Papers by Philipp Novales-Li
Biomedical Chromatography, 1995
Optimal purification of immunoglobulin M (IgM) grown as spent tissue culture supernatant can be a... more Optimal purification of immunoglobulin M (IgM) grown as spent tissue culture supernatant can be achieved by a straightforward size-exclusion chromatography procedure. Preparative isolation of IgM was achieved by precipitation with saturated ammonium sulphate to achieve a 45% solution. IgM was then purified by gel filtration chromatography with a solution of 100 mM Tris + 150 mM NaCl at pH 8. Denaturing electrophoresis and Western immunoblotting revealed two prominent bands at 80 and 25 kDa, indicative of the heavy and light chains of IgM respectively. The specific immunoreactivity of this IgM was assessed by a modified enzyme antigen capture assay. In contrast to affinity and ion-exchange chromatographies, gel filtration allows retention of IgM immunoreactivity.
International Symposium on the Chemistry of Natural Products, May 29, 1988
Procedures for the preparative purification of human acetylcholinesterase (AChE; EC 3.1.1.7) have... more Procedures for the preparative purification of human acetylcholinesterase (AChE; EC 3.1.1.7) have been examined. Chromatofocussing showed caudate nucleus and cerebrospinal fluid (CSF) AChE eluting between isoelectric points (pi) of 5.2 - 5.57, whilst isoelectric focussing (IEF) revealed heterogeneous AChE isoforms migrating between pI 4.5 - 6. Affinity chromatography proved to be better in yielding highly pure samples. The ligands used were either acridinium or procainamide, which yielded brain AChE recoveries of 10.1% and 42.8%, respectively. Various other methods were used such as gel filtration and ion-exchange chromatography on a Pharmacia SMART System. Purified brain AChE was used as antigen to generate anti-AChE monoclonal antibodies (mAbs) by in vivo immunization. BMS-3E4, -7G10, and -9F4 reacted with erythrocyte (G2) AChE, whilst BMS-6D6 bound to brain (G4) AChE in dot blot, titration, and sedimentation experiments. Conversely, the four mAbs recognized only the G4 form and not the G 2 form on immunoblotted IEF and non-denaturing Clarke gels. Deglycosylation studies suggest that the four mAbs recognize a carbohydrate epitope linked to AChE. Four additional mAbs were raised by the novel method of in vitro immunization, using a synthetically-produced C-terminus peptide as antigen. BMS-5, -6, -7, and -8 are all IgMs which recognize soluble brain and not erythrocyte nor membrane-bound AChE in dot blots, IEF, and sedimentation analyses. Although immunocytochemistry of the above-mentioned mAbs did not demonstrate any positive cholinergic binding, the present mAbs may still offer potential clinical and biochemical applications.
Biomedical Chromatography, Nov 1, 1994
With the view of purifying soluble human brain acetylcholinesterase (AChE) into its separate isof... more With the view of purifying soluble human brain acetylcholinesterase (AChE) into its separate isoforms, various preparative chromatographic procedures were compared. Chromatofocusing of cerebrospinal fluid (CSF) AChE revealed two major activity peaks, whilst that of caudate nucleus AChE showed one major peak. Both CSF and caudate nucleus AChE eluted at isoelectric points (pI) of between 5.5 and 5.2. Chromatofocusing failed to separate AChE into its individual isoforms, based on qualitative isoelectric focusing. Preparative purification by affinity chromatography showed a better AChE yield with the use of procainamide as a ligand, vis-à-vis acridinium. Maximum recovery for CSF and caudate nucleus AChE was 10 and 43% using acridinium and procainamide, respectively. Qualitative analysis by SDS-PAGE of affinity-purified AChE revealed four major bands between 50 and 62 kDa, corresponding to the catalytic subunits of AChE as verified by an anti-AChE polyclonal antibody. A size-exclusion column also allowed brain AChE purification, with the latter eluting at a putative molecular mass of 310 kDa. Unfortunately, cation-exchange using the state-of-the-art SMART system failed to separate AChE into its isoforms. AChE aggregation is given as one major obstacle precluding good resolution of isoforms.
Immunopharmacology, Nov 1, 1996
The present study sought to examine the immunopharmacologic effects of suramin on splenocytes fro... more The present study sought to examine the immunopharmacologic effects of suramin on splenocytes from experimental allergic encephalomyelitis (EAE)-induced mice, taken in line with a previous observed action of suramin in ameliorating EAE. Suramin, a polysulfonated napthylurea, decreased the proliferation of T cells, in the presence of various stimuli, such as guinea pig myelin basic protein (MBP), mouse spinal cord homogenate (MSCH), bovine proteolipid protein (PLP), P1 (synthetic peptide 139-151 of PLP), and Mycobacterium tuberculosis (MTB). Suramin inhibited T cell proliferation in a dose-dependent manner. However, cytokine assays revealed that suramin increased antigen-induced levels of IL-4, whilst IFN-gamma levels were decreased. Using various doses of suramin (25, 15, and 5 micrograms/g), its cytokine modulatory effect displayed a consistent dose-dependent activity in vivo. This cytokine modulation commenced on week 2 after immunization and persisted all throughout the drug administration period, up to the 4th week. These results indicate that the prospects of using suramin in the treatment of multiple sclerosis may be feasible.
Hybridoma, Feb 1, 1995
Four murine monoclonal antibodies (MAbs) of the IgM class were raised against human acetylcholine... more Four murine monoclonal antibodies (MAbs) of the IgM class were raised against human acetylcholinesterase (AChE; Ec 3.1.1.7). The MAbs BMS-3E4, BMS-7G10, and BMS-9F4 all recognized human erythrocyte AChE, while BMS-6D6 bound specifically to human soluble brain AChE, on the basis of immunobinding assays. Dose-response studies gave an ELISA EDS0 titer of 4.5 x 10"4 M for BMS-6D6, while BMS-3E4 gave the best titer at 8.8 x 10 4 M. Sucrose density gradients demonstrated sedimentation of antigen-antibody complexes, consistent with earlier findings (i.e., BMS-6D6 bound with brain AChE while BMS-3E4 preferred erythrocyte (AChE). No cross-reactivity between the two MAbs against the two antigens was noted.
European Journal of Pharmacology, Nov 1, 1987
The present study aimed to further elucidate the pharmacological features, with respect to sensit... more The present study aimed to further elucidate the pharmacological features, with respect to sensitivity to L-BHGA agonists, of the receptors sensitive to/3-hydroxy-L-glutamic acid (L-BHGA) in five Achatina giant neurones: PON (periodically oscillating neurone), d-RPLN (dorsal-right parietal large neurone), VIN (visceral intermittently firing neurone), RAPN (right anterior pallial neurone) and v-RCDN (ventral-right cerebral distinct neurone). Of these neurones, d-RPLN and RAPN were depolarized by L-BHGA, while PON, VIN and v-RCDN were inhibited. Threo-B-hydroxy-DL-aspartic acid markedly depolarized d-RPLN and RAPN (effective potency quotient (EPQ) in relation to the more effective L-BHGA isomer: 1 for d-RPLN and 0.3 for RAPN). This compound produced only slight inhibitory effects on PON, VIN and v-RCDN with EPQs calculated to be < 0.03, < 0.03 and 0.03, respectively. On the other hand, erythro-/3-hydroxy-DL-aspartic acid at 10-3 M was almost ineffective, except on v-RCDN where it elicited some slight inhibitory effects (EPQ: 0.01). L-Aspartic and D-aspartic acid at 10-3 M, also had almost no effect except for slight effects of D-aspartic acid on d-RPLN (EPQ: 0.1). N-Methyl-Land N-methyl-D-aspartic acid were slightly effective only on v-RCDN (EPQ: < 0.01 and 0.01 respectively). The other compounds, including /3-hydroxypyrroglutamic acid (cyclic BHGA) and proline derivatives, were almost ineffective at 10-3 M; very weak effects were occasionally observed on some neurones. These results have a twofold meaning: the relative sensitivities of these neurones to the erythro-and threo-L-BHGA stereoisomers do not correspond to the sensitivities of the stereoisomers of/3-hydroxy-DL-aspartic acid and the zwitterionic character of the a-amino group, the presence of a fl-hydroxyl group and a C4 or C5 carboxyl group are necessary to produce the effects on these neurones.
Comparative Biochemistry and Physiology Part C: Comparative Pharmacology, 1989
To elucidate the conformation-activity relationships of P-hydroxy-L-glutamic acid (L-BHGA). the e... more To elucidate the conformation-activity relationships of P-hydroxy-L-glutamic acid (L-BHGA). the effects of four stereoisomers of the novel L-BHGA analogues conformationally fixed in extended or folded form, L-r-(carboxycyclopropyl)glycine (L-ACCG). were tested on five Achatina neurones, PON, VIN. D-RPLN, RAPN and V-RUIN. 2. The L-BHGA receptors were classified into BHG (L-BHGA receptor type) I, 2 and 3. Further. these were also subclassified into Ex (extended), Fd (folded) and LJn (undetermined) types. 3. BHG IEx and IFd (in PON and VIN) are inhibited by L-BHGA. BHG 1Ex are activated by erythro-L-extended compounds. whereas BHG IFd by threo-L-folded. 4. BHG 2Un and 2Fd (in D-RPLN and RAPN) are excited by L-BHGA. BHG 2Un are activated by erythro-L-compounds, while BHG 2Fd by threo-L-folded. 5. BHG 3Un (in V-RCDN) are inhibited only by threo-L-BHGA.
Biochemical and Biophysical Research Communications, Sep 1, 1994
Immunology Letters, Jul 1, 1996
The in vitro immunopharmacologic effect of suramin, a polysulfonated napthylurea, was examined on... more The in vitro immunopharmacologic effect of suramin, a polysulfonated napthylurea, was examined on splenocytes and T helper (Th) 1 and 2 clones. Cytokine capture assays revealed that suramin inhibited the production of IFN-gamma, whilst IL-4 is increased in splenocytes. To examine whether suramin preferentially acts on Th cells, clones SP39A1 and SP41D5 were used. The former is a Th1 clone, whereas the latter is Th2. Suramin also inhibited IFN-gamma production by SP39A1, whilst IL-4 production by SP41D5 was enhanced. The action of suramin was of a dose-dependent nature. With regards to T cell proliferation, suramin inhibited the proliferation of both SP39A1 and SP41D5. The ability of suramin to manipulate Th subset cytokine balance can render it a feasible therapeutic agent in autoimmune disorders such as multiple sclerosis.
European Journal of Pharmacology, Mar 1, 1990
The blocking effects of d-diltiazem, its metabolites, deacetyl-d-diltiazem (d-M1), deacetyl-N-dem... more The blocking effects of d-diltiazem, its metabolites, deacetyl-d-diltiazem (d-M1), deacetyl-N-demethyl-d-diltiazem (d-M2), deacetyl-O-demethyl-d-diltiazem (d-M4) and deacetyl-N, O-demethyl-d-diltiazem (d-M6) and 1-diltiazem were investigated on the voltage-gated calcium current (ICa) of an Achatina neurone. Based on the IC50 values, the order of potency was: d-diltiazem (0.426 mM), d-M2 (0.456), d-M1 (0.491), 1-diltiazem (0.759) greater than d-M4 (1.212) greater than d-M6 (greater than 2.000). Assuming that the IC50 reflects the KD for resting Ca2+ channels (Kr), steady state activation studies gave KD values for the inactivated channels (Ki) and Kr/Ki ratios of 0.122 mM (Ki) and 3.52 (Kr/Ki) (d-diltiazem), 0.112 and 6.98 (1-diltiazem), 0.083 and 6.07 (d-M1) and 0.156 and 2.97 (d-M2). All drugs tested showed a certain degree of voltage-dependence. The further percentage reduction in the normalized ICa after high frequency stimulation demonstrated the use-dependence of: 1-diltiazem (27.5%), d-diltiazem (26.3) greater than d-M2 (19.2) greater than d-M1 (16.7) greater than d-M6 (9.8). The voltage- and use-dependence of these drugs are consistent with their Ca2+ antagonistic properties.
The Philippine journal of science, 1997
Chelerythrine (chloride) has previously been documented to be a potent and selective inhibitor of... more Chelerythrine (chloride) has previously been documented to be a potent and selective inhibitor of the serine/threonine-specific protein kinase C (PKC). In this study, it was shown that 10 microM chelerythrine completely inhibited serotonin secretion and partially inhibited phosphatidic acid formation in human blood platelets activated by thrombin (1U/ml). However, there was no effect on PKC activity as assessed by the level of phosphorylation of the 47K protein. Therefore, chelerythrine has been shown not to be a specific inhibitor of PKC. Without specifically affecting PKC activity, it is nevertheless capable of completely inhibiting platelet secretion, indicating that it may affect the signal transduction pathway responsible for platelet secretion at a point downstream or independent of PKC.
Procedures for the preparative purification of human acetylcholinesterase (AChE; EC 3.1.1.7) have... more Procedures for the preparative purification of human acetylcholinesterase (AChE; EC 3.1.1.7) have been examined. Chromatofocussing showed caudate nucleus and cerebrospinal fluid (CSF) AChE eluting between isoelectric points (pi) of 5.2 - 5.57, whilst isoelectric focussing (IEF) revealed heterogeneous AChE isoforms migrating between pI 4.5 - 6. Affinity chromatography proved to be better in yielding highly pure samples. The ligands used were either acridinium or procainamide, which yielded brain AChE recoveries of 10.1% and 42.8%, respectively. Various other methods were used such as gel filtration and ion-exchange chromatography on a Pharmacia SMART System. Purified brain AChE was used as antigen to generate anti-AChE monoclonal antibodies (mAbs) by in vivo immunization. BMS-3E4, -7G10, and -9F4 reacted with erythrocyte (G2) AChE, whilst BMS-6D6 bound to brain (G4) AChE in dot blot, titration, and sedimentation experiments. Conversely, the four mAbs recognized only the G4 form and n...
Immunopharmacology, 1996
The present study sought to examine the immunopharmacologic effects of suramin on splenocytes fro... more The present study sought to examine the immunopharmacologic effects of suramin on splenocytes from experimental allergic encephalomyelitis (EAE)-induced mice, taken in line with a previous observed action of suramin in ameliorating EAE. Suramin, a polysulfonated napthylurea, decreased the proliferation of T cells, in the presence of various stimuli, such as guinea pig myelin basic protein (MBP), mouse spinal cord homogenate (MSCH), bovine proteolipid protein (PLP), P1 (synthetic peptide 139-151 of PLP), and Mycobacterium tuberculosis (MTB). Suramin inhibited T cell proliferation in a dose-dependent manner. However, cytokine assays revealed that suramin increased antigen-induced levels of IL-4, whilst IFN-gamma levels were decreased. Using various doses of suramin (25, 15, and 5 micrograms/g), its cytokine modulatory effect displayed a consistent dose-dependent activity in vivo. This cytokine modulation commenced on week 2 after immunization and persisted all throughout the drug administration period, up to the 4th week. These results indicate that the prospects of using suramin in the treatment of multiple sclerosis may be feasible.
Comparative Biochemistry and Physiology Part C: Comparative Pharmacology, 1989
To elucidate the conformation-activity relationships of P-hydroxy-L-glutamic acid (L-BHGA). the e... more To elucidate the conformation-activity relationships of P-hydroxy-L-glutamic acid (L-BHGA). the effects of four stereoisomers of the novel L-BHGA analogues conformationally fixed in extended or folded form, L-r-(carboxycyclopropyl)glycine (L-ACCG). were tested on five Achatina neurones, PON, VIN. D-RPLN, RAPN and V-RUIN. 2. The L-BHGA receptors were classified into BHG (L-BHGA receptor type) I, 2 and 3. Further. these were also subclassified into Ex (extended), Fd (folded) and LJn (undetermined) types. 3. BHG IEx and IFd (in PON and VIN) are inhibited by L-BHGA. BHG 1Ex are activated by erythro-L-extended compounds. whereas BHG IFd by threo-L-folded. 4. BHG 2Un and 2Fd (in D-RPLN and RAPN) are excited by L-BHGA. BHG 2Un are activated by erythro-L-compounds, while BHG 2Fd by threo-L-folded. 5. BHG 3Un (in V-RCDN) are inhibited only by threo-L-BHGA.
The Tokai journal of experimental and clinical medicine, 1996
Chelerythrine (chloride) has previously been documented to be a potent and selective inhibitor of... more Chelerythrine (chloride) has previously been documented to be a potent and selective inhibitor of the serine/threonine-specific protein kinase C (PKC). In this study, it was shown that 10 microM chelerythrine completely inhibited serotonin secretion and partially inhibited phosphatidic acid formation in human blood platelets activated by thrombin (1U/ml). However, there was no effect on PKC activity as assessed by the level of phosphorylation of the 47K protein. Therefore, chelerythrine has been shown not to be a specific inhibitor of PKC. Without specifically affecting PKC activity, it is nevertheless capable of completely inhibiting platelet secretion, indicating that it may affect the signal transduction pathway responsible for platelet secretion at a point downstream or independent of PKC.
Hybridoma, 1995
Four murine monoclonal antibodies (MAbs) of the IgM class were raised against human acetylcholine... more Four murine monoclonal antibodies (MAbs) of the IgM class were raised against human acetylcholinesterase (AChE; Ec 3.1.1.7). The MAbs BMS-3E4, BMS-7G10, and BMS-9F4 all recognized human erythrocyte AChE, while BMS-6D6 bound specifically to human soluble brain AChE, on the basis of immunobinding assays. Dose-response studies gave an ELISA EDS0 titer of 4.5 x 10"4 M for BMS-6D6, while BMS-3E4 gave the best titer at 8.8 x 10 4 M. Sucrose density gradients demonstrated sedimentation of antigen-antibody complexes, consistent with earlier findings (i.e., BMS-6D6 bound with brain AChE while BMS-3E4 preferred erythrocyte (AChE). No cross-reactivity between the two MAbs against the two antigens was noted.
Immunology Letters, 1996
The in vitro immunopharmacologic effect of suramin, a polysulfonated napthylurea, was examined on... more The in vitro immunopharmacologic effect of suramin, a polysulfonated napthylurea, was examined on splenocytes and T helper (Th) 1 and 2 clones. Cytokine capture assays revealed that suramin inhibited the production of IFN-gamma, whilst IL-4 is increased in splenocytes. To examine whether suramin preferentially acts on Th cells, clones SP39A1 and SP41D5 were used. The former is a Th1 clone, whereas the latter is Th2. Suramin also inhibited IFN-gamma production by SP39A1, whilst IL-4 production by SP41D5 was enhanced. The action of suramin was of a dose-dependent nature. With regards to T cell proliferation, suramin inhibited the proliferation of both SP39A1 and SP41D5. The ability of suramin to manipulate Th subset cytokine balance can render it a feasible therapeutic agent in autoimmune disorders such as multiple sclerosis.
European Journal of Pharmacology, 1990
The blocking effects of d-diltiazem, its metabolites, deacetyl-d-diltiazem (d-M1), deacetyl-N-dem... more The blocking effects of d-diltiazem, its metabolites, deacetyl-d-diltiazem (d-M1), deacetyl-N-demethyl-d-diltiazem (d-M2), deacetyl-O-demethyl-d-diltiazem (d-M4) and deacetyl-N, O-demethyl-d-diltiazem (d-M6) and 1-diltiazem were investigated on the voltage-gated calcium current (ICa) of an Achatina neurone. Based on the IC50 values, the order of potency was: d-diltiazem (0.426 mM), d-M2 (0.456), d-M1 (0.491), 1-diltiazem (0.759) greater than d-M4 (1.212) greater than d-M6 (greater than 2.000). Assuming that the IC50 reflects the KD for resting Ca2+ channels (Kr), steady state activation studies gave KD values for the inactivated channels (Ki) and Kr/Ki ratios of 0.122 mM (Ki) and 3.52 (Kr/Ki) (d-diltiazem), 0.112 and 6.98 (1-diltiazem), 0.083 and 6.07 (d-M1) and 0.156 and 2.97 (d-M2). All drugs tested showed a certain degree of voltage-dependence. The further percentage reduction in the normalized ICa after high frequency stimulation demonstrated the use-dependence of: 1-diltiazem (27.5%), d-diltiazem (26.3) greater than d-M2 (19.2) greater than d-M1 (16.7) greater than d-M6 (9.8). The voltage- and use-dependence of these drugs are consistent with their Ca2+ antagonistic properties.
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Papers by Philipp Novales-Li