Biomedical subjects

G Chakraborty

Publications and source records attributed to G Chakraborty.

29 records · Page 2Linked to original sources

PubMed1990

The site of amino acid addition to posttranslationally modified proteins of regenerating rat sciatic nerves.

The posttranslational modification of proteins by amino acids has been described in a variety of biological systems. These reactions occur at low levels in intact sciatic nerves of rats but are increased 10-fold following nerve injury and during subsequent regeneration of the nerve. While it has been shown in brain and liver that the site of addition of Arg is to the N-terminus, there is no information on the location at which the other amino acids add on to targeted proteins nor the site of addition of Arg in regenerating nerves. In the present study, we have used manual micro-Edman degradation combined with HPLC, and digestion with carboxypeptidase A and B to determine the site of addition of various amino acids to targeted proteins. Of the 3H-labelled amino acids incorporated posttranslationally into proteins of regenerating sciatic nerves (Arg, Lys, Leu, Phe, Val, Ala, Pro and Ser), only [3H]Arg was found to be present at the N-terminus. To determine whether amino acid additions were occurring at the C-terminus, proteins modified by two of the amino acids incorporated in greatest amounts (Lys and Leu) were incubated with specific carboxypeptidases. [3H]Leucine was not liberated following incubation with carboxypeptidase, suggesting that Leu is not added at the C-terminus of modified proteins. Under similar conditions, some [3H]Lys was liberated, but in amounts not significantly different from controls incubated without carboxypeptidase, indicating a non-specific degradation of Lys modified proteins rather than a specific release of Lys from the C-terminus. These experiments show that in regenerating sciatic nerves of rats, Arg is the only amino acid added posttranslationally to the amino terminus of target proteins, and that Leu, and probably Lys, are not conjugated to proteins at the C-terminus.

Amino Acids↗

PubMed1990

Amino acid modification of proteins in regenerating sciatic nerves of rats.

Recent experiments have shown that Arg, Lys, and Leu can be incorporated posttranslationally into proteins of regenerating sciatic nerves of rats. The present experiments investigate a mixture of 15 radioactive amino acids to determine if additional amino acids can be conjugated posttranslationally to proteins of regenerating nerves. Proteins of regenerating sciatic nerves of rats were able to incorporate Arg, Lys, Leu, Pro, Val, Ala, Phe, and Ser in relatively large amounts and Asp, Glu, Thr, Gly, Ile, His, and Tyr in relatively low or undetectable amounts, in the most advanced portion of the regenerating nerves. Two-dimensional SDS PAGE showed incorporation of the amino acid mixture into distinct radioactive peaks with molecular weights in the 80-90 kD, 53-66 kD, 22-46 kD, and 17 kD ranges with isoelectric points between 5.0 and 7.9. Most of the amino acids were incorporated into proteins in all of the molecular weight ranges. But Ser was incorporated in highest amounts in the 17 kD range, and Val was most abundant in the 22-46 kD range. In some cases results indicated that single proteins were modified by several amino acids. While we do not yet know which amino acids modify specific nerve proteins or the function of the modifications in nerve regeneration, these studies demonstrate the participation of some but not all amino acids in posttranslational modification reactions and the selective modification of specific groups of nerve proteins by these amino acids.

Amino Acids↗

PubMed1990

Regulation of the post-translational conjugation of amino acids to rat brain proteins.

Post-translational conjugation of arginine (but not other amino acids) to proteins has been reported to occur in a high speed supernatant fraction of rat brain homogenates from which molecules of less than 5000 mol. wt have been removed. In the present study we report that removal of molecules of less than 1000 mol. wt by dialysis, does not result in incorporation of arginine into protein in amounts significantly different than in the undialysed supernatant. The addition of molecules with molecular weights greater than 1000 and less than 5000 to the active fraction, inhibits the incorporation of arginine into proteins in a concentration dependent manner suggesting that the post-translational incorporation of arginine into brain is regulated by a molecule(s) of greater than 1000 and less than 5000 mol. wt. Incorporation of lysine into proteins did not occur following removal of molecules of less than 5000 mol. wt, but did occur in the void volume fraction of a Sephacryl S-200 column (molecular weight cut-off 125,000), suggesting that the incorporation of lysine into proteins is regulated by molecules retained by the S-200 column but greater than 5000 mol. wt. When experiments were repeated using the void volume of a Sephacryl S-300 column (molecular weight exclusion, approximately 200 k), leucine and proline were incorporated in amounts similar to arginine and lysine and serine, alanine, valine, phenylalanine and histidine were incorporated at lower but measurable levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1988

Comparison of posttranslational protein modification by amino acid addition after crush injury to sciatic and optic nerves of rats.

Posttranslational protein modifications by the addition of amino acids are reactions which occur in intact sciatic and optic nerves of rats. The nerves differ, however, in that 2 h after crush injury these reactions are activated in sciatic but not in optic nerves. As sciatic nerves will eventually regenerate, whereas optic nerves will not, we have proposed that the activation of these reactions is correlated with the ability of a nerve to regenerate. The current experiments examined the posttranslational addition of amino acids to proteins at times greater than 2 h after nerve crush, during sciatic nerve regeneration and optic nerve degeneration. We also examined the optic nerve for morphologic correlates to changes in protein modification and partially characterized the proteins modified by [3H]Lys in the regenerating sciatic nerve using two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). In a segment of sciatic nerve taken from a region just proximal to the site of crush, protein modification by covalent addition of [3H]Arg, [3H]Lys and [3H]Leu increased during both posttraumatic (2 h postcrush) and regenerative (6 days and 14 days postcrush) stages. Two-dimensional PAGE of [3H]Lys modified sciatic nerve proteins 6 days after crush injury showed labeling of proteins having molecular masses in the 18,000- to 20,000-, 30,000- to 40,000-, and 80,000- to 100,000-Da ranges, with neutral or basic isoelectric points (pI 7.1 to 8.0). In the retinal portion of the crushed optic nerve, incorporation of the same amino acids was unchanged or depressed to 21 days postcrush, except at 6 days postcrush when the incorporation of all three amino acids into proteins was increased threefold. These increases correlated with the appearance of terminal end bulbs in the portion of nerve analyzed. Histological examination of each nerve 2 h postcrush showed marked edema in the optic but not the sciatic nerve, a condition which may be related to the ability of sciatic and inability of optic nerves to activate protein modification reactions.

Amino Acids↗

PubMed1987

Posttranslational protein modification by polyamines in intact and regenerating nerves.

A 150,000-g supernatant from axoplasm of the giant axon of the stellate nerve of the squid and from rat sciatic and goldfish optic nerves was found to be able to incorporate covalently [3H]putrescine and [3H]spermidine into an exogenous protein (N,N'-dimethylcasein). Incorporation of radioactivity was inhibited by CuSO4, a specific inhibitor of transglutaminases, the enzymes mediating these reactions in other tissues. Analysis of pH and temperature range and enzyme kinetics displayed characteristics predicted for transglutaminase-mediated reactions. Transglutaminase activity increased during regeneration of both vertebrate nerves, but greater activity was found in segments of nerve containing no intact axons than in either intact segments or in segments containing regenerating axons. Polyacrylamide gel electrophoresis of endogenous modified proteins (in the absence of N,N'-dimethylcasein) showed labeling of 18-, 46- and 200-kilodalton proteins by both [3H]putrescine and [3H]spermidine. Analysis of the protein-bound radioactivity from intact and regenerating rat sciatic nerves demonstrated it to be predominantly in the form of the parent radioactive polyamine. These experiments demonstrate the covalent modification of proteins by polyamines at low levels in squid axoplasm and at relatively higher levels in rat sciatic and goldfish optic nerves. In the latter two cases, the activity of these modification reactions may be due in part to the modification of axonal proteins, but the majority of the activity occurs in nonneuronal cells of the nerve.

Animals↗

PubMed1986

Protein modification by amino acid addition is increased in crushed sciatic but not optic nerves.

Rat optic and sciatic nerves were crushed, and 10 minutes to 3 days later nerve segments between the crushed site and the cell body were removed and assayed for posttranslational protein modification by amino acid addition. Protein modification was comparable in intact optic and sciatic nerves, but in sciatic nerves increased to 1.6 times control levels 10 minutes after crushing and reached a maximum of ten times control levels by 2 hours. In optic nerves activity was decreased throughout the time course studied. The results indicate that, in a nerve which is capable of regeneration (sciatic), protein modification by the addition of amino acids increases immediately after injury, but a nerve incapable of regeneration (optic) is incapable of activating the modification reaction. These findings may be important in understanding the reasons for the lack of a regenerative response after injury to central mammalian nerves.

Amino Acids↗

PubMed1986

Posttranslational protein modification by amino acid addition in regenerating optic nerves of goldfish.

Previous experiments have demonstrated that 4S RNA, (tRNA), is transported axonally during the reconnection and maturation of regenerating optic nerves of goldfish. The present experiments were performed to determine if tRNA is transported axonally during elongation of these regenerating nerves and whether, as has been demonstrated in other systems, it participates in posttranslational protein modification (PTPM). [3H]Uridine was injected into both eyes of fish with intact optic nerves and 0, 2, 4, or 8 days after bilateral optic nerve cut. Fish were killed 2 days after injection, and [3H]RNA was isolated from retinae and nerves by phenol extraction and ethanol precipitation. [3H]RNA was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although the percentage of [3H]4S RNA remained constant in all retinal and control nerve samples, regenerating nerves showed a twofold increase by 6 days after injury, suggesting that [3H]4S RNA is transported axonally in regenerating nerves as early as 6 days after injury. In other experiments, the 150,000-g supernatant of optic nerves was analyzed for incorporation of 3H-amino acids into proteins. No incorporation of 3H-amino acid was found in the soluble supernatant, but when the supernatant was passed through a Sephacryl S-200 column (removing molecules less than 20,000 daltons), [3H]Arg, [3H]Lys, and [3H]Leu were incorporated into proteins. This posttranslational addition of amino acids was greater (1.4-5 times for Lys and 2-13 times for Leu) in regenerating optic nerves than nonregenerating nerves, and the growing tips of regenerating nerves incorporated 5-15 times more [3H]Lys and [3H]Leu into proteins than did the shafts.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1984

Posttranslational protein modification by amino acid addition in intact and regenerating axons of the rat sciatic nerve.

Experiments were performed to determine whether posttranslational addition of amino acids to axonal proteins occurs in axons of the rat sciatic nerve. Two ligatures were placed 1 cm apart on sciatic nerves. Six days later, segments proximal to each ligature were removed, homogenized, centrifuged at 150,000 X g, and analyzed for the ability to incorporate 3H-amino acids into proteins. No incorporation of amino acids into proteins was found in the high-speed supernatant, but when the supernatant was passed through a Sephacryl S-200 chromatography column (removing molecules less than 20 kD), [3H]arginine, lysine, leucine and aspartic acid were incorporated into proteins in both proximal and distal nerve segments. Small but consistently greater amounts of radioactivity were incorporated into proteins in proximal segments compared with distal segments, indicating that the components necessary for the reaction are transported axonally. This reaction represents the posttranslational incorporation of a variety of amino acids into proteins of rat sciatic nerve axons. Other experiments showed that the incorporation of amino acids into proteins is by covalent bonding, that the amino acid donor is likely to be tRNA, and that the reaction is inhibited in vivo by a substance whose molecular mass is less than 20 kD. This inhibition is not affected by incubation with physiological concentrations of unlabeled amino acids, by boiling, or by treatment with Proteinase K. When the axonally transported component of the reaction was determined in regenerating nerves, the amount of incorporation of amino acids into protein was 15-150 times that in intact nerves.(ABSTRACT TRUNCATED AT 250 WORDS)

Acylation↗

PubMed1983

Incorporation of 3H-amino acids into proteins in a partially purified fraction of axoplasm: evidence for transfer RNA-mediated, post-translational protein modification in squid giant axons.

Transfer RNA (tRNA) has been demonstrated to be present in axons of both invertebrates and the higher vertebrates, but nothing is known of its role in the metabolism of the axon. The present experiments were performed to determine whether tRNA functions in axons as a participant in post-translational protein modification of endogenous proteins. RNA was extracted from the axoplasm of squid giant axons and incubated with a variety of 3H-amino acids, aminoacyl-tRNA synthetases (obtained from squid optic lobe), and an appropriate reaction mixture. All of the amino acids tested were bound to an RNA fraction, but this reaction did not occur when samples were incubated in the presence of ribonuclease or in the absence of axoplasmic RNA. When radioactive RNA was chromatographed by polyacrylamide gel electrophoresis, the radioactivity comigrated with known tRNA markers, suggesting the presence of 3H-aminoacylated tRNA. Aminoacylation of RNA could also be demonstrated by incubating fresh axoplasm with labeled amino acids and a reaction mixture, minus exogenous aminoacyl-tRNA synthetases. These findings indicate the presence in axoplasm of a variety of species of aminoacyl-tRNAs as well as their corresponding synthetase enzymes. In the latter experiment no radioactivity was found associated with the protein fraction. This was also the finding when 3H-aminoacylated tRNA was either injected directly into the axon or incubated with extruded axoplasm. Thus, under the conditions described above, there is no evidence of transfer of amino acids from tRNA to proteins. In other experiments, axoplasm was pooled to a volume of 50 to 100 microliters, homogenized gently, and centrifuged at 150,000 X g for 1 hr. Some of the high speed supernatant was incubated with labeled amino acids and an appropriate reaction mixture, and the remainder was passed through an S-200 Sephacryl column before incubation with the same reaction mixture. There was no incorporation of amino acids into protein in the high speed supernatant fraction. However, in the S-200 purified fraction 3H-labeled Arg, Lys, Tyr, Leu, and Asp were all incorporated into proteins in amounts of 44, 30, 7, 5 and 3.5 times heat-inactivated controls. The reaction is not inhibited by Ca2+ or Ca2+-activated proteases, but appears to be dependent on the presence of tRNA. The addition of amino acids to protein is not protein synthesis since the reactions occurred in a partially purified fraction of the 150,000 X g supernatant, a fraction devoid of ribosomes and free amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗

PubMedDate not supplied

Toxoplasmosis in women of child bearing age and infant follow up after in-utero treatment.

A total of 540 women (including 70 pregnant cases) of child bearing age with bad obstetrical history were tested serologically for anti-toxoplasma antibody using microlatex agglutination test. Forty two women including 5 cases of pregnancy were found to be seropositive in a titre of 1:32 or more. Maximum prevalence (10.2%) and highest titer of anti-toxoplasma antibodies were observed in women of 35-42 years age group. The overall prevalence of toxoplasmosis in these women was 7.7%, whereas it was 7.1% in pregnant women. Further studies are needed to estimate the exact rate of prevalence of infection. Of the 70 pregnant women, 5 were seropositive and two of them acquired infection during pregnancy which was detected by IgM immunosorbent assay. Seropositive pregnant women were treated using combined regimen of sulfadiazine and pyrimethamine. Four infected women with pregnancy were followed up and one did not turn up subsequently. There was spontaneous abortion in one case and in 3 other cases full term normal babies were delivered. Incidence of toxoplasmosis in women is low because of infrequent and uncommon practices of ingesting undercooked or uncooked food stuff specially meat by a substantial number of the population surveyed.

Adolescent↗

PubMedDate not supplied

Neonatal candidemia.

Fifty five clinically diagnosed cases of neonatal septicaemia of the nursery ward, Medical College Hospital Calcutta were subjected to blood culture on two consecutive days in all cases. Bacterial isolates were found in 34 (61.8%) cases as pure growth and candida species from 9 (16.4%) as pure culture. Remaining 12 samples did not yield any growth. Species distribution of candida showed C. albicans 6, C. parapsilosis 2, and C. guillermondii 1. Asphyxia neonatorum was the common feature in all cases of candidaemia. All neonates with candidal infection were low-birth-weight and premature. C. albicans, the predominant species (66.6%) recovered, was responsible for a localised outbreak of infection in the nursery.

Candidiasis↗