adsorption of many proteins. As a covalently bound capillary coating, it displays good resistance to the adsorption of cationic proteins, providing clean separations of a mixture of lysozyme, cytochrome c, ribonuclease A, and myoglobin for more than 200 consecutive runs. Electroosmotic flow (EOF) was measured as a function of pH; the coated capillary retains significant cathodal EOF, with roughly 50% of the EOF of an uncoated capillary at neutral pH, making this coating promising for applications requiring some EOF. The EOF was reasonably stable, with a 2.9% relative standard deviation during a 24 h period consisting of 72 consecutive separations of cationic proteins. Efficiencies for cationic protein separations were moderate, in the range of 190 000-290 000 theoretical plates per meter. The coating procedure was simple, requiring only a standard cleaning procedure followed by a rinse with the silane reagent at room temperature. No buffer additives are required to maintain the stability of the coating, making it flexible for a range of applications, potentially including capillary electrophoresis-mass spectrometry (CE-MS).

Control and analysis of protein aggregation is an increasing challenge to pharmaceutical research and development. Due to the nature of protein interactions, protein aggregation may occur at various points throughout the lifetime of a protein and may be of different quantity and quality such as size, shape, morphology. It is therefore important to understand the interactions, causes and analyses of such aggregates in order to control protein aggregation to enable successful products. This review gives a short outline of currently discussed pathways and induction methods for protein aggregation and describes currently employed set of analytical techniques and emerging technologies for aggregate detection, characterization and quantification. A major challenge for the analysis of protein aggregates is that no single analytical method exists to cover the entire size range or type of aggregates which may appear. Each analytical method not only shows its specific advantages but also has its limitations. The limits of detection and the possibility of creating artifacts through sample preparation by inducing or destroying aggregates need to be considered with each method used. Therefore, it may also be advisable to carefully compare analytical results of orthogonal methods for similar size ranges to evaluate method performance. ß

tein is used in several areas such as clinical analysis, food science and technology, biochemistry, protein chemistry, physiology. Five spectrophotometric methods are mostly used: biuret, Lowry, Bradford, Smith and UV absorption. In this review a general overview of these methods is presented (interferences, applications); other methodologies are also discussed.

Study compares the quality and quantity of sericin obtained from four sources, namely mulberry silk cocoons, silk flats, reeling silk waste and woven silk fabric. Sericin has been extracted using the conventional HTHP machine as well as IR heating machine. Results show that among the four sources, the maximum yield of sericin (28%) is obtained from silk fabric followed by 25% sericin obtained from silk flats. IR machine extraction gives ~13 times higher yield of sericin as compared to that obtained by HTHP at 100 0 C for 60 min. Sample obtained from IR extraction is also compared with the standard sample in terms of its physical properties, morphological structure, protein quality, protein structure, molecular weight and thermal behaviour using various analytical methods. The study proposes a protocol for assessing the quality of sericin protein. Since the source as well as the method of extraction can affect the properties of sericin, this protocol can be used to assess and determine the quality parameters of various sericin samples.

Electrospray ionisation (ESI) is a soft technique used in mass spectrometry (MS) analysis able to introduce a wide variety of analytes in the gas phase. Briefly, it consists in the application of a highvoltage to a solution to spray it through a small orifice. Hence, charged droplets and ions produced in the gas phase are directed towards a counter electrode. Based on the electrospray process itself, it is possible to carry out diverse experiments by adding functions at three different levels. The first one, taking advantage of the microfluidic nature, incorporates operation units such as mixers, reactors or chromatographic elements into the emitter. In this part, biphasic electrospray ionisation (BESI) sources will also be presented. The electrochemical properties of ESI provide the second level of functionalities. Electrochemistry, being inherent to the ESI process, has proved to be useful for different on-line purposes such as protein electrolysis or electrochemical-induced chemical derivatisation. Finally, the third level is the aerosol zone where a fine mist of charged droplets is formed. In this zone, desorption and extraction reactions can happen for different substrates and vapours. Hence, desorption and extractive electrospray ionisation (DESI and EESI, respectively) will be presented with an emphasis on the advantages brought by these methods. The present review does not intend to focus on the electrospray ionisation technique in itself but rather on the introduction of functional emitters for ESI-MS. The first part covers basic concepts required for developing the other sections. Then, the different options available at the three different levels are reviewed in order to highlight the potential of functional ESI in the growing field of mass spectrometry.

Capillary isoelectric focusing (CIEF) is a high resolution technique for protein separation. The on-column single point detector requires a mobilization step which lengthens the analysis time and causes an uneven resolution along the separation column. The real time and whole column imaging detection has been developed for performing CIEF without mobilization. Three types of imaging detection systems have been developed: optical absorption, refractive index gradient, and laser induced fluorescence. This technique provides a fast analysis speed (about 6 min) and a good resolution of 0.03 pH unit level. Using the absorption imaging detector, ampholyte-free IEF in tapered capillary is being demonstrated, which eliminates the interference of the expensive carrier ampholytes for protein detection in UV region. Recent advancements in this imaged CIEF technique as well as its applications are reviewed.

The purpose of the presented study here was to evaluate the prevalence of hepatitis C and B infections in patients diagnosed with or suspected of hepatic carcinoma or any other co-morbid with the objective of correlating diagnostic efficacy of alpha-fetoprotein (AFP). The study covers the period of January 1998 to January 2008. Study requirements such as medical history, reports and related information of patients from both genders (n = 93) with 50 males and 43 females were gathered for analysis and assessment ensuring that diagnostic confirmation of HCC existed with lab test of alpha-feto proteins available. Results shows the distribution analysis of hepatitis infections, the related co-morbid, the high-level AFP results and positive hepatitis profile as; hepatic carcinoma with HCV origin 33.33% (n = 31) and with HBV origin 41.93% (n = 39). For HCC of cirrhotic origin only, 10.75% (n = 10) was noted with no-infection whereas HCC of chronic liver disease [CLD] was around 13.97% (n = 13). As per medical setting (e.g. cirrhosis and chronic liver disease) and hepatic infections (B and C), 50 males patients were segregated as carcinoma patients with HCV and HBV, n = 15 and 24, whereas with cirrhosis and CLD, 6 and 5 respectively. Forty three female patients were also segregated in similar manner, thus resulting in groups of N = 14, 17, 5 and 7, respectively for HCV and HBV related carcinoma and non-malignant co-morbid such as cirrhosis and chronic liver disease. Regarding AFP levels, in males, 10.00% (n = 10) patients have AFP in the highest range of > 300.00 to < 768.00 ng/ml in HCV group; 46.00% (n = 23) in the range of > 150 to < 300 ng/ml in HBV and cirrhosis groups and 8% (n = 4) in the range of >300 to <520 ng/ml in HBV alone group. In females, 20.90% (n = 9) showed AFP ranges between >300 to 612 ng/ml in HCV group; and Address for Correspondence 18.60% (n = 8) in range of >150 to <300 ng/ml in HBV and CLD groups. Nonetheless the present studies neither irrevocably instate direct correlation between presence of hepatitis infections and formation of HCC nor ascertain that hepatitis infection were the 'only' cause of HCC. However efficacy of AFP has been found to be significant as a diagnostic tool where HCC was related to the hepatitis infections. The analytical data which was assessed after combining all factors is demonstrated here according to medical conditions diagnosed, risk factors obtained and the etiological factors related to the carcinoma and alpha-feto protein concentration characteristics.

Thirty-one clones of sweetpotatoes collected from some parts of Zimbabwe were used as inoculum sources to mechanically inoculate 13 experimental hosts: Chenopodium amaranticolor, C. quinoa, Cucumis sativus, Datura stramoniumitalic, Gomphrena globosa, Ipomoea purpurea, I. quamoclit, I. rubrocorulea, Nicotiana benthamiana, N. clevelandii, N. glutinosa, N. rustica, and N. tabacum. Systemic vein clearing was observed in N. benthamiana inoculated with buffered sap from nine clones. Purification of the vein clearing inducing agent from one of the sweetpotato clones gave yields ranging from 2 to 17 mg/kg and the A260nm/A280nm was around 1.2. Electron microscopy revealed flexuous filamentous particles with a modal length of 830 nm. Protein analysis of purified virus preparations by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a major protein band of 40 kDa, and this was assumed to be the viral coat protein. Minor protein bands of 27, 37, and 46 kDa were also observed. ...

Carrier ampholyte-based capillary electrophoresis (CABCE) has recently been introduced as an alternative to CE (CZE) in the classical buffers. In this study, isoelectric BGEs were obtained by fractionation of Servalyt pH 4-9 carrier ampholytes to cuts of typical width of 0.2 pH unit. CABCE feasibility was examined on a series of insect oostatic peptides, i.e. proline-rich di-to decapeptides, and phosphinic pseudopeptides -tetrapeptide mimetics synthesized as a mixture of four diastereomers having the -P(O)(OH)-CH 2 -moiety embedded into the peptide backbone. With identical selectivity, the separation efficiency of CABCE proved to be as good as classical CE for the insect oostatic peptides and better for diastereomers of the phosphinic pseudopeptides. In addition, despite the numerous species present in the narrow pH cuts of carrier ampholytes, CABCE seems to be free of system zones that could hamper the analysis. Peak symmetry was good for moderately to low mobile peptides, whereas some peak distortion due to electromigration dispersion, was observed for short peptides of rather high mobility.

tein is used in several areas such as clinical analysis, food science and technology, biochemistry, protein chemistry, physiology. Five spectrophotometric methods are mostly used: biuret, Lowry, Bradford, Smith and UV absorption. In this review a general overview of these methods is presented (interferences, applications); other methodologies are also discussed.

one of the first steps in characterizing heparan sulfate (Hs) and its close relative heparin is to conduct disaccharide composition analysis. this provides an overall picture of the structure of the polysaccharide in terms of its constituent disaccharides. this is of importance, for example, in the initial characterization of spatially and temporally regulated structures. two protocols for conducting disaccharide analysis are presented here, both exploiting exhaustive digestion of the polysaccharide, yielding constituent disaccharides, by bacterial heparin lyases. the first method, suitable for microgram quantities of material, relies on the separation of the disaccharides by high-performance liquid chromatography (Hplc) coupled to ultraviolet absorbance detection and can be performed in 2 d. the second exploits reducing end-labeling with the fluorophore BoDIpY hydrazide, separation by Hplc, and subsequent fluorescence detection and quantitation. the latter is a high-sensitivity method that requires nanograms of starting material and has a detection limit in the low fmol range, and is thus the most sensitive method for disaccharide compositional analysis of Hs yet reported. Fluorescence detection can be routinely carried out in 3 d.

We introduce a framework for predicting novel protein-protein interactions (PPIs), based on Fisher's method for combining probabilities of predictions that are based on different data sources, such as the biomedical literature, protein domain and mRNA expression information. Our method compares favorably to our previous method based on text-mining alone and other methods such as STRING. We evaluated our algorithms through the prediction of experimentally found protein interactions underlying Muscular Dystrophy, Huntington's Disease and Polycystic Kidney Disease, which had not yet been recorded in protein-protein interaction databases. We found a 1.74-fold increase in the mean average prediction precision for dysferlin and a 3.09-fold for huntingtin when compared to STRING. The top 10 of predicted interaction partners of huntingtin were analysed in depth. Five were identified previously, and the other five were new potential interaction partners. The full matrix of human protein pairs and their prediction scores are available for download. Our framework can be extended to predict other types of relationships such as proteins in a complex, pathway or related disease mechanisms.

The dichloromethane extracts of the leaves, stem bark, bark and the roots of the three species of the primitive endemic genus Hortonia, H. angustifolia, H. floribunda and H. ovalifolia, collected from nine geographical locations ranging from lower elevations (84-420 m) to higher (2000 m) showed comparable HPLC profiles and mosquito larvicidal and antifungal activities; protein analysis of the leaves of the three species of Hortonia showed identical peaks and bands. The two major metabolites (4S)-4-methyl-2-(11-dodecynyl)-2-butenolide (2) and (4S)-4-methyl-2-(11-dodecenyl)-2-butenolide (3), which were previously reported from all three plants, showed potent larvicidal activities. Compound 2 was excessively high in the extracts of the stem bark and the roots of all three species amounting to approximately 38 and 60%, respectively. A minor new butenolide (4), (4S)-4-methyl-2-((2R)-hydroxy-11-dodecenyl)-2-butenolide, with much reduced larvicidal activity and ishwarane (1), which showed ...

Conformational clusters and consensus sequences for protein loops have been derived by computational analysis of their structures in a nonredundant set of 233 proteins with less than 25% sequence homology (Xray resolution better than 2.5 A Ê). Loops have been classi®ed into ®ve types (a± a, b± b links, b± b hairpins, a± b and b ± a) according to the secondary structures they embrace. Four variables have been used to describe the loop geometry, three angles and one distance between the secondary structure elements embracing the loop. Ramachandran angles (f, c) are used to de®ne the loop conformations within each brace geometry. All loops from the non-redundant set have been clustered by means of these geometric features. A total of 56 classes (9 a ±a, 11 b±b links, 14 b ±b hairpins, 13 a ± b and 9 b ± a) were identi®ed with consensus Ramachandran angles in the loops. These classes were divided into subclasses based on the brace geometry. This clustering procedure captures most of the clusters analysed by predominantly visual inspection methods and ®nds other clusters that have hitherto not been described. Consensus sequence patterns were identi®ed for the subclasses. An extensive characterisation of loop conformations has therefore been achieved and the computational approach is readily open to the incorporation of information from newly determined structures. These clusters should also enhance model building by comparison studies.

Here we describe a protocol for using force-clamp spectroscopy to precisely quantify the effect of force on biochemical reactions. a calibrated force is used to control the exposure of reactive sites in a single polyprotein substrate composed of repeated domains. the use of polyproteins allows the identification of successful single-molecule recordings from unambiguous mechanical unfolding fingerprints. Biochemical reactions are then measured directly by detecting the length changes of the substrate held at a constant force. We present the layout of a force-clamp spectrometer along with protocols to design and conduct experiments. these experiments measure reaction kinetics as a function of applied force. We show sample data of the force dependency of two different reactions, protein unfolding and disulfide reduction. these data, which can be acquired in just a few days, reveal mechanistic details of the reactions that currently cannot be resolved by any other technique.

Angelwing clam (Pholas orientalis) hydrolysate was prepared by hydrolysis using bromelain. The hydrolysate named as bromelain hydrolysate (BH) was then treated with β-cyclodextrin in the ratio of 1:0.8 (v/w) by physical mixing and kneading methods producing the physical mixed hydrolysate (PMH) and kneaded method hydrolysate (KMH), respectively. The masking effect of β-cyclodextrin on bitterness was evaluated based on sensory analysis, amino acid analysis and determination of flavor compound by gas chromatography-mass spectrometry (GC-MS) and field emission scanning electron microscope (FESEM). Sensory analysis showed that KMH has least bitter taste compared to BH. Amino acids analysis showed that hydrophobic amino acids content that contributed to the bitter taste were lower in KMH and PMH compared to BH. GC-MS analysis also showed that benzothiazole compounds were present in KMH. The absence of benzene, 1-phenyl-4-2-(2-cyano-2-phenylethyl) in KMH and PMH indicated that phenylalanine in BH had been masked by β-cyclodextrin. FESEM showed that the new solid phase formed by kneading method has a crystal structure which was completely different from the original morphology of BH and β-cyclodextrin. Therefore, the bitterness in BH had successfully been masked by β-cyclodextrin, thus indicates its potential to be used as food ingredient..

Prions are proteinaceous infectious agents responsible for the transmission of prion diseases. The lack of a procedure for cultivating prions in the laboratory has been a major limitation to the study of the unorthodox nature of this infectious agent and the molecular mechanism by which the normal prion protein (PrP C ) is converted into the abnormal isoform (PrP Sc ). Protein misfolding cyclic amplification (PMCA ), described in detail in this protocol, is a simple, fast and efficient methodology to mimic prion replication in the test tube. PMCA involves incubating materials containing minute amounts of infectious prions with an excess of PrP C and boosting the conversion by cycles of sonication to fragment the converting units, thereby leading to accelerated prion replication. PMCA is able to detect the equivalent of a single molecule of infectious PrP Sc and propagate prions that maintain high infectivity, strain properties and species specificity. A single PMCA assay takes little more than 3 d to replicate a large amount of prions, which could take years in an in vivo situation. Since its invention 10 years ago, PMCA has helped to answer fundamental questions about this intriguing infectious agent and has been broadly applied in research areas that include the food industry, blood bank safety and human and veterinary disease diagnosis.

Six major Hepatitis C virus (HCV) genotypes and hundreds of subtypes have been identified globally. All these genotypes are generally studied for epidemiology, their vaccine development and clinical management. This article comments the frequency distribution of various HCV genotypes circulate in different areas/districts of Khyber Pakhtoonkhaw Province of Pakistan. Sum of 415 HCV RNA PCR positive sera samples were tested by a molecular genotyping assay. Data analysis revealed that out of these 415 HCV RNA positive patients 243 were males and 172 were females. Distribution breakup of the patients was 135, 58, 51, 51, 36, 32, 6, 7and 9 patients come from the districts of Abbottabad, Mardan, Pehawar, Swat, Haripure, Swabi and Dera Ismail Khan, respectively. Out of the tested samples, genotype specific PCR fragments were observed in 299 (74.82%) patient serum samples. The distribution of genotypes of the typeable samples was as fallows: 3 patients (0.72%) each were infected with genotype 1a and genotype 1b; 240 patients (80.26%) of genotype 3a; 25 patients (6.00%) genotype 3b; and 28 patients (6.73%) were observed as with mixed genotypic infection. Sums of 116 serum samples (27.88%) were still found untypeable by the used molecular genotyping system. In conclusion, HCV genotypes 1a, 1b, 3a and 3b are distributed in various parts of KPK among which the genotype 3a is the most frequent genotype.

Capillary isoelectric focusing (CIEF) is a high resolution technique for protein separation. The on-column single point detector requires a mobilization step which lengthens the analysis time and causes an uneven resolution along the separation column. The real time and whole column imaging detection has been developed for performing CIEF without mobilization. Three types of imaging detection systems have been developed: optical absorption, refractive index gradient, and laser induced fluorescence. This technique provides a fast analysis speed (about 6 min) and a good resolution of 0.03 pH unit level. Using the absorption imaging detector, ampholyte-free IEF in tapered capillary is being demonstrated, which eliminates the interference of the expensive carrier ampholytes for protein detection in UV region. Recent advancements in this imaged CIEF technique as well as its applications are reviewed.

Antimicrobial peptides are components of innate immunity that is the first-line defense against invading pathogens for a wide range of organisms. Here, we describe the isolation, biological characterization and amino acid sequencing of a novel neutral Glycine/Leucinerich antimicrobial peptide from skin secretion of Leptodactylus pentadactylus named leptoglycin. The amino acid sequence of the peptide purified by RP-HPLC (C 18 column) was deduced by mass spectrometric de novo sequencing and confirmed by Edman degradation: GLLGGLLGPLLGGGGGGGGGLL. Leptoglycin was able to inhibit the growth of Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli and Citrobacter freundii with minimal inhibitory concentrations (MICs) of 8 mM, 50 mM, and 75 mM respectively, but it did not show antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus and Enterococcus faecalis), yeasts (Candida albicans and Candida tropicalis) and dermatophytes fungi (Microsporum canis and Trichophyton rubrum). No hemolytic activity was observed at the 2-200 mM range concentration. The amino acid sequence of leptoglycin with high level of glycine (59.1%) and leucine (36.4%) containing an unusual central proline suggests the existence of a new class of Gly/Leu-rich antimicrobial peptides. Taken together, these results suggest that this natural antimicrobial peptide could be a tool to develop new antibiotics.

Size-exclusion chromatography (SEC), also termed gel filtration (GF) and gel permeation chromatography (GPC), separates solutes according to decreasing size. Typical applications are desalting (buffer exchange), fractionation (purification) and size or size-heterogeneity analysis (mass determination). The theory of SEC is well established and simulations of experiments may readily be performed to aid in optimisation of experimental conditions, although optimisation often is not needed. Unlike many other chromatographic techniques separation using SEC allows a limited volume of sample since it is not concentrated on the column. The acceptable volume depends on column size and mode of separation. SEC is possibly the most common final step in protein purification because of its simplicity and its power to remove not only impurities but also aggregates of the target protein. Buffer exchange of protein preparations by SEC is a good alternative to ultrafiltration or dialysis methods becau...

Angelwing clam (Pholas orientalis) hydrolysate was prepared by hydrolysis using bromelain. The hydrolysate named as bromelain hydrolysate (BH) was then treated with β-cyclodextrin in the ratio of 1:0.8 (v/w) by physical mixing and kneading methods producing the physical mixed hydrolysate (PMH) and kneaded method hydrolysate (KMH), respectively. The masking effect of β-cyclodextrin on bitterness was evaluated based on sensory analysis, amino acid analysis and determination of flavor compound by gas chromatographymass spectrometry (GC-MS) and field emission scanning electron microscope (FESEM). Sensory analysis showed that KMH has least bitter taste compared to BH. Amino acids analysis showed that hydrophobic amino acids content that contributed to the bitter taste were lower in KMH and PMH compared to BH. GC-MS analysis also showed that benzothiazole compounds were present in KMH. The absence of benzene, 1-phenyl-4-2-(2-cyano-2-phenylethyl) in KMH and PMH indicated that phenylalanine...

The germination of loquat seeds faces certain problems. The present research was designed to study the promotion of the germination of loquat seeds by moist-chilling and GA 3 applications. The results showed that loquat seeds display an endogenous dormancy that can be released by moistchilling treatment for a certain period. In this respect, the best treatment was moist-chilling for 3 weeks at 5 AE 1 8C or 1 week of moist-chilling followed by soaking in 250 ppm GA 3 solution for 20 h. These treatments significantly increased germination percentage (88 and 85%, respectively) and decreased time to 50% germination (T50) (31.5 and 40.7 days, respectively) compared to control (51% and 56 days, respectively). Also, the characteristics of the obtained seedlings were much better than the control seedlings. In addition, the 3-week moist-chilled seeds contained the highest soluble protein concentration and were characterized by the synthesis of new protein band of 161.7 kDa that was absent in all other treatments. This treatment lead to the absence of five polypeptides bands (222.5, 201.5, 109.5, 71.1 and 49.3 kDa), which were synthesized in GA 3 treatment, and the presence of a higher number of polypeptide bands compared with those of other moist-chilling periods and the control treatments. However, increasing the moist-chilling period over 3 weeks significantly decreased both germination percentage and T50. The combination between GA3 and moist-chilling treatments produced differential effects on seed germination, soluble protein and the number of protein bands depending on the length of the moist-chilling period. Although GA3 application on unchilled seeds resulted in more synthesis of protein bands than other tested treatments, it did not improve the germination process. The concentration of soluble inorganic phosphorus of the tested seeds was negatively (r = À0.57 *) and the concentration of soluble organic phosphorus positively www.elsevier.com/locate/scihorti Scientia Horticulturae 105 (2005) 331-342

For on-line monitoring of bioprocesses present applications are mainly restricted to gas analysis, but several techniques have been improved recently: membrane probes, the application of MS/MS techniques, methods of correlating available on-line data like gas reaction rates with bioprocess characteristics using stoichiometric models and other empirical correlations. New ionization and ion separation methods for biomolecules are developing dramatically. Most striking developments in this area are improved desorption techniques, electrospray, the renaissance of time-of-flight instruments and new challenges in ion trap techniques. Enormous progress is made in the analysis of peptides and other biopolyreefs. Combinations with new separation techniques like capillary electrophoresis and capillary HPLC show new horizons in biomolecule analysis.

It is well known that the structure of BSA explains its capacity to interact both hydrophobically and hydrophilically with other biochemical species. In the native state the BSA molecule takes such a conformation that the polar residues of amino-acids are oriented towards the ...

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Six structurally similar and strongly cationic peptides belonging to the brevinin-1 family were isolated from skin secretions of the plains leopard frog Lithobates blairi and the lowland leopard frog Lithobates yavapaiensis on the basis of their antimicrobial activities. Brevinin-1BLc (FLPIIAGIAAKFLPKIFCTISKKC) from L. blairi represented the most potent peptide (MIC ¼ 25 mM Escherichia coli, MIC ¼ 1.5 mM Staphylococcus aureus, MIC ¼ 3 mM Candida albicans, LC 50 ¼ 9 mM human erythrocytes and LC 50 ¼ 6 mM HepG2 human hepatoma-derived cells). The appreciably lower antimicrobial potencies of brevinin-1Ya and-1Yc from L. yavapaiensis correlate with the decreases in cationicity produced by the amino acid substitutions Lys 11 / Asn (brevinin-1Ya) and Pro 14 / Glu (brevinin-1Yc). In addition, a peptide isolated from the skin secretions of L. yavapaiensis belonging to the ranatuerin-2 family (ranatuerin-2Ya; GLMDTIKGVAKTVAASWLDKLKCKIT GC) inhibited the growth of E. coli (MIC ¼ 50 mM) and S. aureus (MIC ¼ 50 mM). In contrast to brevinin-1BLc, ranatuerin-2Ya showed appreciably greater cytolytic activity against HepG2 cells (LC 50 ¼ 20 mM) than against erythrocytes (LC 50 > 100 mM).

The development and function of the vertebrate nervous system depend on specific interactions between different cell types. Two examples of such interactions are synaptic transmission and myelination. LGI1-4 (leucine-rich glioma inactivated proteins) play important roles in these processes. They are secreted proteins consisting of an LRR (leucine-rich repeat) domain and a so-called epilepsy-associated or EPTP (epitempin) domain. Both domains are thought to function in protein-protein interactions. The first LGI gene to be identified, LGI1, was found at a chromosomal translocation breakpoint in a glioma cell line. It was subsequently found mutated in ADLTE (autosomal dominant lateral temporal (lobe) epilepsy) also referred to as ADPEAF (autosomal dominant partial epilepsy with auditory features). LGI1 protein appears to act at synapses and antibodies against LGI1 may cause the autoimmune disorder limbic encephalitis. A similar function in synaptic remodelling has been suggested for L...

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METHODS. Spectrophotometric determination of total protein is used in several areas such as clinical analysis, food science and technology, biochemistry, protein chemistry, physiology. Five spectrophotometric methods are mostly used: biuret, Lowry, Bradford, Smith and UV absorption. In this review a general overview of these methods is presented (interferences, applications); other methodologies are also discussed.

The present study showed the occurrence of three species of Siphonaria, namely, S. ashgar, S, belcheri and S. kurracheensis on the rocky coast of Mubarak village, Karachi, Pakistan. Keeping in view the biomedical interest in the high molecular mass hemocyanin of the marine mollusc, the study provides for the first time, the basic knowledge about the presence and chemical composition of oxygen transport protein hemocyanin in Siphonaria species. The tissue homogenates of S. ashgar, S. belcheri and S. kurracheensis when subjected to polyacrylmaide gel electrophoresis under dissociating conditions showed one major band (ca. 400kDa) which corresponds to two hemocyanin subunits (named as Siphonaria subunit S1 and S2). Under reducing conditions this band splits into three major bands, one major approximately 400kDa corresponding to the subunit S1, while S2 splits to produce two i.e. a 280kDa fragment S2.1 (corresponding to six-FUs) and the other 100-120kDa fragment S2.2 (corresponding to two-FUs). The size exclusion and ion-exchange chromatography results further revealed two specific major peaks corresponding to the two hemocyanin subunits. We conclude that the subunit composition of hemocyanin from Siphonaria resembles to the hemocyanin of other reported gastropods and thus could also be subjected for further immunological investigations for their possible biomedical application.

Substantial research has been conducted on the immediate early 1 (ie-1) genes from the prototype baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) and the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV). In both cases ie-1 gene products have been implicated in transcriptional activation and repression. In this study an ie-1 homolog was identified from Trichoplusia ni single nucleocapsid polyhedrosis virus (TniSNPV). Nucleotide sequence analysis indicated that the TniSNPV ie-1 gene consists of a 2217 nucleotide open reading frame (ORF), encoding a protein with a molecular mass of 84.464 kDa. This represents the largest baculovirus ie-1 gene characterised to date. Of the seven ie-1 homologs identified to date, the TniSNPV ie-1 shared most sequence similarity with the ie-1 gene of Spodoptera exigua MNPV (SeMNPV) (41%). At the nucleotide level, expected TATA and CAGT motifs were found to precede each ie-1 ORF. At the protein level, it was confirmed that the N-termini are poorly conserved, but share the characteristic of having a high proportion of acidic amino acids. In addition it was found that N-terminal regions significantly matched the SET domain in the Swiss-Prot prosite database. The C-terminal regions of the deduced IE-1 sequences were found to be substantially more conserved than the N-termini. Several conserved motifs were identified in the C-terminal sequences. A phylogenetic tree of nine baculovirus IE-1 proteins was constructed using maximum parsimony analysis. The phylogenetic estimation of the ie-1 genes shows that TniSNPV is a member of the previously described lepidopteran NPV group II and it is most closely related to SeMNPV.

Here we describe protocols for the expression of human antibody fragments in Escherichia coli. antigen-specific clones are identified by soluble fragment elIsa and concentrated by periplasmic preparation. they are then further purified by affinity chromatography. this article provides an overview of expression and purification strategies for human antibody fragments, as well as detailed protocols for the identification of high-affinity binders and for affinity maturation.

Six major Hepatitis C virus (HCV) genotypes and hundreds of subtypes have been identified globally. All these genotypes are generally studied for epidemiology, their vaccine development and clinical management. This article comments the frequency distribution of various HCV genotypes circulate in different areas/districts of Khyber Pakhtoonkhaw Province of Pakistan. Sum of 415 HCV RNA PCR positive sera samples were tested by a molecular genotyping assay. Data analysis revealed that out of these 415 HCV RNA positive patients 243 were males and 172 were females. Distribution breakup of the patients was 135, 58, 51, 51, 36, 32, 6, 7and 9 patients come from the districts of Abbottabad, Mardan, Pehawar, Swat, Haripure, Swabi and Dera Ismail Khan, respectively. Out of the tested samples, genotype specific PCR fragments were observed in 299 (74.82%) patient serum samples. The distribution of genotypes of the typeable samples was as fallows: 3 patients (0.72%) each were infected with genotype 1a and genotype 1b; 240 patients (80.26%) of genotype 3a; 25 patients (6.00%) genotype 3b; and 28 patients (6.73%) were observed as with mixed genotypic infection. Sums of 116 serum samples (27.88%) were still found untypeable by the used molecular genotyping system. In conclusion, HCV genotypes 1a, 1b, 3a and 3b are distributed in various parts of KPK among which the genotype 3a is the most frequent genotype.

Prions are proteinaceous infectious agents responsible for the transmission of prion diseases. The lack of a procedure for cultivating prions in the laboratory has been a major limitation to the study of the unorthodox nature of this infectious agent and the molecular mechanism by which the normal prion protein (PrP C ) is converted into the abnormal isoform (PrP Sc ). Protein misfolding cyclic amplification (PMCA ), described in detail in this protocol, is a simple, fast and efficient methodology to mimic prion replication in the test tube. PMCA involves incubating materials containing minute amounts of infectious prions with an excess of PrP C and boosting the conversion by cycles of sonication to fragment the converting units, thereby leading to accelerated prion replication. PMCA is able to detect the equivalent of a single molecule of infectious PrP Sc and propagate prions that maintain high infectivity, strain properties and species specificity. A single PMCA assay takes little more than 3 d to replicate a large amount of prions, which could take years in an in vivo situation. Since its invention 10 years ago, PMCA has helped to answer fundamental questions about this intriguing infectious agent and has been broadly applied in research areas that include the food industry, blood bank safety and human and veterinary disease diagnosis.

Antimicrobial peptides are components of innate immunity that is the first-line defense against invading pathogens for a wide range of organisms. Here, we describe the isolation, biological characterization and amino acid sequencing of a novel neutral Glycine/Leucinerich antimicrobial peptide from skin secretion of Leptodactylus pentadactylus named leptoglycin. The amino acid sequence of the peptide purified by RP-HPLC (C 18 column) was deduced by mass spectrometric de novo sequencing and confirmed by Edman degradation: GLLGGLLGPLLGGGGGGGGGLL. Leptoglycin was able to inhibit the growth of Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli and Citrobacter freundii with minimal inhibitory concentrations (MICs) of 8 mM, 50 mM, and 75 mM respectively, but it did not show antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus and Enterococcus faecalis), yeasts (Candida albicans and Candida tropicalis) and dermatophytes fungi (Microsporum canis and Trichophyton rubrum). No hemolytic activity was observed at the 2-200 mM range concentration. The amino acid sequence of leptoglycin with high level of glycine (59.1%) and leucine (36.4%) containing an unusual central proline suggests the existence of a new class of Gly/Leu-rich antimicrobial peptides. Taken together, these results suggest that this natural antimicrobial peptide could be a tool to develop new antibiotics.

Background: Based on clinical representation of disease symptoms multiple sclerosis (MScl) patients can be divided into two major subtypes; relapsing remitting (RR) MScl (85-90%) and primary progressive (PP) MScl (10-15%). Proteomics analysis of cerebrospinal fluid (CSF) has detected a number of proteins that were elevated in MScl patients. Here we specifically aimed to differentiate between the PP and RR subtypes of MScl by comparing CSF proteins.