PubMed Health⌕ Search

Biomedical subjects

G Chakraborty

Publications and source records attributed to G Chakraborty.

At least 19 recordsLinked to original sources
PubMed2003

Interleukin-2 receptors and interleukin-2-mediated signaling in myelin: activation of diacylglycerol kinase and phosphatidylinositol 3-kinase.

Myelin was previously shown to possess neurotransmitter and cytokine receptors that trigger well-defined signaling mechanisms within the multilamellar structure. The present study reveals the presence of an interleukin-2 (IL-2) receptor in isolated mouse CNS myelin that responds to recombinant mouse IL-2 by activating diacylglycerol kinase (DAGK) and phosphoinositide 3-kinase (PI3K); additional evidence suggests participation by protein tyrosine kinase. Activation of myelin DAGK by IL-2 occurred in brain stem tissue mince and was blocked by chelerythrin chloride, indicating an essential role for myelin-localized protein kinase C. Two inhibitors of PI3K, wortmannin and LY294002, blocked endogenous PI3K as well as that enhanced by IL-2. Activation of PI3K by IL-2 was also blocked by tyrphostin A25, a selective inhibitor of PTK, suggesting activation of the latter by IL-2 is upstream to PI3K activation. This reaction resulted in tyrosine phosphorylation of a protein tentatively identified as the p85 subunit of PI3K. Developmental changes were noted in that receptor density and signaling activity were robust during the period of rapid myelination and declined rapidly thereafter.

Animals↗
PubMed2001

Intraneuronal N-acetylaspartate supplies acetyl groups for myelin lipid synthesis: evidence for myelin-associated aspartoacylase.

Despite its growing use as a radiological indicator of neuronal viability, the biological function of N-acetylaspartate (NAA) has remained elusive. This is due in part to its unusual metabolic compartmentalization wherein the synthetic enzyme occurs in neuronal mitochondria whereas the principal metabolizing enzyme, N-acetyl-L-aspartate amidohydrolase (aspartoacylase), is located primarily in white matter elements. This study demonstrates that within white matter, aspartoacylase is an integral component of the myelin sheath where it is ideally situated to produce acetyl groups for synthesis of myelin lipids. That it functions in this manner is suggested by the fact that myelin lipids of the rat optic system are well labeled following intraocular injection of [14C-acetyl]NAA. This is attributed to uptake of radiolabeled NAA by retinal ganglion cells followed by axonal transport and transaxonal transfer of NAA into myelin, a membrane previously shown to contain many lipid synthesizing enzymes. This study identifies a group of myelin lipids that are so labeled by neuronal [14C]NAA, and demonstrates a different labeling pattern from that produced by neuronal [14C]acetate. High performance liquid chromatographic analysis of the deproteinated soluble materials from the optic system following intraocular injection of [14C]NAA revealed only the latter substance and no radiolabeled acetate, suggesting little or no hydrolysis of NAA within mature neurons of the optic system. These results suggest a rationale for the unusual compartmentalization of NAA metabolism and point to NAA as a neuronal constituent that is essential for the formation and/or maintenance of myelin. The relevance of these findings to Canavan disease is discussed.

Amidohydrolases↗
PubMed1999

The phosphoinositide signaling cycle in myelin requires cooperative interaction with the axon.

Previous studies on the origin of myelin phosphoinositides involved in signaling mechanisms indicated axon to myelin transfer of phosphatidylinositol followed by myelin-localized incorporation of axon-derived phosphate groups into phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate. This is in agreement with other studies showing the presence of phosphorylating activity in myelin that converts phosphatidylinositol into the mono-and diphospho derivatives. It was also found that the second messenger, inositol 1,4,5-trisphosphate, is hydrolyzed to inositol 1,4-bisphosphate by a myelin-localized enzyme. The present study was undertaken to determine the locus of the remaining reactions leading to formation of free inositol and completion of the cycle by resynthesis of phosphatidylinositol. The latter reaction was found to occur preferentially in isolated axons, and to a limited extent if at all in myelin. On the other hand, hydrolytic reactions which sequentially convert inositol 1,4,5-trisphosphate to inositol 1,4-bisphosphate, inositol 1-phosphate, and free inositol were found to occur more prominently in myelin. Thus, restoration of phosphoinositides following signal-induced breakdown of PIP2 in myelin is seen as requiring metabolic interplay between myelin and axon.

Animals↗
PubMed1999

Using choice-based conjoint to determine the relative importance of dental benefit plan attributes.

The purpose of this study was to use conjoint analysis to determine the importance of specific dental benefit plan features for University of Iowa (UI) staff and to build a model to predict enrollment. From a random sample of 2000 UI staff, 40 percent responded (N = 773). The survey instrument was developed using seven attributes (five dental benefit plan features and two facility characteristics) each offered at three levels (e.g., premium = $20, $15, $10/month). Pilot testing was used to find a realistic range of plan options. Twenty-seven hypothetical dental benefit plans were developed using fractional factorial combinations of the three levels for each of the seven attributes. For all of the hypothetical plans, dental care was to be provided in the UI predoctoral dental clinic. Plan profiles were arranged four per page by combining the existing plan with three hypothetical plans, for a total of nine pages. Respondents' task was to select one plan from each set of four. A regression-like statistical model (Multinomial Logit) was used to estimate importance of each attribute and each attribute level. Relative importance (and coefficients) for each of the seven attributes are as follows: maximum annual benefit (.98), orthodontic coverage (.72), routine restorative (.70), major restorative (.67), time to complete treatment (.61), clinic hours of operation (.47), premium (.18). For each attribute, relative importance of each of three levels will also be presented. These coefficients for each level are used to predict enrollment for plans with specific combinations of the dental benefit plan features.

Algorithms↗
PubMed1998

Cytokines, signal transduction, and inflammatory demyelination: review and hypothesis.

The mechanism of focal demyelination in multiple sclerosis has been a long-standing enigma of this disorder. Cytokines, a diverse family of signalling molecules, are viewed as potential mediators of the process based on clinical observations and studies with animal models and tissue/cell culture systems. Myelin and oligodendrocyte (OL) destruction occur in cultured preparations subjected to cytokines such as tumor necrosis factor-alpha (TNF alpha) and lymphotoxin (LT). Many studies have shown these and other cytokines to be elevated at lesion sites and in the CSF of multiple sclerosis (MS) patients, with similar findings in animal models. Some variability in the nature of MS lesion formation has been reported, both OLs and myelin being primary targets. To account for myelin destruction in the presence of apparently functional OLs we hypothesize that cytokines such as TNF alpha and LT alpha contribute to myelin damage through triggering of specific reactions within the myelin sheath. We further propose that neutral sphingomyelinase (SMase) is one such enzyme, two forms of which have been detected in purified myelin. An additional event is accumulation of cholesterol ester, apparently a downstream consequence of cytokine-induced SMase. The resulting lipid changes are viewed as potentially destabilizing to myelin, which may render it more vulnerable to attack by invading and resident phagocytes.

Animals↗
PubMed1998

Serology and immunoglobulin profile in rheumatoid arthritis.

One hundred and twenty cases of clinically diagnosed rheumatoid arthritis, 80 non-rheumatoid cases suffering from various other diseases and 40 healthy individuals were investigated for the presence of rheumatoid factor, quantitation of serum immunoglobulin, demonstration of ANA and LE cell phenomenon. Microlatex agglutination test of serum for rheumatoid factor showed 56.6% positivity in rheumatoid group and 3.7% positivity in non-rheumatoid group. All three serum immunoglobulins (IgG, IgM, IgA) were raised in serum in significant titre in cases of rheumatoid arthritis, whereas only IgA lever was elevated in the group of non-rheumatoid diseases. ANA and LE cell phenomenon were observed in 11.7% and 4.4% cases of rheumatoid arthritis who had severe underlying disease. In non-rheumatoid group, only one of 6 cases of systemic lupus erythematosus showed rheumatoid factor and that too in an insignificant titre (less than 1:20). Synovium and synovial fluid contained plenty of plasma cells and lymphocytes. It has been observed that RF appears first in synovial fluid and it may take several months to a year to attain detectable level in serum.

Adolescent↗
PubMed1997

Myelin contains neutral sphingomyelinase activity that is stimulated by tumor necrosis factor-alpha.

Purified myelin from mouse brain was found to contain two forms of neutral sphingomyelinase, one Mg2+ dependent and the other Mg2+ independent. The former had a pH optimum of 7.5 and Km of 0.35 mM, whereas the corresponding values for the latter were pH 8.0 and Km 3.03 mM. Specific activity of the Mg(2+)-dependent enzyme showed a rostral-caudal gradient, ranging from 75 nmol/mg protein/hr in myelin from cerebral hemispheres to 21 nmol/mg protein/hr in myelin from spinal cord. Relative specific activity was approximately 20% that of brain stem or cerebral hemisphere homogenate. Treatment of myelin with taurocholate or high salt concentration did not significantly reduce activity of the Mg(2+)-dependent enzyme. The activity of that enzyme did not change with time or in the presence or absence of protease inhibitors; by contrast, that of Mg(2+)-independent enzyme decreased sharply in the absence of protease inhibitors but rose in their presence. To test for the effect of tumor necrosis factor-alpha (TNF alpha) on myelin sphingomyelinase, mouse brain myelin was labeled in vivo by intracerebral injection of [3H]acetate into 18-20-day-old mice. After 40 hr, brain stems were removed, minced, and treated with TNF alpha in Krebs-Ringer solution, after which myelin was immediately isolated. Separation and counting of individual lipids revealed TNF alpha treatment to cause increased labeling of myelin ceramide and cholesterol ester with concomitant decrease in myelin sphingomyelin. Western blotting of myelin proteins using antibodies to the two TNF alpha receptors as probes revealed the presence of the p75 receptor. Implications of these findings in relation to possible mechanisms of autoimmune demyelination are discussed.

Acetates↗
PubMed1996

Characterization of guanylyl cyclase in purified myelin.

This study was undertaken to characterize the enzymatic properties of the particulate guanylyl cyclase previously shown to be present at a high level of activity in purified rat brain myelin. Significant activation was achieved by both Lubrol-PX and Triton X-100, the latter being somewhat more effective. A pH optimum of 7.8 was observed, compared to 7.4 for microsomes. Employing 1.2 mM GTP with 1% Triton X-100, linearity of response was observed up to 60 min and approximately 1.2 mg of myelin protein. Kinetic analysis revealed Km values of 0.258mM and 0.486mM for myelin and microsomes, respectively, similar values being obtained by Lineweaver-Burke analysis or Direct Linear Plot. Vmax values were 20 and 266 pmol/mg protein/min for myelin and microsomes, respectively. Washing of the myelin with 0.5 M NaCl or 0.1% Na taurocholate did not remove a significant amount of guanylyl cyclase activity, indicating the enzyme to be intrinsic to the myelin sheath.

Animals↗
PubMed1994

Serine protease inhibitors block N-terminal arginylation of proteins by inhibiting the arginylation of tRNA in rat brains.

The tRNA mediated, posttranslational, N-terminal arginylation of proteins occurs in all eukaryotic cells. In nervous tissue, these reactions can be inhibited by endogenous molecules with a molecular weight of between one thousand and five thousand. In the present experiments, exogenous serine protease inhibitors (10(-5) M or less) but not other types of protease inhibitors, were found to be able to block the arginylation of protein in extracts of rat brain homogenates. Inhibition was not by the usual mode of action of protease inhibitors, but by interfering (non-competitively) with the charging of tRNA. Since arginylated proteins are rapidly ubiquitinated and degraded by cytosolic proteases, serine protease inhibitors may act to stabilize proteins by a dual mechanism of inhibiting arginylation as well as inhibiting serine proteases.

Animals↗
PubMed1994

How consumers choose health insurance.

The authors used choice-based conjoint analysis to model consumers' decision processes when evaluating and selecting health insurance in a multiplan environment. Results indicate that consumer choice is affected by as many as 19 attributes, some of which have received little attention in previous studies. Moreover, the importance of the attributes varies across different demographic segments, giving marketers several targeting opportunities.

Adult↗
PubMed1993

N-terminal arginylation of proteins in explants of injured sciatic nerves and embryonic brains of rats.

Posttranslational modification of proteins by arginine and lysine has been demonstrated in crude extracts of vertebrate nerves and brain but not in intact cells. In the present experiments we have exploited the fact that Arg is added posttranslationally only at the N-terminus of target proteins, to demonstrate these reactions in intact cells of sciatic nerves and embryonic brains of rats. Sciatic nerves were crushed in anaesthesized rats and 2 hrs later segments of nerve, including the site of the crush, were removed and incubated in media containing [3H]Arg. Incorporation of [3H]Arg into total proteins was analyzed by acid precipitation and the presence of label at the N-terminus was determined by a modification of the Edman degradation procedure. Approximately 25% of protein bound [3H]Arg was released from the N-terminus by the Edman reaction indicating that it was added posttranslationally rather than through protein synthesis. N-terminal labeling was not detectable in nerves not crushed prior to explant and incubation. Slices of embryonic day 20 visual cortex, when incubated under similar conditions as injured sciatic nerves, also showed approximately 25% of the protein incorporated [3H]Arg at the N-terminus, while arginylation was not detectable in adult rat brain slices. Since Lys is not added posttranslationally to the N-terminus, we have attempted to observe lysylation of proteins in intact cells by using cycloheximide (Cx) to block protein synthesis without interfering with protein modification. The posttranslational incorporation of Arg/Lys into proteins was found to be insensitive to up to 2.0 mM Cx in tissue extracts (in vitro).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

N-terminal arginylation and ubiquitin-mediated proteolysis in nerve regeneration.

Damaged sciatic nerves of rats respond to injury within minutes by activating reactions that result in the transfer RNA-mediated posttranslational addition of several amino acids to a variety of cytoplasmic proteins. For the most part, the site of addition of individual amino acids and the identity of the target proteins is not known. However, arginine, one of the amino acids added in greatest amounts, has been shown to be covalently linked to the N-terminus of acceptor proteins. In other simpler eukaryotic cells, N-terminal arginylation results in degradation of the arginylated proteins via the ubiquitin proteolytic pathway. Recent experiments have shown that when proteins, obtained from sciatic nerves 2 h after injury, are arginylated in vitro, they form high molecular weight aggregates. Other experiments have shown that these arginylated proteins are immunoreactive to a monoclonal antibody to ubiquitin. These findings suggest that following injury to the sciatic nerve, proteins which are arginylated are candidates for ubiquitin mediated proteolysis. Injury to a nerve incapable of regeneration without experimental intervention, the rat optic nerve, does not result in activation of the arginylation reactions until 6 days following injury. Based on the temporal differences in response to injury of sciatic and optic nerves (2 h vs. 6 days), we propose that the lack of arginylation following injury to the CNS is related to its inability to mount a regenerative response. The association of Arg modification of damaged proteins with the ubiquitin-mediated degradation of those proteins, suggests that regenerative failure in the CNS may be related, in part, to a failure to degrade intracellular proteins at the site of injury.

Animals↗
PubMed1993

Guanylyl cyclase activity in rat brain myelin and white matter.

Purified myelin from rat brainstem was found to have an appreciable level of guanylyl cyclase activity, as seen in the formation of 3',5'-cyclic GMP from [3H]GTP at a rate approximately 45% that of whole brainstem. Freshly isolated myelin from pooled rat brainstems was incubated with GTP in an appropriate mixture. This gave rise to 29.9 +/- 3.6 pmol of 3',5'-cyclic GMP/mg of protein/min measured by HPLC and a similar result (26.7 +/- 2.6 pmol/mg/min) with 125I-3',5'-cyclic GMP radioimmunoassay. The latter method applied to the reaction product from whole brainstem gave a value of 56.6 +/- 3.4 pmol/mg/min. In analyzing brainstem products by HPLC we observed in most trials concurrent formation of a second radiolabeled product that comigrated with 2',3'-cyclic GMP but that, on further examination, proved not to be that product. Its identity remains unknown.

Animals↗
PubMed1993

Understanding consumers' preferences for dental service.

Previous researchers typically have used attitude and opinion surveys to investigate consumers' perceived importance of dental services attributes. These approaches do not require consumers to trade-off among different attributes, however. The authors use conjoint analysis to analyze consumers' preferences for dental services described by a set of 24 attributes, examining the relative importance as well as the trade-off among these attributes. They also illustrate how dental care providers can use the results from conjoint analysis to tailor their offerings to meet the needs of different segments.

Age Factors↗
PubMed1992

Ubiquitin is associated with aggregates of arginine modified proteins in injured nerves.

Crush injury to rat sciatic nerves results in a 10-fold increase in the post-translational arginylation of proteins. In other systems, N-terminal arginylation leads to ubiquitination and proteolysis of the arginylated proteins. In the present experiments, proteins obtained from the 150 kg supernatant of crushed sciatic nerves were posttranslationally modified by 3H-arginine. These arginine modified proteins formed aggregates (precipitated at 20 kg) which then partially separated by SDS-PAGE were immunoreactive to a monoclonal antibody to ubiquitin. The results indicate that following injury to sciatic nerves, certain proteins are arginylated and ubiquitinated, probably targeting them for degradation. It is likely that these reactions help to rid cells of proteins damaged by the crush which would otherwise be cytotoxic.

Animals↗
PubMed1992

Evidence that axonal tRNAs are resistant to RNase and ATPase and can be aminoacylated in the absence of exogenous ATP.

A high molecular weight (HMW) fraction of the 150,000 g supernatant of rat brain homogenates contains protein-tRNA complexes which are able to incorporate [3H]Arg and [3H]Lys into tRNA. The aminoacylation of tRNA(Arg) was found to be dependent on ATP and inhibited by RNase. Conversely, the aminoacylation of tRNA(Lys) did not require exogenous ATP and was resistant to RNase and ATPase. In HMW fractions of regenerating rat sciatic nerves, the charging of both tRNA(Arg) and tRNA(Lys) was resistant to RNase and ATPase and did not require exogenous ATP. Because sciatic nerves are rich in axoplasm and tRNAs are known to be present in axons, we tested the hypothesis that degradative enzyme-resistant, ATP-tRNA complexes were of axonal origin. In HMW fractions from rat liver (containing no axons), both tRNA(Arg) and tRNA(Lys) were sensitive to RNase and required exogenous ATP for charging. But, in similar fractions of axoplasm obtained from the giant axon of squid, both tRNAs were insensitive to RNase and ATPase and did not require exogenous ATP for charging. These results suggest that tRNAs in axons are present in protected HMW complexes and contain endogenous stores of ATP. The presence of ATP in the HMW complexes was demonstrated by the luciferase-luciferin assay for ATP. The nature of the protection of tRNAs from RNases was examined by dissociating proteins from HMW complexes by boiling, treating with proteinase K, or overhomogenizing the tissue. These procedures failed to render brain tRNA(Lys) susceptible to RNase. But phenol-extracted, ethanol-precipitated brain tRNA(Lys) was sensitive to RNase, suggesting that the protection of tRNA(Lys) may be by a protease- and heat-resistant polypeptide or by a nonproteinaceous mechanism.

Adenosine Triphosphatases↗