Biomedical subjects

R Nagaraj

Publications and source records attributed to R Nagaraj.

At least 55 records · Page 3Linked to original sources

PubMed1996

Specific antimicrobial and hemolytic activities of 18-residue peptides derived from the amino terminal region of the toxin pardaxin.

Peptides are part of the host defense system against bacteria and fungi in species right across the evolutionary scale. However, endogenous antibacterial peptides are often composed of 25 residues or more and, therefore, are not ideal for therapeutic use. Hence it is of considerable interest to design and engineer short peptides having antimicrobial activity. Peptides composed of 18 amino acids, derived from the N-terminal region of the 33-residue toxin pardaxin (PX), GFFALIPKIISSPLFKTLLSAVGSALSSSGEQE, were synthesized and examined for biological activities. Peptide corresponding to the 1-18 stretch of PX exhibited antimicrobial activity only against Escherichia coli and not against Gram-positive microorganisms. The peptide also did not possess hemolytic activity. Replacement of P7 by A resulted in a peptide possessing both antibacterial and hemolytic activity. Substitution of both K residues by Q in the 'A' analog resulted in a peptide having only hemolytic activity. Conformational analysis of these peptides and investigation of their model membrane permeabilizing activities indicated that selective activity can be explained by their biophysical properties. Hence, by a rational design approach based on biophysical principles, it should be possible to generate short peptides having specific biological activity.

Amino Acid Sequence↗

PubMed1996

Acylation of proteins: recent advances.

The biochemistry and cell biology of covalent attachment of the fatty acids palmitic and myristic to proteins has been the subject of extensive investigations during the past fifteen years. While the site of attachment of fatty acids and the primary structure of proteins around the acylation site have been extensively documented, the exact role of the fatty acids have only been speculated upon. Since fatty acids would prefer to be associated with the lipid bilayer of membranes, it has been assumed that the role of the fatty acid is to provide a stable membrane anchor. This review discusses recent reports in the area of fatty acylation which suggests roles for the fatty acid other than that of a stable membrane anchor.

Acylation↗

PubMed1995

Advanced Maillard reaction and crosslinking of corneal collagen in diabetes.

Diabetes mellitus is associated with a number of changes in the cornea. These include increased corneal autofluorescence, thickening and enhanced endothelial cell permeability. In this study we have investigated the biochemical changes of corneal collagen due to advanced Maillard reaction and lysyl oxidase mediated crosslinking in diabetes. Advanced Maillard reaction was estimated by collagen-bound fluorescence and pentosidine. Hydroxypyridinium (a trifunctional fluorescent crosslink) was estimated as an index of lysyl oxidase mediated crosslinking. Both fluorescence (p < 0.05) and pentosidine were present at higher levels in diabetic corneas when compared with age-matched control corneas. Hydroxypyridinium levels were only marginally increased in diabetes. These results suggest that corneal collagen is modified in diabetes by advanced Maillard reaction and that such modifications may have effect on corneal thickening, endothelial cell permeability and other abnormalities of the cornea in diabetes.

Antioxidants↗

PubMed1995

Conformations of peptides corresponding to fatty acylation sites in proteins. A circular dichroism study.

Fatty acid acylation is a posttranslational modification found in membrane proteins that have hydrophobic sequences serving as transmembrane segments as well as those that do not have them. The fatty acids myristate and palmitate are linked through an amide bond to N-terminal glycine and SH of cysteine via a thioester bond, respectively. In order to elucidate whether or how fatty acid acylation would modulate peptide structure, especially in hydrophobic environment, we have carried out circular dichroism studies on synthetic peptides both hydrophobic and hydrophilic in nature, corresponding to fatty acylation sites and their fatty acyl derivatives. The hydrophilic peptides were approximately 12 residues in length as studies on proteins modified by site-directed mutagenesis indicated that a peptide segment of approximately 12 residues is sufficient to direct acylation as well as membrane association, especially when the fatty acid is myristic acid. The peptide corresponding to a transmembrane segment composed of 31 residues as well as its palmitoyl derivative was found to adopt alpha-helical structure. Acylation appeared to favor increased partitioning into miscelles even in the case of a hydrophobic peptide. The hydrophilic peptides and their myristoyl or palmitoyl derivatives showed very little ordered structure in micelles. Our results suggest that the myristoyl and the palmitoyl moieties do not have the ability to "force" a hydrophilic peptide segment into a hydrophobic micellar environment. Thus, the mere presence of a fatty acid moiety may not be sufficient for membrane binding and recycling as is assumed especially in proteins in which no hydrophobic segment is present.

Acylation↗

PubMed1995

Interaction of peptides corresponding to fatty acylation sites in proteins with model membranes.

In recent years, a large number of proteins having covalently linked myristic and palmitic acids have been discovered. It is assumed that fatty acid acylation serves to anchor proteins to membranes. However, it is not clear whether fatty acids modulate orientation of peptide chain in membranes or help in associating hydrophilic segments of peptides with membranes. We have examined the aggregation properties and membrane association of peptides corresponding to myristoylation and palmitoylation regions of proteins by fluorescence spectroscopy. Both acylated and non-acylated peptides were used for investigation. Binding of the peptides to lipid vesicles was assessed by monitoring the fluorescence of tryptophan as well as the quenching of its fluorescence in the presence of quenchers like I- and acrylamide. Our results indicate that in the peptide corresponding to a transmembrane segment, palmitoylation results in a change in the orientation of the peptide chain in the lipid bilayer. In the case of peptides that do not have a hydrophobic segment, acylation with palmitic or myristic acid does not appear to result in increased binding to lipid bilayer. Our results suggest that (i) the primary role of myristoylation may not be an anchor for membrane attachment as assumed, (ii) palmitoylation in the case of proteins having transmembrane segments may serve to realign the transmembrane segment from the normal orientation perpendicular to the bilayer surface, (iii) in the case of proteins where there is no hydrophobic segment, palmitoylation may not serve as a membrane anchor and could be involved in interaction with other membrane-bound proteins.

Acylation↗

PubMed1995

Seminal plasmin.

The importance of seminal plasma in fertilization was appreciated as early as 1677 and would thus hardly seem a source for the search of antibacterial agents. The observation that seminal plasma had the ability to inhibit the growth of microorganisms in 1940 led to a systematic search for molecules possessing antimicrobial activity in addition to factors that might have a role in reproductive physiology. Extensive investigations led to the discovery in bovine seminal fluid of a 47-residue peptide, possessing potent antimicrobial activity as well as calcium transport modulatory properties in bovine sperm. We describe in this article the two, apparently unrelated, biological activities of this peptide.

Amino Acid Sequence↗

PubMed1995

Generation of analogs having potent antimicrobial and hemolytic activities with minimal changes from an inactive 16-residue peptide corresponding to the helical region of Staphylococcus aureus delta-toxin.

The delta-toxin is a 26-residue peptide from Staphylococcus aureus with the sequence formyl-MAQDIISTIGDLVKWIIDTVNKFTKK. NMR studies indicate that the segment IISTIGDLVKWIIDTV occurs in an alpha-helical conformation in the toxin. A synthetic peptide corresponding to this segment, although helical, did not exhibit hemolytic activity. Since charged residues like D and K are likely to modulate cytolytic activity, analogs of the 16-residue peptide were synthesized where D was systematically replaced by K. Analogs in which the first D and both Ds were replaced by K showed potent antimicrobial and hemolytic activities. The analog in which the second D was replaced by K was relatively less active. However, all the peptides showed an alpha-helical structure with similar helical content. The activities of the peptides were found to correlate directly with their ability to permeabilize model membranes. Thus, by minimal judicious replacement of charged amino acids, it should be possible to generate cytolytic peptides from short segments of peptide toxins.

Amino Acid Sequence↗

PubMed1995

Structural and charge requirements for antimicrobial and hemolytic activity in the peptide PKLLETFLSKWIG, corresponding to the hydrophobic region of the antimicrobial protein bovine seminalplasmin.

Several analogs of the 13-residue antimicrobial and hemolytic peptide PKLLETFLSKWIG (SPF), which is the most hydrophobic region of the 47-residue antimicrobial protein seminalplasmin [Sitaram, N. & Nagaraj, R. (1990) J. Biol. Chem. 265, 10438-10442] have been synthesized. The antimicrobial and hemolytic properties of the peptides were investigated with a view to gain a insight into the structural and charge requirements for these activities of SPF. Peptides in which E was replaced by K exhibited considerably improved antimicrobial activity with no concomitant increase in hemolytic activity. A peptide in which the aromatic amino acids were replaced by leucine exhibited antimicrobial activity like those of the peptides which had aromatic amino acids. Interchange in the positions of E and K and total replacement of K by E resulted in complete loss of activity. The peptides having antimicrobial activity like those of the peptides which had aromatic amino acids. Interchange in the positions of E and K and total replacement of K by E resulted in complete loss activity. The peptides having antimicrobial activities showed appreciable helical content in a hydrophobic environment, whereas inactive peptides did not. Thus, by suitable 'engineering' the biological activity of a short 13-residue peptide can be altered by yield peptides specifically having only antimicrobial activity with increased potency.

Amino Acid Sequence↗

PubMed1995

Design of 16-residue peptides possessing antimicrobial and hemolytic activities or only antimicrobial activity from an inactive peptide.

We have explored the possibility of generating peptides having antimicrobial and hemolytic activities or only antimicrobial activity, from a 16-residue peptide, GFFALIPKIISSPLFK, corresponding to the N-terminal region of the toxin pardaxin. This peptide does not exibit these activities, although it can permeabilize model membranes. Peptides were synthesized wherein either A4 or P7 were substituted by K and S11 replaced by K. Peptides in which P7 and S11 were replaced with K, (AK) and A4 and S11 replaced with K and A instead of P at position 7, (KA) showed potent antimicrobial and hemolytic activities. However, the peptide where S11 and A4 were replaced with K, (KP) showed pronounced antimicrobial activity with very weak hemolytic activity. Circular dichroism studies indicated that peptides AK and KA had a strong propensity to occur in a helical conformation, whereas KP did not. Peptides AK and KA were very effective in permeabilizing model membranes, whereas KP was relatively ineffective. Our studies thus suggest the requirements for a peptide to have only antimicrobial activity and also that selectivity in activity can be rationalized on the basis of biophysical principles. Thus, by judicious positioning of amino acids, especially positively charged ones, it should be possible to generate biologically active peptides without taking recourse to a combinatorial approach.

Amino Acid Sequence↗

PubMed1994

Sex- and tissue-specific Bkm(GATA)-binding protein in the germ cells of heterogametic sex.

The ZZ male/ZW female system of sex determination (female heterogamety) is found in snakes and birds whereas XY male/XX female system of sex determination (male heterogamety) operates in mammals including humans. The W and Y chromosomes are largely heterochromatic and undergo cycles of condensation and decondensation in the germ cells of ovary and testis, respectively, whereas they remain highly condensed and transcriptionally inactive in all somatic cells. Both chromosomes have enriched stretches of GATA repeats along their entire length (which is identified as banded krait minor satellite DNA and called Bkm) that are highly conserved through widely separated orders of eukaryotes. Here we report the existence of a factor, which specifically binds to Bkm, in the germ cells of the heterogametic sex (ovary in female heterogamety and testis in male heterogamety) where decondensation (activation) of the W and Y chromosomes, respectively, occurs; it has been purified as a polypeptide of 57.5 kDa from the rat snake ovary and designated as Bkm-binding protein (BBP) by virtue of its binding to GATA repeats of Bkm. Such a sex- and tissue-specific BBP is also present in the ovary of other species of snakes and in the testis of mouse and human where the Y chromosome is highly decondensed. We suggest that GATA repeats of Bkm brings about a coordinated decondensation of the W and Y sex chromosomes in the germ cells of the heterogametic sex in response to BBP which may serve as a "switch" for the activation of the genes present on the W and Y chromosomes.

Animals↗

PubMed1994

Cell-lytic and antibacterial peptides that act by perturbing the barrier function of membranes: facets of their conformational features, structure-function correlations and membrane-perturbing abilities.

Almost all hemolytic and antimicrobial peptides form part of the defense mechanism of species widely distributed across the evolutionary scale. Although these peptides are of varying lengths and composition, they form amphiphilic structures in a hydrophobic environment. They also have the ability to form channels in natural and model membranes. Hemolytic peptides have proven to be very useful in studying the mechanism of hemolysis and the permeability properties of red blood cells. Preliminary investigations indicate that these peptides may also be useful in the investigation of complex cellular phenomena like exocytosis and neurotransmission. Although molecules like vancomycin, bacitracin and penicillins have been extensively used as antibiotics for therapeutic purposes, most species throughout the evolutionary scale use peptides as antimicrobial agents. These peptides exert their activity by altering the permeability properties of the bacterial plasma membrane and do not interfere with macro molecular synthesis like the other antibiotics that are presently used in therapies. Hence it is likely that resistance to peptide antibacterial agents may not develop easily. Since the problem of antibiotic resistance is presently a particularly severe one, peptide antibiotics may be the drugs of choice in the future.

Amino Acid Sequence↗

PubMed1994

Banded krait minor-satellite (Bkm)-associated Y chromosome-specific repetitive DNA in mouse.

The mouse Y chromosome remains highly condensed in all somatic tissues but decondenses extensively in testis. We have isolated a mouse Y chromosome-specific repeat M34 (11.5 kb) and shown that this is distributed along the Y chromosome except the sex-determining region (the Y short arm) in which GATA repeats are predominantly concentrated. It has 32 copies of GATA repeats in a 2.7 kb fragment. About 200-300 copies of M34 on the Y chromosome are interspersed among other sequences. A 1.2 kb fragment (p3) of M34, containing GATA repeats, also has scaffold attachment region (SAR) motifs which bind to nuclear matrices. A strong affinity of histone H1 to SAR motifs is implicated in maintaining the condensed state of the Y chromosome in somatic tissues. The probable significance of molecular organization of the Y chromosome is discussed.

Animals↗

PubMed1994

Increased redness in turkey breast muscle induced by fusarial culture materials.

Short-term feeding trials with experimentally moldy rice (10% of diet) or corn (12.5%) were carried out on turkey poults to screen for toxigenic Fusarium, Penicillium, and Aspergillus, which were recovered from corn or commercial turkey feed. A major finding was that culture materials of some fusaria increased the redness of turkey breast muscle without causing obvious hemorrhages either in the musculature or in the internal organs. Corn culture but not rice culture of Fusarium moniliforme (M-1325) depressed body weight gains, feed utilization, and heart weights (P = .01). The increased redness (reddish discoloration) could lead to the depreciation of fresh turkey muscle product.

Animal Feed↗

PubMed1993

Interaction of hydrophobic peptides with model membranes: slow binding to membranes and not subtle variations in pore structure is responsible for the gradual release of entrapped solutes.

Investigation of the mechanism of action of membrane-perturbatory peptides often involves monitoring the release of entrapped solutes from small unilamellar vesicles. Complete release of vesicular contents can take 15 min or more. Theoretical calculations suggest that the process should be of the order of seconds and not minutes. We have investigated the membrane-perturbatory abilities of hydrophobic peptides corresponding to regions of pardaxin that are important for toxin action. Peptide-induced release of entrapped carboxyfluorescein (CF) from lipid vesicles under various conditions was monitored by fluorescence spectroscopy. Several minutes were required for the complete release of CF. This has been shown to be due to lack of instantaneous and complete association by all the added peptide with the lipid vesicles. In addition, for a given peptide/lipid molar ratio, an increase in lipid concentration causes an increase in the rate of CF-release. It is likely that increased binding following a greater number of collisions between peptide and vesicles is responsible for this observation. A Fast Protein Liquid Chromatography assay confirms that a significant amount of peptide remains unbound from the vesicles. Other investigators have reported the requirement for a similar time span for the complete release of vesicular contents by pardaxin and several other membrane-perturbatory peptide toxins. The proposed reason for the delay in lysis may therefore be applicable to a large variety of membrane-perturbants. Thus, the assay of peptide-induced release of vesicular contents is likely to predominantly reflect only the rate of association of peptide with the membrane, and not more subtle variations in the nature of the pore formed.

Amino Acid Sequence↗

PubMed1993

Identification of a second membrane-active 13-residue peptide segment in the antimicrobial protein, bovine seminalplasmin.

Seminalplasmin (SPLN) is a 47-residue protein from bovine seminalplasma having broad-spectrum antibacterial activity. The protein has no hemolytic activity. SPLN interacts with lipid vesicles and its antibacterial activity appears to stem from its ability to permeabilize the bacterial plasma membrane. Analysis of SPLN's primary structure, with respect to its relative hydrophobicity and hydrophilicity, revealed a segment, PKLLETFLSKWIG, more hydrophobic than the rest of the protein. A synthetic peptide corresponding to this region had not only antibacterial activity but also hemolytic properties. Analysis of the SPLN sequence based on hydrophobic moment plots has revealed a second segment, SLSRYAKLANRLA, which could be membrane active. A synthetic peptide corresponding to this region shows only antibacterial activity with no hemolytic activity.

Amino Acid Sequence↗

PubMed1993

Conformations of peptide fragments comprising the amino-terminal, central, and carboxyl-terminal regions of a membrane-active polypeptide. Build-up of secondary structure in pardaxin.

The conformations of synthetic peptides of different lengths corresponding to the amino-terminal, central, and carboxyl-terminal regions of pardaxin (GFFALIPKIISSPLFKTLLSAVGSALSSSGEQE) have been studied by circular dichroism spectroscopy. The peptides GFFALIPKIISSPLF-OMe, GFFALIPKIISSPLFK-OMe corresponding to the amino-terminal region, as well as peptides KIISSPLFKTLLSAV and IISSPLFKTLLSAV corresponding to the central region of the toxin have a marked tendency to adopt helical conformation. Ordered conformation is also discernible in the 8- and 7-residue peptides GFFALIPK-OMe and IISSPLF-OMe. Peptides corresponding to the central segments KTLLSAV, LSAVGSAL, and the carboxyl-terminal segment SSSGEQE, however, exhibit very little secondary structure. The peptide segments that adopt ordered conformation show similar conformation when present in the entire toxin as suggested by proton magnetic resonance data (Zagorski, M. G., Norman, D. G., Barrow, C. J., Iwashita, T., Tachibana, K., and Patel, D. J. (1991) Biochemistry 30, 8009-8017). The observation that peptide segments corresponding to the amino-terminal and central regions of the toxin adopt ordered conformations compared to the carboxyl-terminal segment in isolation as well as in the toxin, indicates a role for these regions in initiating and maintaining ordered conformation of pardaxin.

Amino Acid Sequence↗

PubMed1993

Interaction of wild-type signal sequences and their charged variants with model and natural membranes.

The interaction of synthetic peptides corresponding to wild-type signal sequences, and their mutants having charged amino acids in the hydrophobic region, with model and natural membranes has been studied. At high peptide concentrations, i.e. low lipid/peptide ratios, the signal peptides cause release of carboxyfluorescein (CF) from model membranes with lipid compositions corresponding to those of translocation-competent as well as translocation-incompetent membranes. Interestingly, mutant sequences, which were non-functional in vivo, caused considerable release of CF compared with the wild-type sequences. Both wild-type and mutant signal sequences perturb model membranes even at lipid/peptide ratios of 1000:1, as indicated by the activities of phospholipases A2, C and D. These studies indicate that such mutant signals are non-functional not because of their inability to interact with membranes, but due to defective targeting to the membrane. The signal peptides inhibit phospholipase C activity in microsomes, uncouple oxidative phosphorylation in mitochondria and increase K+ efflux from erythrocytes, and one of the mutant sequences is a potent degranulator of the mast cells. Both wild-type and mutant signal sequences have the ability to perturb vesicles of various lipid compositions. With respect to natural membranes, the peptides do not show any bias towards translocation-competent membranes.

Amino Acid Sequence↗

PubMed1993

Interaction of the 47-residue antibacterial peptide seminalplasmin and its 13-residue fragment which has antibacterial and hemolytic activities with model membranes.

The interaction of seminalplasmin (SPLN), a 47-residue antibacterial peptide, and its 13-residue fragment (SPF), which has antibacterial and hemolytic activities, with model membranes has been investigated. The fluorescence characteristics of the single Trp residue in these peptides indicate strong binding to lipid vesicles. SPLN binds more strongly to dioleoylphosphatidylglycerol vesicles compared to dioleoylphosphatidylcholine and phosphatidylserine vesicles. Localization studies using fluorescence quenchers like NO3-, I-, and acrylamide indicate that the Trp residues in both of the peptides are located away from the head group region and are associated with the hydrophobic core. Both peptides cause release of carboxyfluorescein from zwitterionic as well as anionic vesicles. The biological activities of SPLN and SPF have been rationalized in terms of lipid-peptide interactions. It is proposed that the specificity in biological activity arises due to differences in the manner in which the peptides associate with the bacterial and red blood cell surfaces.

Acrylamide↗