Lipid-induced recognition of a conformational determinant (residues 65 to 83) in myelin basic protein

The precipitation by antibodies to intact myelin basic protein (BP) and to synthetic peptides containing a sequence based on the region 65 to 83 of bovine BP, S82, S81, S79, and S24, of intact BP in solution or bound to lipid vesicles was compared, using 125I-BP or 14C-DPPC-labeled lipid-BP vesicles. The antipeptide antibodies were shown earlier to recognize conformational determinants which are not expressed in the intact protein in solution. Several anti-BP antibodies precipitated more of the BP free in solution than when bound to lipid vesicles, suggesting that some of the determinants recognized by these antibodies were either sequestered in the bilayer or were altered in conformation. In contrast, one anti-peptide antisera, which had a high titer for the conformational determinant in two of these peptides, S82 and S81, precipitated the protein to a significant degree when it was bound to PG vesicles, even though it did not react with the intact protein in solution. These results indicated that PG was able to confer on the protein the unique peptide conformation recognized by this antibody. PS was less effective, and other lipids were ineffective at conferring this conformation on the protein, supporting earlier results which showed that the conformation of the protein is influenced by the lipid composition of its environment. None of the other anti-peptide antibodies studied bound to the protein either in solution or in lipid vesicles. These results indicate that the lipid environment can sequester or alter the conformation of some antigenic determinants, preventing recognition by some anti-BP antibodies, and can expose or generate other conformational determinants, allowing recognition by an anti-peptide antiserum.     

A common TCR V-D-J sequence in V beta 13.1 T cells recognizing an immunodominant peptide of myelin basic protein in multiple sclerosis.

T cell responses to the immunodominant peptide (residues 83-99) of myelin basic protein are potentially associated with multiple sclerosis (MS). This study was undertaken to examine whether a common sequence motif(s) exists within the TCR complementarity-determining region (CDR)-3 of T cells recognizing the MBP83-99 peptide. Twenty MBP83-99-reactive T cell clones derived from patients with MS were analyzed for CDR3 sequences, which revealed several shared motifs. Some V beta 13.1 T cell clones derived from different patients with MS were found to contain an identical CDR3 motif, V beta 13.1-LGRAGLTY. Oligonucleotides complementary to the shared CDR3 motifs were used as specific probes to detect identical target CDR3 sequences in a large panel of T cell lines reactive to MBP83-99 and unprimed PBMC. The results revealed that, in contrast to other CDR3 motifs examined, the LGRAGLTY motif was common to T cells recognizing the MBP83-99 peptide, as evident by its expression in the majority of MBP83-99-reactive T cell lines (36/44) and PBMC specimens (15/48) obtained from randomly selected MS patients. The motif was also detected in lower expression in some PBMC specimens from healthy individuals, suggesting the presence of low precursor frequency of T cells expressing this motif in healthy individuals. This study provides new evidence indicating that the identified LGRAGLTY motif is preferentially expressed in MBP83-99-reactive T cells. The findings have important implications in monitoring and targeting MBP83-99-reactive T cells in MS.     

Induction of a non-encephalitogenic type 2 T helper-cell autoimmune response in multiple sclerosis after administration of an altered peptide ligand in a placebo-controlled, randomized phase II trial. The Altered Peptide Ligand in Relapsing MS Study Group

In this 'double-blind', randomized, placebo-controlled phase II trial, we compared an altered peptide ligand of myelin basic protein with placebo, evaluating their safety and influence on magnetic resonance imaging in relapsing-remitting multiple sclerosis. A safety board suspended the trial because of hypersensitivity reactions in 9% of the patients. There were no increases in either clinical relapses or in new enhancing lesions in any patient, even those with hypersensitivity reactions. Secondary analysis of those patients completing the study showed that the volume and number of enhancing lesions were reduced at a dose of 5 mg. There was also a regulatory type 2 T helper-cell response to altered peptide ligand that cross-reacted with the native peptide.     

Phosphorylation of U24 from Human Herpes Virus type 6 (HHV-6) and its potential role in mimicking myelin basic protein (MBP) in multiple sclerosis

Myelin basic protein (MBP) from multiple sclerosis (MS) patients contains lower levels of phosphorylation at Thr97 than normal individuals. The significance of phosphorylation at this site is not fully understood, but it is proposed to play a role in the normal functioning of MBP. Human Herpesvirus Type 6 encodes the protein U24, which has tentatively been implicated in the pathology of MS. U24 shares a 7 amino acid stretch encompassing the Thr97 phosphorylation site of MBP: PRTPPPS. We demonstrate using a combination of mass spectrometry, thin layer chromatography and autoradiography, that U24 can be phosphorylated at the equivalent threonine. Phospho-U24 may confound signalling or other pathways in which phosphorylated MBP may participate, precipitating a pathological process.     

Partitioning of myelin basic protein into membrane microdomains in a spontaneously demyelinating mouse model for multiple sclerosis.

We have characterized the lipid rafts in myelin from a spontaneously demyelinating mouse line (ND4), and from control mice (CD1 background), as a function of age and severity of disease. Myelin was isolated from the brains of CD1 and ND4 mice at various ages, and cold lysed with 1.5% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulphonate). The lysate was separated by low-speed centrifugation into supernatant and pellet fractions, which were characterized by Western blotting for myelin basic protein (MBP) isoforms and their post-translationally modified variants. We found that, with maturation and with disease progression, there was a specific redistribution of the 14-21.5 kDa MBP isoforms (classic exon-II-containing vs exon-II-lacking) and phosphorylated forms into the supernatant and pellet. Further fractionation of the supernatant to yield detergent-resistant membranes (DRMs), representing coalesced lipid rafts, showed these to be highly enriched in exon-II-lacking MBP isoforms, and deficient in methylated MBP variants, in mice of both genotypes. The DRMs from the ND4 mice appeared to be enriched in MBP phosphorylated by MAP kinase at Thr95 (murine 18.5 kDa numbering). These studies indicate that different splice isoforms and post-translationally modified charge variants of MBP are targeted to different microdomains in the myelin membrane, implying multifunctionality of this protein family in myelin maintenance.     

Reaction of peptide 89-169 of bovine myelin basic protein with 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine.

The C-terminal half of the bovine myelin basic protein, peptide 89-169, was treated with BNPS-skatole [2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine], and the products were isolated by repeated gel filtration through Sephadex G-50. They consisted of uncleaved peptide 89-169 in which approximately 30% of the tyrosine had been monobrominated and the tryptophan converted to oxindolealanine, peptide 116-169 modified by partial bromination (30%) of the tyrosine, and two chromatographic forms of peptide 89-115. The major form contained the lactone of dioxindolealanine at the C terminus; the minor form contained the uncyclized oxidation product. Each form of peptide 89-115 was resolved into several components by electrophoresis in polyacrylamide gels (10%, w/w) containing 1 M acetic acid and 8 M urea. The presence of three of these components could be explained by partial deamidation of Asn-91 and Gln-102. Studies on the oxidation of tryptophan-containing model peptides by BNPS-skatole indicated that the reaction can also include partial bromination of the dioxindole and its lactone and partial cleavage at the amino peptide bond of the tryptophan.     

Identity of myelin basic protein from multiple sclerosis and human control brains: discovery of a genetic variant.

Abstract— Myelin basic protein was isolated from the brains of 7 multiple sclerosis and 5 control patients. When acid extracts of the delipidated brains were chromatographed on carboxymethylcellulose at alkaline pH the elution profiles were the same for the two groups of patients. Component I, the most basic species of the protein, from 2 multiple sclerosis and one control brains was fragmented by limited pepsin digestion. Tryptic peptide maps were prepared from the three major products, fragment 1–38, 39–89 plus 45–89 and 90–170. The amino acid compositions of corresponding peptides were identical except for a 50:50 substitution of serine for glycine in tryptic peptide 44–49 from one (N.L.) of the 2 patients with multiple sclerosis. Peptide 44–49. isolated from intact component 1 from the other 6 multiple sclerosis and 5 control brains, did not show this substitution. In both multiple sclerosis and control basic proteins phosphorus was present only in fragment 90–170 of component 3 in the amount of 0.22 mol of phosphorus/mol of protein. These data suggest that there is no difference in either the amino acid sequence or in the modification of basic protein from control and multiple sclerosis patients. The amino acid substitution in patient N.L. represents the first example of a mutation in basic protein.     

Mucin gene (MUC1) transfected dendritic cells as vaccine: results of a phase I/II clinical trial

Dendritic cells (DC) derived from peripheral blood monocytes are currently being investigated in clinical trials for their role in stimulating the immune system. We performed a phase I/II clinical trial using human autologous DC transfected with cDNA of the human tumor antigen mucin (MUC1) as a vaccine in 10 patients with advanced breast, pancreatic or papillary cancer. After liposomal transfection, flow cytometry testing showed that 2% to 53% of the DC expressed mucin epitopes. Patients were immunized two or three times with 1 million transfected DC injected subcutaneously (s.c.). A vaccine-specific delayed-type hypersensitivity (DTH) reaction was observed in 3 out of 10 patients. After vaccination, 4 patients showed a 2- to 10-fold increase in the frequency of mucin-specific interferon-gamma (IFN-gamma)-secreting CD8+ T cells. We demonstrated the feasibility and safety of a vaccine consisting of autologous gene-transfected DC, and that immunologic responses could be induced even in patients with pretreated and advanced disease.     

Structure of a human autoimmune TCR bound to a myelin basic protein self-peptide and a multiple sclerosis-associated MHC class II molecule

Multiple sclerosis is mediated by T-cell responses to central nervous system antigens such as myelin basic protein (MBP). To investigate self-peptide/major histocompatibility complex (MHC) recognition and T-cell receptor (TCR) degeneracy, we determined the crystal structure, at 2.8 A resolution, of an autoimmune TCR (3A6) bound to an MBP self-peptide and the multiple sclerosis-associated MHC class II molecule, human leukocyte antigen (HLA)-DR2a. The complex reveals that 3A6 primarily recognizes the N-terminal portion of MBP, in contrast with antimicrobial and alloreactive TCRs, which focus on the peptide center. Moreover, this binding mode, which may be frequent among autoimmune TCRs, is compatible with a wide range of orientation angles of TCR to peptide/MHC. The interface is characterized by a scarcity of hydrogen bonds between TCR and peptide, and TCR-induced conformational changes in MBP/HLA-DR2a, which likely explain the low observed affinity. Degeneracy of 3A6, manifested by recognition of superagonist peptides bearing substitutions at nearly all TCR-contacting positions, results from the few specific interactions between 3A6 and MBP, allowing optimization of interface complementarity through variations in the peptide.     

Treatment of experimental allergic encephalomyelitis (EAE) induced by guinea pig myelin basic protein epitope 72-85 with a human MBP(87-99) analogue and effects of cyclic peptides

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory and demyelinating disease of the central nervous system and is an animal model of multiple sclerosis (MS). In the present report, a linear analogue and a series of cyclic semi-mimetic peptides were designed and synthesized based on the human myelin basic protein (MBP(87-99)) epitope (Val87-His-Phe-Phe-Lys-Asn-Ile-Val-Thr-Pro-Arg-Thr-Pro90) and on Copolymer I (a mixture of random polymers of Ala, Gln, Lys and Tyr used to treat MS). These analogues were designed looking for suppressors of EAE induced by guinea pig MBP(72-85) epitope (Gln-Lys-Ser-Gln-Arg-Ser-Gln-Asp-Glu-Asn-Pro-Val) in Lewis rats. The linear analogue [Arg91,Ala96]MBP(87-99), in which Arg substitutes Lys91 and Ala substitutes Pro96, was found to be a strong inhibitor which when administered to Lewis rats together with the encephalitogenic agonist MBP(72-85) completely prevented the induction of EAE. In contrast, three N- and C-termini amide-linked cyclic semi-mimetic peptides, [cyclo-Phe-Arg-Asn-Ile-Val-Thr-Ala-Acp (1), cyclo-Phe-Ala-Arg-Gln-Acp (2), cyclo-Tyr-Ala-Lys-Gln-Acp (3)] as well as a Lys side chain and C-terminous cyclic semi mimetic peptide cyclo(Lys, Acp)-Phe-Lys-Asn-Ile-Val-Thr-Ala-Acp (4) which contain segments of MBP(87-99) or are constituted from immunophoric residues of copolymer 1, were ineffective in inducing or inhibiting EAE in Lewis rats. However co-injection of cyclic analogues with MBP(72-85) delayed the onset of EAE indicating a modulatory effect on the EAE activity of MBP(72-85). These findings suggest that molecule length, size of cyclic moiety and backbone conformation are important elements for immunogenic activity. Moreover blockade of MBP(72-85) induced EAE by the unrelated peptide [Arg91,Ala56]MBP(87-99) could indicate that the mechanism of inhibition is not due to binding competition but rather due to the delivery of a negative signal by the antagonist which overcomes the agonist response possibly through the activation of antigen specific regulatory T cells.     

Suppression of epimerization by cupric (II) salts in peptide synthesis using free amino acids as amino components.

An epimerization-free system for coupling N-protected peptides with free amino acids was developed. A number of inorganic substances were tested as epimerization suppressant additives during the coupling by various methods (carbodiimide plus additives, uronium salts, Woodward's reagent-K, isobutyl-chloroformate, etc.). Some of them (ZnCl2, RbClO4, LiCl, SnCl4, AlCl3, etc.) in combination with some coupling methods can guarantee coupling with minimal epimerization (D-epimer < 1%). But only a simultaneous use of 1-hydroxybenzotriazole and Cu2+ ions as additives in carbodiimide-mediated peptide couplings appeared to give a standard result (D-epimer < 0.1%). There was no epimerization even in the case when N-methyl amino acid (sarcosine) was used as an amino component, while in the absence of Cu2+ ions an unacceptable level of epimerization was observed (D-epimer, 22% for carbodiimide with the 1-hydroxybenzotriazole method). So far it has been considered that Cu2+ ions prevent obtaining peptides in high yields (< 90%) by various coupling methods. We have found that the use of 1-hydroxybenzotriazole, CuCl2 and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide instead of dicyclohexylcarbodiimide provides a possible method for obtaining the desired peptides in 90-99% yields without epimerization. All these results were shown by employing several model peptide couplings with free amino acids as amino components dissolved in an effective solvent system which readily dissolved them.     

An Arg/Lys-->Gln mutant of recombinant murine myelin basic protein as a mimic of the deiminated form implicated in multiple sclerosis

The degree of post-translational enzymatic deimination (conversion of arginyl to citrullinyl residues) of myelin basic protein (MBP) is correlated with the severity of the human autoimmune disease multiple sclerosis (MS). It is difficult to obtain large quantities of deiminated MBP from natural sources (autopsy material), and in vitro deimination using peptidylarginine deiminase (EC 3.5.3.15) is both non-specific and irreproducible. Since there is no known codon for citrulline, we have constructed a mutant form of recombinant murine MBP (rmMBP) in which 5 Arg and 1 Lys residues have been replaced by Gln as the most reasonable analogue of Cit. The residues were chosen to correspond to the 6 Arg residues in human MBP which are most commonly deiminated in chronic MS. The mutant species, rmMBP-qCit(6) where the "q" represents "quasi-," was probed by numerous biochemical and biophysical techniques. Highly homogeneous protein preparations were obtained using a modified expression system which minimised spurious misincorporation of Lys for Arg, as ascertained by electrospray ionisation mass spectrometry. The mutant form rmMBP-qCit(6) had a reduced ability to aggregate lipid vesicles, a slightly greater susceptibility to digestion by cathepsin D, a greater proportion of random secondary structure, and different conformational responses to lipids, compared with the unmodified rmMBP. Overall, the mutant protein's properties were consistent with the effects of deimination and support its use as a model for evaluating the effects of this modification.     

NMR and molecular dynamics studies of an autoimmune myelin basic protein peptide and its antagonist: structural implications for the MHC II (I-Au)-peptide complex from docking calculations.

Experimental autoimmune encephalomyelitis can be induced in susceptible animals by immunodominant determinants of myelin basic protein (MBP). To characterize the molecular features of antigenic sites important for designing experimental autoimmune encephalomyelitis suppressing molecules, we report structural studies, based on NMR experimental data in conjunction with molecular dynamic simulations, of the potent linear dodecapeptide epitope of guinea pig MBP, Gln74-Lys75-Ser76-Gln77-Arg78-Ser79-Gln80-Asp81-Glu82-Asn83-Pro84-Val85 [MBP(74-85)], and its antagonist analogue Ala81MBP(74-85). The two peptides were studied in both water and Me(2)SO in order to mimic solvent-dependent structural changes in MBP. The agonist MBP(74-85) adopts a compact conformation because of electrostatic interactions of Arg78 with the side chains of Asp81 and Glu82. Arg78 is 'locked' in a well-defined conformation, perpendicular to the peptide backbone which is practically solvent independent. These electrostatic interactions are, however, absent from the antagonist Ala81MBP(74-85), resulting in great flexibility of the side chain of Arg78. Sequence alignment of the two analogues with several species of MBP suggests a critical role for the positively charged residue Arg78, firstly, in the stabilization of the local microdomains (epitopes) of the integral protein, and secondly, in a number of post-translational modifications relevant to multiple sclerosis, such as the conversion of charged arginine residues to uncharged citrullines. Flexible docking calculations on the binding of the MBP(74-85) antigen to the MHC class II receptor site I-A(u) using haddock indicate that Gln74, Ser76 and Ser79 are MHC II anchor residues. Lys75, Arg78 and Asp81 are prominent, solvent-exposed residues and, thus, may be of importance in the formation of the trimolecular T-cell receptor-MBP(74-85)-MHC II complex.     

Treatment of experimental allergic encephalomyelitis (EAE) by a rationally designed cyclic analogue of myelin basic protein (MBP) epitope 72-85

In this report the rational design, synthesis and pharmacological properties of an amide-linked cyclic antagonist analogue of the guinea pig myelin basic protein epitope MBP_72–85 are described. Design of the potent cyclic analogue was based on 2D NOESY nuclear magnetic resonance and molecular dynamics studies carried out in the linear antagonist Ala⁸¹MBP_72–85. The cyclic antagonist completely prevented the induction of experimental allergic/autoimmune encephalomyelitis when coinjected with linear and cyclic agonist analogues MBP_72–85 and cyclo(2–9)MBP_72–85.     

Some mixed-ligand palladium(II) complexes of 2,2'-bipyridine and amino acids as potential anticancer agents

Eight new palladium complexes of the formula [Pd(bipy)(AA)]Cl 1 or 2 H2O (where bipy is 2,2'-bipyridine and AA is an anion of glycine, L-alanine, L-leucine, L-proline, L-serine, L-lysine, L-asparagine, or L-glutamine) have been synthesized by reaction of [Pd(bipy)Cl2] with an appropriate mono sodium salt of amino acid in water. These complexes have been characterized by chemical analysis and by visible, infrared, and 1H NMR spectroscopy. The detailed 1H NMR and infrared spectral studies of these complexes ascertain the mode of binding of amino acids to palladium through nitrogen of terminal -NH2 group and oxygen of terminal -COO- group. The molar conductance values of these complexes in water suggest them to be 1:1 electrolytes. These complexes have also shown growth inhibition against L1210 lymphoid leukemic, P388 lymphocytic leukemic, Sarcoma 180, and Ehrlich ascitic tumor cells. Some of these complexes show better 50% inhibitory dose values than cis-diamminedichloroplatinum(II).     

T cell specificity for class II (I-A) and the encephalitogenic N-terminal epitope of the autoantigen myelin basic protein

The role of class II restriction in T cell recognition of an epitope of the autoantigen myelin basic protein (MBP) has been investigated. Encephalitogenic PL/J(H-2u) and (PL/J X SJL/J(H-2s))F1 ((PLSJ)F1) clones, isolated after immunization with intact MBP, recognize the N-terminal 11 amino acid residues of MBP in association with I-Au class II molecules. The synthetic peptide MBP 1-11 has been tested in vivo for induction of EAE. Clinical and histological EAE occurs in PL/J and (PLSJ)F1 mice but not SJL/J. The class II restriction of T cells primed with MBP 1-11 has been examined in primary cultures in vitro. Similar to encephalitogenic T cell clones, isolated after continuous selection in vitro, the population of MBP 1-11-specific proliferative PL/J and (PLSJ)F1 T cells, recognize this epitope in association with I-Au class II molecules. Not all MBP-specific T cell clones which are restricted to I-Au class II molecules cause autoimmune encephalomyelitis. The specificity of these non-encephalitogenic clones has been examined in this report. These clones also recognize MBP 1-11. Thus recognition of an encephalitogenic T cell epitope is not sufficient for induction of EAE.     

Photolabeling of myelin basic protein in lipid vesicles with the hydrophobic reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine

The hydrophobic photolabel 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine([125I]TID) was used to label myelin basic protein or polylysine in aqueous solution and bound to lipid vesicles of different composition. Although myelin basic protein is a water soluble protein which binds electrostatically only to acidic lipids, unlike polylysine it has several short hydrophobic regions. Myelin basic protein was labeled to a significant extent by TID when in aqueous solution indicating that it has a hydrophobic site which can bind the reagent. However, myelin basic protein was labeled 2-4-times more when bound to the acidic lipids phosphatidylglycerol, phosphatidylserine, phosphatidic acid, and cerebroside sulfate than when bound to phosphatidylethanolamine, or when in solution in the presence of phosphatidylcholine vesicles. It was labeled 5-7-times more than polylysine bound to acidic lipids. These results suggest that when myelin basic protein is bound to acidic lipids, it is labeled from the lipid bilayer rather than from the aqueous phase. However, this conclusion is not unequivocal because of the possibility of changes in the protein conformation or degree of aggregation upon binding to lipid. Within this limitation the results are consistent with, but do not prove, the concept that some of its hydrophobic residues penetrate partway into the lipid bilayer. However, it is likely that most of the protein is on the surface of the bilayer with its basic residues bound electrostatically to the lipid head groups.     

Susceptibility of myelin proteins to a neutral endoproteinase: The degradation of myelin basic protein (MBP) and P2 protein by purified bovine brain multicatalytic proteinase complex (MPC)

Multicatalytic proteinase complex (MPC) was isolated from bovine brain and the susceptibility of myelin basic protein (MBP) and P₂ protein of bovine central and peripheral nervous system was examined. SDS-polyacrylamide electrophoretic analysis of purified MPC revealed protein bands of molecular weight ranging from 22–35 kDa. The enzyme is activated by SDS at a concentration less than 0.01%. Upon incubation with MPC, purified MBP and P₂ proteins were degraded into smaller fragments. There was a 57% and 100% loss of MBP at 2 and 6 hours of incubation. The P₂ protein which is not susceptible to any endogenous non-lysosomal enzyme thus far studied was digested into small peptide fragments only in the presence of SDS (0.01%) and not in its absence. These results indicate that MPC which is active at physiological conditions may have a role in the turnover of myelin proteins and in demyelinating diseases.     

Identification of the ATP.Mg-dependent protein phosphatase activator (FA) as a myelin basic protein kinase in the brain

The activating factor FA of the ATP.Mg-dependent protein phosphatase FcM was purified to near homogeneity from pig brain by a procedure involving chromatography on phosphocellulose, phosvitin-Sepharose 4B, and Blue Sepharose CL-6B. A specific myelin basic protein (MBP) kinase was found to co-purify with FA in a constant ratio throughout purification. It also proved impossible to separate the two activities on nondenaturing gel electrophoresis and 5-20% sucrose density gradient ultracentrifugation. Kinetic study indicated that MBP, presumably a substrate for FA, could compete with FcM for FA and thereby prevent the FA-mediated activation of the FcM activity. All the results taken together demonstrate that MBP kinase and FA are localized on the same protein. This, together with the data that FA, by activating the ATP.Mg-dependent phosphatase, promotes the dephosphorylation of [32P]MBP, phosphorylated by FA itself, suggests the evidence for a protein bearing two opposing activities involved in the regulation of brain functions. Moreover, since FA is tightly associated with the purified brain myelin membrane, the results further support the notion that FA may well be an endogenous protein kinase responsible for the cyclic phosphorylation-dephosphorylation of the central nervous system myelin.     

Synthesis, characterization and antimicrobial activities of some mixed ligand complexes of Co(II) with thiosemicarbazones and N-protected amino acids

The reaction of cobalt(II) chloride with a new class of thiosemicarbazones viz; cis-3,7-dimethyl-2,6-octadienthiosemicarbazone(CDOTSC; L(1)H) and 3,7-dimethyl-6-octenethiosemicarbazone (DOTSC; L(2)H) and N-phthaloyl derivative of DL-glycine(A(1)H), L-alanine(A(2)H) or L-valine(A(3)H) in 1:1:1 molar ratio in dry refluxing ethanol have been studied. All the isolated complexes have the general composition [Co(L)(A)]. Tentative structures are proposed for these complexes based upon elemental analysis, electrical conductances, magnetic moment, molecular weight determination and spectral (IR, electronic) studies.The ligands and Co(II) complexes have been tested for their antibacterial and antifungal activities against three bacterial strains S. aureus, B. subtilis, E. coli and two fungal strains F. moniliformae and M. phaseolina. Attempts have been made to establish a correlation between the antibacterial and antifungal activity and the structures of products.