Routine primary immunization: status in two district of West Bengal.

The study was conducted at Birbhum and Purba Medinipur districts of West Bengal to assess the routine primary immunization coverage following 40 cluster sampling technique was used to study 320 children in each of the districts. BCG coverage was found to be 79.69% at Birbhum and 84.38% at Purba Medinipur. Only 62.81% children at Birbhum and 67.81% children at Purba Medinipur received all the three primary doses of DPT. Regarding OPV, coverage with three primary doses were only 65% and 66.88% at Birbhum and Purba Medinipur respectively. Measles vaccine coverage was very poor at both the districts, 55.94% at Birbhum and 62.5% at Purba Medinipur. Full primary immunization was observed 53.13% and 61.56% in Birbhum and Purba Medinipur respectively. High drop-out rate was identified as a major deficiency in both the districts. Of the children who received at least one routine vaccine, more than 1/3rd at Birbhum and more than 1/4 th at Purba Medinipur did not turn up later for completion of their primary vaccine doses. It is evident that routine immunization coverage was poor in both the districts and it seems there has been no improvement in situation for last few years. It will influence not only the child morbidity and mortality situation but also will jeopardize the paralytic polio eradication programme. Urgent intervention should be undertaken to address the large number of non-immunized children as well as high proportion of drop-outs.

A role for the alpha 113 (GH1) amino acid residue in the polymerization of sickle hemoglobin. Evaluation of its inhibitory strength and interaction linkage with two fiber contact sites (alpha 16/23) located in the AB region of the alpha-chain.

A cluster of amino acid residues located in the AB-GH region of the alpha-chain are shown in intra-double strand axial interactions of the hemoglobin S (HbS) polymer. However, alphaLeu-113 (GH1) located in the periphery is not implicated in any interactions by either crystal structure or models of the fiber, and its role in HbS polymerization has not been explored by solution experiments. We have constructed HbS Twin Peaks (betaGlu-6-->Val, alphaLeu-113-->His) to ascertain the hitherto unknown role of the alpha113 site in the polymerization process. The structural and functional behavior of HbS Twin Peaks was comparable with HbS. HbS Twin Peaks polymerized with a slower rate compared with HbS, and its polymer solubility (C(sat)) was found to be about 1.8-fold higher than HbS. To further authenticate the participation of the alpha113 site in the polymerization process as well as to evaluate its relative inhibitory strength, we constructed HbS tetramers in which the alpha113 mutation was coupled individually with two established fiber contact sites (alpha16 and alpha23) located in the AB region of the alpha-chain: HbS(alphaLys-16-->Gln, alphaLeu-113-->His), HbS(alphaGlu-23-->Gln, alphaLeu-113-->His). The single mutants at alpha16/alpha23 sites were also engineered as controls. The C(sat) values of the HbS point mutants involving sites alpha16 or alpha23 were higher than HbS but markedly lower as compared with HbS Twin Peaks. In contrast, C(sat) values of both double mutants were comparable with or higher than that of HbS Twin Peaks. The demonstration of the inhibitory effect of alpha113 mutation alone or in combination with other sites, in quantitative terms, unequivocally establishes a role for this site in HbS gelation. These results have implications for development of a more accurate model of the fiber that could serve as a blueprint for therapeutic intervention.

An enigmatic peptide ligation reaction: protease-catalyzed oligomerization of a native protein segment in neat aqueous solution.

We report an enigmatic peptide ligation reaction catalyzed by Glu-specific Staphylococcus aureus V8 protease that occurs in neat aqueous solution around neutral pH utilizing a totally unprotected peptide substrate containing free alpha-carboxyl and alpha-amino groups. V8 protease catalyzed a chain of ligation steps between pH 6 and 8 at 4 degrees C, producing a gamut of covalent oligomers (dimer through octamer or higher) of a native protein segment TAAAKFE (S39) derived from ribonuclease A (RNAse A). Size-exclusion chromatography suggested the absence of strong interaction between the reacting peptides. The circular dichroism spectra of monomer through pentamer showed length-dependent enhancement of secondary structure in the oligomers, suggesting that protease-catalyzed ligation of a monomer to an oligomer resulted in a product that was more structured than its precursor. The relative conformational stability of the oligomers was reflected in their ability to resist proteolysis, indicating that the oligomerization reaction was facilitated as a consequence of the "conformational trapping" of the product. The ligation reaction proceeded in two phases-slow formation and accumulation of the dimer followed by a fast phase of oligomerization, implying that the conformational trap encountered in the oligomerization reaction was a two-step process. The Gly substitution at any position of the TAAAKFE sequence was deleterious, suggesting that the first step of the conformational trap, namely the dimerization reaction, that proceeded very slowly even with the parent peptide, was quite sensitive to amino acid sequence. In contrast, the oligomerization reaction of an Ala analog, AAAAKFE, occurred in much the same way as S39, albeit with faster rate, suggesting that Ala substitution stabilized the overall conformational trapping process. The results suggest the viability of the product-directed "conformational trap" as a mechanism to achieve peptide ligation of totally unprotected peptide fragments in neat aqueous solution. Further, the study projects the presence of considerable innate synthetic potential in V8 protease, baring rich possibilities of protein engineering of this enzyme to generate a "V8 peptide ligase."

Scavenger receptor-mediated delivery of muramyl dipeptide activates antitumor efficacy of macrophages by enhanced secretion of tumor-suppressive cytokines.

We showed that muramyl dipeptide (MDP) conjugated to maleylated bovine serum albumin (MBSA) was internalized by macrophages (Mphi) through scavenger receptor (SCR)-mediated endocytosis, which leads to 50-fold higher cytotoxic activity against non-Mphi tumor cells compared with that elicited by free MDP-treated Mphi. The enhanced cytotoxic effect of MBSA-MDP was found to be a result of higher secretion of interleukin (IL)-1, IL-6, tumor necrosis factor alpha (TNF-alpha), and nitric oxide (NO) because the addition of antibodies directed against IL-1, IL-6, or TNF-alpha in combination with Mphi cultures totally abrogated the tumoricidal activity of MBSA-MDP. It is interesting to note that MBSA-MDP triggers the secretion of IL-12, whereas IL-10, a Mphi suppressor cytokine, could be detected only on free MDP treatment. The cytotoxic activity of MBSA-MDP was inhibited by indomethacin, indicating a regulatory role for prostaglandin E2 (PGE2). Efficient SCR-mediated intracellular delivery of MDP leading to elimination of cancer cells suggests the immunotherapeutic potential of this approach for treatment of neoplasia.

Selective activation of antitumor activity of macrophages by the delivery of muramyl dipeptide using a novel polynucleotide-based carrier recognized by scavenger receptors.

We have shown that muramyl dipeptide (MDP) conjugated to a 10-mer polyguanylic acid (PolyG) is specifically internalized by macrophages through scavenger receptor (SCR)-mediated endocytosis. Macrophages activated by PolyG-MDP displayed about 20-fold higher cytotoxic activity against nonmacrophage tumor cells compared to that elicited by free MDP. The PolyG-MDP was found to trigger the secretion of higher levels of interleukin-6, interleukin-1alpha, TNF-alpha, and nitric oxide in comparison to free MDP. Addition of antibodies directed against IL-6 and TNF-alpha to macrophage culture completely abrogated the tumoricidal response of PolyG-MDP, indicating that these two cytokines are primarily responsible for bioefficacy. This general approach of PolyG as a vehicle may find wide application in the delivery of genes and antisense oligonucleotides to macrophages.

Oligonucleotides tethered to a short polyguanylic acid stretch are targeted to macrophages: enhanced antiviral activity of a vesicular stomatitis virus-specific antisense oligonucleotide.

The poor membrane permeability of oligonucleotides is one of the major problems of antisense technology. Here we report the construction of designer oligonucleotides for targeted delivery to macrophages. The oligonucleotides tethered to a 10-mer poly(G) sequence at their 3' ends were recognized by scavenger receptors on macrophages and were taken up about 8- to 10-fold as efficiently as those oligonucleotides that either lacked a poly(G) tail or that contained a 10-mer poly(C) tail instead of the poly(G) tail. The enhanced uptake of poly(G) constructs was inhibited in the presence of poly(G) and other known ligands of the scavenger receptor. The bioefficacy of poly(G)-mediated targeting of antisense oligonucleotides (ANS) was demonstrated by using vesicular stomatitis virus (VSV) as a model system. The ability of ANS directed against the translation initiation site of N protein mRNA of VSV to inhibit virus replication was assessed. The ANS with the 10-mer poly(G) sequences (ANS-G) brought about significant inhibition of VSV replication in J774E cells (a murine monocyte/macrophage cell line) and Chinese hamster ovary (CHO) cell transfectants expressing scavenger receptors. The ANS lacking a 10-mer poly(G) stretch were ineffective. The inhibition of VSV replication due to ANS-G was completely abrogated in the presence of 10-mer poly(G), indicating that the antisense effect of the ANS-G molecule was a consequence of scavenger receptor-mediated enhanced uptake. Importantly, antisense molecules linked exclusively by natural phosphodiester bonds were as bioeffective as those synthesized with a mixed backbone of phosphodiester and phosphorothioate. Taken together, these results suggest that macrophage-directed designer ANS against infective agents may simply be obtained by adding a short stretch of guanylic acid sequence to the desired specific ANS during solid-phase synthesis. This nucleic acid-based strategy, which utilizes homogeneous preparation of ANS, may find applications in directed manipulation of macrophage metabolism for a variety of purposes as well as in therapy of a broad spectrum of macrophage-related disorders amenable to the antisense approach.

Helix-enhancing propensity of fluoro and alkyl alcohols: influence of pH, temperature and cosolvent concentration on the helical conformation of peptides.

We have analyzed the effects of trifluoroethanol (TFE) and three other alcohols(1-propanol, 2-propanol and hexafluoro-2-propanol) on S-peptide (residues 1-20) of ribonuclease A, an analog of S-peptide (QHM-->AAA, Sa-peptide) and TC-peptide (residues 295-316) of thermolysin to assess the helix-enhancing propensity of fluoro and alkyl alcohols under different environmental conditions of cosolvent concentration, pH and temperature by circular dichroism (CD). The dependence of cosolvent concentration on helix-induction showed a plateauing effect in all cases. 1-Propanol and 2-propanol were as effective as TFE in all the three peptides. Hexafluoro-2-propanol (HFIP) was a better helix enhancer in all cases however, the relative effectiveness varied with the peptide sequence. The alcohol transitions were analyzed assuming a two-state transition. The free energy decreased linearly in the cosolvent concentration range of 0-5 m for all the three peptides. The m-value (constant of proportionality) varied between peptides but was similar for any given peptide for TFE, 1-propanol or 2-propanol. The m-values of HFIP for all three peptides was much higher compared to other cosolvents. The isothermal cosolvent helix-induction curves for the three peptides exhibited similar features of shape and character for 1-propanol, 2-propanol and TFE. The additivity of cosolvent-induced helix formation was observed for different blends of alkyl and/or fluoro cosolvents. The pH-dependence of helix formation was observed in both TFE and 1-propanol solutions for S-peptide and TC-peptide, respectively, while in Sa-peptide, which was designed to perturb the pH-effect, helix formation was unaffected. The overall results provide some insight into the mechanism of cosolvent-mediated helix-enhancement in protein segments and are likely to facilitate optimization of conditions for cosolvent usage in chemistry and biology.

Hemoglobin endocytosis in Leishmania is mediated through a 46-kDa protein located in the flagellar pocket.

Four lines of evidence indicate that a specific high affinity binding site on the surface of Leishmania donovani promastigotes mediates rapid internalization and degradation of hemoglobin. 1) Binding and uptake of 125I-hemoglobin by Leishmania followed saturation kinetics and were competed by unlabeled hemoglobin but not by globin or hemin or other heme- or iron-containing proteins. 2) Immunogold labeling studies revealed that, at 4 degreesC, hemoglobin binding was localized in the flagellar pocket of the promastigotes. Indirect immunofluorescence assays showed that, at 37 degreesC, the bound hemoglobin in such cells entered an endocytic compartment within 2 min and dispersed throughout the cell body by 15 min. 3) After incubation with hemoglobin-gold conjugates at 25 degreesC or 37 degreesC, the particles accumulated in discrete intracellular vesicles. 4) A single biotinylated protein of 46 kDa was revealed when solubilized membranes from surface biotinylated intact Leishmania adsorbed by hemoglobin-agarose beads were subjected to SDS-polyacrylamide gel electrophoresis and Western blotting with avidin-horseradish peroxidase. Considered together, these data indicate that this 46-kDa protein on the cell surface of L. donovani promastigotes mediates the binding of hemoglobin and its rapid internalization through a vesicular pathway characteristic of receptor-mediated endocytosis.

B cell responses to a peptide epitope. V. Kinetic regulation of repertoire discrimination and antibody optimization for epitope.

The influence of imposing various conformational constraints on immune responses to a model epitope within a synthetic peptide immunogen was examined in mice. Although overall immunogenicity was affected, the model epitope (sequence DPAF) remained the predominant recognition site regardless of the conformation in which it was presented. A comparison of anti-DPAF mAbs obtained in response to two analogue peptides, PS1CT3 and CysCT3, in which the DPAF segment was either unconstrained or held within a cyclic loop, respectively, revealed a significant homology in the paratope composition. At one level a subset of anti-PS1CT3 and anti-CysCT3 mAbs was found to share a common heavy chain variable region. In addition, nucleotide sequence homology comparisons of both heavy and light chain variable regions identified the presence of anti-PS1CT3 and anti-CysCT3 mAbs that collectively appeared to derive from a common progenitor, but with nonidentical somatic mutations. Interestingly, however, no bias toward homologous Ag could be discerned on measurement of relative affinities of the mAbs for the two peptides. In contrast, mAb binding on-rates clearly discriminated between peptides representing the homologous vs the heterologous confomer of the DPAF epitope. Thus, it would appear that the kinetics of Ag recognition dominate over equilibrium binding criteria both in epitope-driven repertoire selection and Ab maturation in a humoral response.

Inhibition of sickle beta-chain (betaS)-dependent polymerization by nonhuman alpha-chains. A superinhibitory mouse-horse chimeric alpha-chain.

Horse alpha-chain inhibits sickle beta-chain-dependent polymerization; however, its inhibitory potential is not as high as that of mouse alpha-chain. Horse alpha-(1-30) and alpha-(31-141) segments make, respectively, minor and major contributions to the inhibitory potential of horse alpha-chain. The sum of the inhibitory potential of the two segments does not account for the inhibitory potential of the full-length horse alpha-chain. Although the polymerization inhibitory potential of horse alpha-chain is lower than mouse alpha-chain, the inhibitory potential of horse alpha-(31-141) is comparable to that of mouse alpha-(31-141). When mouse alpha-(1-30) is stitched to horse alpha-(31-141), the product is a chimeric alpha-chain with an inhibitory potential greater than mouse alpha-chain. In contrast, the stitching of horse alpha-(1-30) with mouse alpha-(31-141) had no additional inhibitory potential. Molecular modeling studies of HbS containing the mouse-horse chimeric alpha-chain indicate altered side-chain interactions at the alpha1beta1 interface when compared with HbS. In addition, the AB/GH corner perturbations facilitate a different stereochemistry for the interaction of the epsilon-amino group of Lys-16(alpha) with the beta-carboxyl group of Asp-116(alpha), resulting in a decrease in the accessibility of the side chain of Lys-16(alpha) to the solvent. Based on molecular modeling, we speculate that these perturbations by themselves, or in synergy with the altered conformational aspects of the alpha1beta1 interactions, represent the molecular basis of the superinhibitory potential of the mouse-horse chimeric alpha-chains.

Conformationally driven protease-catalyzed splicing of peptide segments: V8 protease-mediated synthesis of fragments derived from thermolysin and ribonuclease A.

We have studied the conformation as well as V8 protease-mediated synthesis of peptide fragments, namely amino acid residues 295-316 (TC-peptide) of thermolysin and residues 1-20 (S-peptide) of ribonuclease A, to examine whether "conformational trapping" of the product can facilitate reverse proteolysis. The circular dichroism study showed cosolvent-mediated cooperative helix formation in TC-peptide with attainment of about 30-35% helicity in the presence of 40% 1-propanol and 2-propanol solutions at pH 6 and 4 degrees C. The thermal melting profiles of TC-peptide in the above cosolvents were very similar. V8 protease catalyzed the synthesis of TC-peptide from a 1:1 mixture of the non-interacting complementary fragments (TC295-302 and TC303-316) in the presence of the above cosolvents at pH 6 and 4 degrees C. In contrast, V8 protease did not catalyze the ligation of S1-9 and S10-20, although S-peptide could assume helical conformation in the presence of the cosolvent used for the semisynthetic reaction. V8 protease was able to synthesize an analog of S-peptide (SA-peptide) in which residues 10-14 were substituted (RQHMD-->VAAAK). While S-peptide exhibited helical conformation in the presence of aqueous propanol solutions, SA-peptide displayed predominantly beta-sheet conformation. SA-peptide showed enhanced resistance to proteolysis as compared with S-peptide. Thus, failure of semisynthesis of S-peptide may be a consequence of high flexibility around the 9-10 peptide bond due to its proximity to the helix stop signal. The results suggest that protease-mediated ligations may be achieved by design and manipulation of the conformational aspects of the product.

B cell responses to a peptide epitope: IV. Subtle sequence changes in flanking residues modulate immunogenicity.

We examined modulation of primary humoral responses to a model synthetic peptide immunogen, peptide PS1CT3, as a consequence of single amino acid substitutions. Two analogues were employed, one in which the amino-terminal histidine (His1, peptide G28CT3) and another in which an internal proline (Pro14, peptide G41CT3) were replaced with glycine residues. Peptide G28CT3 displayed markedly enhanced immunogenicity relative to peptide PS1CT3 in BALB/c mice, whereas peptide G41CT3 was only poorly immunogenic. Nevertheless, in all three cases the mature polyclonal IgG response was predominantly directed against a tetrapeptide segment of sequence Asp-Pro-Ala-Phe between positions 4 and 7 of the sequence. While all three peptides proved equally capable of priming Ag-specific Th cells, they, however, displayed significant differences in their abilities to recall T cell responses. Regardless of the priming immunogen, in vitro challenge with either PS1CT3 or its analogues consistently gave a hierarchy of potencies as G28CT3 > PS1CT3 > G41CT3. This could also be correlated with B cell recall responses in which an identical hierarchy was obtained on restimulation of G41CT3-primed B cells in adoptive transfer experiments. Subsequent studies revealed that peptide-mediated modulation of Th cell recruitment by Ag-primed B cells was probably due to differences in on-rates for engagement of B cell Ag receptor by these analogues. This was despite the fact that all three peptides displayed equally randomized conformations in solution. These studies indicate that even subtle variations in the flanking sequences can markedly influence the immunogenicity of B cell epitopes.

Symmetric interspecies hybrids of mouse and human hemoglobin: molecular basis of their abnormal oxygen affinity.

Interspecies hybrids of HbA and Hb from mouse C57BL/10 [alpha 2M beta 2H and alpha 2H beta 2M (H = human, M = mouse)], representing 19 and 27 sequence differences per alpha beta dimers (as compared with human alpha beta dimer) have been generated in vitro. The efficiency of the assembly of the interspecies hybrids by the alloplex intermediate pathway is about twofold higher than the low-pH-mediated subunit approach. The interspecies hybrids exhibit a cooperative O2 binding. The intrinsic O2 affinity of mouse Hb is slightly lower than HbA, while the 2,3-diphosphoglycerate (DPG) effect is comparable. Interestingly, the interspecies hybrid alpha 2M beta 2H has high O2 affinity (compared to either human or mouse Hb), while the interspecies hybrid alpha 2H beta 2M exhibits a very low O2 affinity. These results suggest that the mouse beta chain generates a tetramer with very low oxygen affinity. However, the complementarity of the mouse alpha and beta chains generates a set of unique interactions that compensate for the low-oxygen-affinity propensity of the mouse beta chain. DPG binds the tetramer in the central cavity formed by the two beta subunits, hence the DPG effects on the interspecies hybrids should be as in the parent molecule. However, the results of the present study demonstrate that the DPG binding pocket is influenced by the nature of the alpha chain present in the tetramer. The mouse alpha chain reduces considerably the DPG right shift of the O2 affinity of the human beta-chain containing hybrid.(ABSTRACT TRUNCATED AT 250 WORDS)

Bissulfosuccinimidyl esters of aliphatic dicarboxylic acids: a new class of 'affinity directed' beta beta crosslinkers of HbA.

The potential of sulfosuccinimidyl ester of suberic acid as an intramolecular crosslinker of HbA, directed to the positive charge rich domain of the protein (DPG pocket) has been investigated. The suberate ester introduced crosslinks between the beta-chains and inhibited the dissociation of HbA into alpha beta dimers. The facile crosslinking reaction seen with this suberate ester as compared to the absence of crosslinking with the diaspirin derivative of suberic acid suggests the strong 'steering' influence of the sulfosuccinimidyl moiety of the reagent to the targeted site. The application of this crosslinking approach in the preparation of Hb based blood substitutes is discussed.

Molecular basis of the inhibition of beta s-chain-dependent polymerization by mouse alpha-chain. Semisynthesis of chimeras of human and mouse alpha-chains.

The transgenic mouse models expressing beta s-globin genes do not fully exhibit the sickling phenotype, primarily as a result of the inhibition of beta s-chain-dependent polymerization by the mouse alpha-chains. The mouse alpha-chain differs from the human alpha-chain at 19 sequence locations. Of these, only alpha 78 and alpha 116 are the known hemoglobin (Hb) S polymer contact sites. To define whether the inhibition of polymerization by the mouse alpha-chain is solely a consequence of the differences at these two sites or additional sites of sequence differences are also involved, we have constructed chimeric alpha-chains by employing the alpha-globin semisynthetic reaction (Sahni, G., Cho, Y. J., Iyer, K. S., Khan, S. A., Seetharam, R., and Acharya, A. S. (1989) Biochemistry 28, 5456-5461). Mouse alpha 1-30 was spliced with human alpha 31-141 using endoproteinase Glu-C to generate a chimeric alpha-globin (alpha MH) containing eight of the 19 sequence differences of mouse alpha-globin. Similarly, human alpha 1-30 was spliced with mouse alpha 31-141 to generate another chimeric alpha-globin (alpha HM) containing 11 sequence differences. The respective chimeric globins were purified, reconstituted with heme and beta s-chain into tetrameric hemoglobin, and the tetramers were purified by ion-exchange chromatography. The inhibitory potential of the chimeric alpha MH-chain on the polymerization is 10-fold lower than that of the mouse alpha-chain. The absence of the alpha 31-141 region of the mouse alpha-chain relieves only a portion of the inhibition. The inhibitory potential of alpha MH contributed by the mouse alpha 1-30 segment is significant although none of the sequence differences in this segment are located at any of the implicated polymer contact sites. The chimeric alpha HM-chain also inhibits the polymerization, but the extent of inhibition is again lower (4-fold) than that of the full-length mouse alpha-chain. The results demonstrate that the inhibitory potential of mouse alpha-chains involves the sequence differences from both the alpha 1-30 and alpha 31-141 regions. Besides, since the sum of the inhibitory potential of either of these chimeric alpha-chains is lower than that of the intact mouse alpha-chains, we speculate that conformational changes that require the copresence of sequence differences in both portions of the mouse alpha-chain also contribute to the inhibitory propensity of the mouse alpha-chain.

Helix formation in enzymically ligated peptides as a driving force for the synthetic reaction: example of alpha-globin semisynthetic reaction.

The alpha-globin semisynthetic reaction, namely, the ligation of the complementary fragments of alpha-globin, alpha 1-30 and alpha 31-141, in the presence of 30% l-propanol that is catalyzed by V8 protease is distinct as compared with the previously studied protease-catalyzed splicing of the discontinuity sites of the fragment complementing systems [Sahni et al. (1989) Biochemistry 28, 5456]. The complementary fragments of alpha-globin do not exhibit noncovalent interaction between them even in the presence of l-propanol, the organic cosolvent used to facilitate the alpha-globin semisynthetic reaction. Besides, a significant portion of the fragment alpha 31-141 does not contribute to the protease-catalyzed splicing reaction. Alpha 1-30 and alpha 31-40 are ligated by V8 protease to yield alpha 1-40 in much the same way as the splicing of alpha 1-30 with either alpha 31-141 or alpha 31-47 to yield alpha-globin or alpha 1-47, respectively. An equimolar mixture of alpha 1-30 and alpha 31-40 does not show any 'complexation' in the presence of 30% l-propanol, the medium used for the synthetic reaction. The splicing junction, i.e., Glu30-Arg31 peptide bond, is located in the middle of the B-helix (residues 20-35) of the parent protein. Most of the residues from the A-helix of the protein could also be deleted from segment alpha 1-30 without influencing the V8 protease-catalyzed splicing reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

The primary structure of xylanase from Thermoascus aurantiacus.

The amino acid sequence of xylanase isolated from the culture medium of Thermoascus aurantiacus was determined. It had 269 amino acid residues with an alpha-N-acetyl group at the amino terminus. The structure of blocked N-terminal 11 amino acid tryptic peptide except for acetylalanine was determined by sequence analysis of peptides derived from partial acid hydrolysis and from thermolysin digestion. The blocked N-terminal amino acid was determined as N-acetylalanine by electron ionization mass spectrometry. The sequence comparison of xylanase from T. aurantiacus with the xylanases of alkalophilic Bacillus sp C-125 and Cryptococcus albidus showed 40% similarity. Xylanase from T. aurantiacus had up to 15% similarity with the other two xylanases known. All the five xylanases showed a higher degree of similarity at the level of secondary structure.

Aldimine to ketoamine isomerization (Amadori rearrangement) potential at the individual nonenzymic glycation sites of hemoglobin A: preferential inhibition of glycation by nucleophiles at sites of low isomerization potential.

The relative roles of the two structural aspects of nonenzymic glycation sites of hemoglobin A, namely the ease with which the amino groups could form the aldimine adducts and the propensity of the microenvironments of the respective aldimines to facilitate the Amadori rearrangement, in dictating the site selectivity of nonenzymic glycation with aldotriose has been investigated. The chemical reactivity of the amino groups of hemoglobin A for in vitro reductive glycation with aldotriose is distinct from that in the nonreductive mode. The reactivity of amino groups of hemoglobin A toward reductive glycation (i.e., propensity for aldimine formation) decreases in the order Val-1(beta), Val-1(alpha), Lys-66(beta), Lys-61(alpha), and Lys-16(alpha). The overall reactivity of hemoglobin A toward nonreductive glycation decreased in the order Lys-16(alpha), Val-1(beta), Lys-66(beta), Lys-82(beta), Lys-61(alpha), and Val-1(alpha). Since the aldimine is the common intermediate for both the reductive and nonreductive modification, the differential selectivity of protein for the two modes of glycation is clearly a reflection of the propensity of the microenvironments of nonenzymic glycation sites to facilitate the isomerization reaction (i.e., Amadori rearrangement). A semiquantitative estimate of this propensity of the microenvironment of the nonenzymic glycation sites has been obtained by comparing the nonreductive (nonenzymic) and reductive modification at individual glycation sites. The microenvironment of Lys-16(alpha) is very efficient in facilitating the rearrangement and the relative efficiency decreases in the order Lys-16(alpha), Lys-82(beta), Lys-66(beta), Lys-61(alpha), Val-1(beta), and Val-1(alpha). The propensity of the microenvironment of Lys-16(alpha) to facilitate the Amadori rearrangement of the aldimine is about three orders of magnitude higher than that of Val-1(alpha) and is about 50 times higher than that of Val-1(beta). The extent of nonenzymic glycation at the individual sites is modulated by various factors, such as the pH, concentration of aldotriose, and the concentration of the protein. The nucleophiles--such as tris, glycine ethyl ester, and amino guanidine--inhibit the glycation by trapping the aldotriose. The nonenzymic glycation inhibitory power of nucleophile is directly related to its propensity to form aldimine. Thus, the extent of inhibition of nonenzymic glycation at a given site by a nucleophile directly reflects the relative role of pKa of the site in dictating the glycation at that site.(ABSTRACT TRUNCATED AT 400 WORDS)