Biomedical subjects

M K Thakur

Publications and source records attributed to M K Thakur.

At least 19 recordsLinked to original sources

PubMed2001

Pyridoxal 5'-phosphate inhibits DNA binding of HNF1.

An efficient Escherichia coli expression system was constructed for the production of a variant form of HNF1 protein having the additional five amino acid residues (Asp-Arg-Trp-Gly-Ser) at the NH(2)-terminal. The cDNA encoding HNF1 was ligated to 6 x His tag and inserted into an inducible bacterial expression vector pRSET A. After expression in E. coli, the recombinant product was purified by Ni-NTA affinity column chromatography. The purified product showed expected NH(2)-terminal sequence and specific binding to the HNF1 site. The effect of pyridoxal 5'-phosphate and its analogues on the binding activity of recombinant HNF1 was examined and found that only pyridoxal 5'-phosphate effectively inhibited the DNA binding. The concentration of pyridoxal 5'-phosphate that inhibited 50% of DNA binding was around 100 microM. Furthermore, we identified Lys197 of HNF1 molecule as the essential residue of DNA binding. These observations suggest that pyridoxal 5'-phosphate directly interacts with tissue-specific transcription factor HNF1 and modulates the binding to DNA.

Amino Acid Sequence↗

PubMed2001

Age- and sex-related expression of norbin in the brain cortex of mice.

Norbin is a novel neuron specific protein that extends the neurites of neuronal cells. It is expressed in neural tissues like brain cortex, hippocampus, spinal cord and cerebellum. In this paper, we have studied the expression of norbin mRNA and protein in the brain cortex of male and female mice of different ages. Northern blot analysis showed that the level of norbin mRNA increased in both sexes during aging. However, Western blotting revealed that the protein increased in male but decreased in female with advancing age. These findings suggest that norbin is involved in brain function which is dependent on age and sex.

Aging↗

PubMed2000

Synthesis and phosphorylation of androgen receptor of the mouse brain cortex and their regulation by sex steroids during aging.

To examine the synthesis and phosphorylation of androgen receptor (AR) and their regulation by sex steroids, adult (24 weeks) and old (65 weeks) male and female mice were gonadectomized and administered with testosterone and estradiol. AR amount, synthesis and phosphorylation were measured in the brain cortex by immunoblotting and immunoprecipitation using antibody raised against rat AR transactivation domain (TAD) which was expressed in E. coli as a fusion protein. We found that the amount of AR was high in adult and declined in old mice of both sexes. Administration of testosterone and estradiol significantly down-regulated the level of AR in old male and adult female. Similarly, the rate of AR synthesis also declined with age. Exogenous treatment of gonadectomized mice with testosterone and estradiol reduced the extent of synthesis significantly in all groups except in old female. No sex-dependent variation was noticed either in the level or synthesis of AR. In contrast, the extent of phosphorylation was higher in old mice of both sexes as compared to their adult counterparts. Testosterone and estradiol supplementation resulted in remarkable increase in AR phosphorylation in all groups. Thus it is evident from our findings that the amount and synthesis of AR decrease but phosphorylation of AR increases in the brain cortex with advancing age of mice and they are regulated by testosterone and estradiol in age- and sex-specific manner.

Aging↗

PubMed2000

Amplification of exons 4 and 5 of androgen receptor gene by testosterone in aged female mouse brain cortex.

We have investigated the effect of testosterone on the amplification of androgen receptor (AR) gene in the brain cortex of aging female mice. For this purpose, high molecular weight (HMW) DNA purified from the brain cortex of intact, gonadectomized, testosterone- and estradiol-treated adult and old female mice was digested with different restriction enzymes and used for Southern hybridization with 32P-labeled AR cDNA fragments representing different domains of AR. The results reveal that only exons 4 and 5 corresponding to amino-terminal part of the hormone binding domain of AR are amplified in testosterone-treated old female but not in adult mice. Densitometric analysis further shows that testosterone increases the copy number of exons 4 and 5 of mouse AR gene by four-fold. Reprobing of slot blots with estrogen receptor and cathepsin D cDNA as probes supports the observation that amplification occurs only in AR gene. The tissue specificity is also confirmed when the slot blot hybridization of mouse liver HMW DNA with AR cDNA fails to show similar amplification. As the restriction map analysis of Southern blots does not show restriction fragment length polymorphism, the possibility of structural rearrangement leading to amplification of AR gene is ruled out. Thus our results suggest that the in vivo induction of mouse AR gene amplification by testosterone is tissue- and age-specific, and might contribute to the progress of genetic instability in the brain of aged female mice.

Aging↗

PubMed1999

Sex steroids modulate the synthesis and phosphorylation of proteins in the brain cortex of aging mice.

We have analysed the synthesis and phosphorylation of total cellular proteins and their modulation by sex steroids (testosterone and 17beta-estradiol) in the brain cortex of adult (25-28 weeks) and old (54-58 weeks) male and female AKR mice. The level of (35S) methionine incorporation in total proteins is comparatively higher in males than females. It declines significantly in older males but shows no difference with age in females. After gonadectomy, the extent of (35S) methionine incorporation decreases in adults but not in the old. The incorporation is induced remarkably by estradiol in males and by both sex steroids in females. Further analysis by fluorography shows several proteins, but only a few (66, 45 and 29 kDa) vary in levels significantly with age, sex and hormonal treatment. The data on phosphorylation of total cellular proteins by (32P) orthophosphate incorporation exhibit no age-dependent variation. However, it is reduced drastically by gonadectomy in adults. After the addition of testosterone, the extent of phosphorylation is enhanced significantly in adults but remains the same in the old of both sexes. Estradiol also increases this modification remarkably in males of both ages and adult females, but shows no effect in old females. These results suggest that both testosterone and estradiol modulate the synthesis and phosphorylation of brain cortex proteins in age- and sex-dependent manner. This leads to alterations in physiological activities of the animal.

Aging↗

PubMed1999

Sex-specific alterations in chromatin conformation of the brain of aging mouse.

Chromatin conformation has been analysed in the brain cortex of adult (24+/-2 weeks) and old (65+/-4 weeks) male and female mice. Nuclei purified from different groups of mice were digested with MNase and DNase I for varying time periods (0-90 min), and with endogenous endonucleases for 1 h. MNase and DNase I digestion kinetics showed that the percentage of acid solubility of chromatin was relatively lower in old than adult and in female than male. This was further supported by electrophoretic analysis of nuclease digested DNA fragments. When the nuclei were incubated with only Ca2+ or Mg2+, no endonuclease digestion was observed. However, under similar conditions, the liver DNA was cleaved substantially. When divalent cations were added together, they activated endogenous endonucleases and digested the brain chromatin. The activity of Ca2+/Mg2+-dependent endogenous endonucleases was higher in male than female. Thus the accessibility of chromatin to MNase, DNase I and endogenous endonucleases was higher in male than female, and MNase as well as DNase I were more active in adult than old. Such sex- and age-dependent conformation of chromatin may attribute to differential expression of genes in the mouse brain.

Aging↗

PubMed1999

Age-dependent degradation of amyloid precursor protein in the post-mortem mouse brain cortex.

We have examined the degradation of amyloid precursor protein (APP) in the brain cortex of adult (24 +/- 2) and old (58 +/- 2) mice at different post-mortem time intervals (0, 1.5, 3, 6, 12 and 24 h). The brain cortex extract was prepared and processed for immunoblotting using antibodies against N-terminal 47-62 amino acids (Asp29) and central 301-316 amino acids containing Kunitz protease inhibitor (KPI) domain (Asp45) of APP. Asp29 (N-terminal) recognizes two bands of 140 and 112 kDa. The amount of 140 kDa is relatively higher in adult than old. The level of 112 kDa is 1.6 times lower in adult than old. It shows no remarkable change with varying post-mortem time. On the other hand, Asp45 (KPI) detects two bands of 110 and 116 kDa. While 116 kDa disappears rapidly after death of the animal, 110 kDa shows no remarkable change with different post-mortem periods. Further incubation of the disrupted tissue at 4 degrees C for 24 h and immunoblot analysis with Asp29 (N-terminal) shows 112 kDa in both ages but 58.5 kDa in adult and 70 kDa in old only. Analysis with Asp45 (KPI) shows only 54 kDa which increases after 3 h in adult but decreases significantly after 1.5 h and becomes undetectable at 24 h in old. Thus the present findings indicate that APP is degraded in a precise pattern and it depends on cellular intactness, post-mortem period and age of the animal.

Age Factors↗

PubMed1999

What causes cancer gallbladder?: a review.

Gallbladder cancer is a common malignancy of the biliary tract. It is the fifth common malignancy of the gastrointestinal tract in United States and third in Northern India. Despite such high prevalence, there is scanty published literature about this disease in indexed journals. Therefore, this article is intended to provide a brief overview of gallbladder cancer risk factors, based mainly on published evidence from analytical epidemiology and recent research findings of biologists and practising oncologists. Furthermore, an attempt has been made to establish an association between different causative factors and the occurrence of the disease.

Age Distribution↗

PubMed1998

Nucleosome positioning and periodicity of satellite DNA in the liver of aging rats. Nucleosome positioning and periodicity of satellite DNA.

The positioning of nucleosomes has been analysed by comparing the pattern of cutting sites of a probing reagent on chromatin and naked DNA. For this purpose, high molecular weight DNA and nuclei from the liver of young (18 +/- 2 weeks) and old (100 +/- 5 weeks) Wistar male rats were digested with micrococcal nuclease (MNase) and hybridized with 32P-labelled rat satellite DNA probe. A comparison of the ladder generated by MNase with chromatin and nuclei indicates long range organization of the satellite chromatin fiber with distinct non-random positioning of nucleosomes. However, the positioning of nucleosomes on satellite DNA does not vary with age. For studying the periodicity and subunit structure of satellite DNA, high molecular weight DNA from the liver of young and old rats were digested with different restriction enzymes. Surprisingly, no noteworthy age-related change is visible in the periodicity and subunit structural organization of the satellite DNA. These results suggest that the nucleosome positioning and the periodicity of liver satellite DNA do not vary with age.

Aging↗

PubMed1998

Purification, characterization and differentiation-dependent expression of a perchloric acid soluble protein from rat kidney.

We have recently reported the presence of a novel perchloric acid soluble protein in rat liver (PSP1) that inhibits cell-free protein synthesis in a rabbit reticulocyte system. While studying the perchloric acid soluble proteins from different tissues of rats, we found that the kidney protein cross-reacted with antibody against the PSP1. In this investigation, we have purified a perchloric acid soluble protein from the rat kidney and studied its characterization and expression. The protein extracted from the postmitochondrial supernatant fraction with 5% perchloric acid was purified by ammonium sulfate fractionation and CM-Sephadex chromatography. By immunoscreening with the rabbit antisera against the PSP1, we detected a cDNA that contained an open reading frame of 411 bp, encoding a 137 amino-acid protein with a molecular mass of 14,149 daltons. The deduced amino acid sequence was completely identical with that of PSP1 from rat liver. The perchloric acid soluble protein from rat kidney (K-PSP1) also inhibited cell-free protein synthesis in the rabbit reticulocyte lysate system in a different manner than RNase A. Immunohistochemistry showed that the expression of K-PSP1 increased from fetal 17th day to postnatal 4th week, and it remained almost the same until the 7th week of postnatal age. Furthermore, the expression of K-PSP1 in the kidney of the nephrotic rat model was shown to be differentiation dependent. On the other hand, the expression of K-PSP1 in renal tumor cells was downregulated as compared with intact tissue. These results suggest that the expression of K-PSP1 is regulated in a differentiation-dependent manner in the kidney.

Amino Acid Sequence↗

PubMed1997

Expression of 112-kDa estrogen receptor in mouse brain cortex and its autoregulation with age.

The mouse brain cortex expresses ER whose size is larger than uterus and level changes with age and hormonal treatment. Western blot analysis shows the presence of 66 kDa ER in uterus but 112 and 116 kDa ER in the brain. Whereas 116 kDa ER is phosphorylated and predominantly present in nuclei, 112 kDa ER is unphosphorylated and present in both cytosol as well as nuclei. Similar to uterus, the level of ER decreases drastically in the brain cortex of old mice. Hormonal regulation of ER shows age-specific pattern. Ovariectomy reduces the level of ER in adult but increases it significantly in old. While testosterone does not alter the level of ER in either age, estradiol decreases it considerably in old but shows no detectable difference in adult. Our results provide evidence for the expression of a novel form of ER in mouse brain cortex and its regulation by sex steroid hormones in age-specific manner.

Age Factors↗

PubMed1997

Nucleosomal organization of the rat liver satellite DNA-containing chromatin during aging.

Nucleosomal organization of the satellite DNA-containing chromatin of the liver of young (18 +/- 2 weeks) and old (100 +/- 5 weeks) rats was examined by nucleases and satellite I DNA probe. The satellite DNA-containing chromatin exhibits lower accessibility to endogenous endonucleases in old rats. The nucleosomal repeat length of this chromatin as investigated from digestion with endogenous endonucleases and MNase differs remarkably from bulk chromatin, though it does not alter with age. However, age-dependent loss of satellite DNA is apparent from hybridization results. Furthermore, DNase I analysis of the satellite DNA-containing chromatin at nucleosomal level reveals a relatively loose organization in young rats than old ones. It also shows an altered 10 bp periodicity as compared to bulk chromatin in both ages. These findings establish organizational differences between rat liver bulk and satellite DNA-containing chromatin. They further show that repeat length and altered 10 bp periodicity are similar in young and old, but accessibility to nucleases declines with age.

Aging↗

PubMed1997

Nuclease susceptibility of the rat liver satellite DNA-containing chromatin decreases with age.

Nuclease susceptibility of the satellite DNA-containing chromatin of the liver of young (18 +/- 2 weeks) and old (100 +/- 5 weeks) rats was analysed using nick-translated rat 185 bp satellite I DNA fragment cloned in pBR322. With increasing concentration of DNaseI and micrococcal nuclease (MNase), multimeric forms of the satellite ladder gradually disappear in both the ages. The rate of disappearance is faster in young rats as compared to old ones. Such age-dependent decrease in the susceptibility of satellite DNA-containing chromatin reflects its condensation towards heterochromatization in old age.

Age Factors↗

PubMed1996

Age-related analysis of EcoRI generated satellite DNA-containing chromatin of rat liver.

EcoRI digestion of nuclei and their subsequent lysis with EDTA solubilizes 45% and 36% of chromatin DNA from the liver of young (18 +/- 2 weeks) and old (100 +/- 5 weeks) rats, respectively. After hybridization with 185 bp rat satellite I DNA, these soluble fractions are found to be enriched in specific DNA sequences such as satellite DNA. Besides regular repeat pattern, a major portion of the satellite chromatin forms higher order organization. Digestion kinetics confirms condensation of satellite DNA-containing chromatin similar to that of bulk chromatin in old age. Furthermore, densitometric scanning of the slot-blot of soluble chromatin fractions reveals loss of satellite DNA in the old. However, an increase in the linker histone H1 and its subfraction H1zero in the satellite DNA-enriched fraction of chromatin from old rats suggests greater compaction. These results provide the first evidence that the satellite DNA-containing chromatin differs in the liver of young and old rats.

Aging↗

PubMed1993

Gene expression and aging.

Considerable amount of data has accumulated during the past few years showing several changes in gene expression as a function of age. However, the basic mechanism of aging still remains poorly understood. In this review, we have mainly analysed the data pertaining to the hypothesis that aging is associated with genetic instability and have attempted further to highlight the gaps that need to be bridged in order to have a clear picture of the aging phenomenon. Extensive investigations employing new and novel approaches are needed in future to elucidate the intricately interwoven patterns of molecular control that underlie the various aspects of gene expression during aging.

Aging↗

PubMed1992

Phenylmethylsulfonyl fluoride stimulates proteolysis of nuclear proteins from chick liver.

The degradation of H1 histone and high mobility group (HMG) nonhistone proteins was stimulated when the homogenate from chick liver was incubated in the presence of phenylmethylsulfonyl fluoride (PMSF). Two proteinase inhibitors, elastatinal and chymostatin, significantly inhibited the PMSF-stimulated degradation of H1 histone and HMG proteins. On the contrary, other proteinase inhibitors like leupeptin, pepstatin, trypsin inhibitor, antipain, o-phenanthroline and EDTA had no effect on the degradation of the nuclear proteins. These results warn the researcher to be cautious while using PMSF for preparation of nuclear proteins such as H1 histone and HMG proteins.

Animals↗

PubMed1992

Estrogen-induced synthesis of uterine proteins declines during aging.

The synthesis of total cellular as well as acid-soluble nuclear proteins and estrogen receptor is high in the uteri of young (22 weeks) and decreases to half in old (104 weeks) rats. Administration of estrogen induces the synthesis of these proteins significantly in young but shows no remarkable effect in old rats. Interestingly, a specific cytosolic protein of 45 kDa is stimulated about two-fold after estrogen injection in young but not in old rats. These findings further establish the reduced responsiveness of uterus to estrogen in old age.

Aging↗

PubMed1991

Analysis of age-associated alteration in the synthesis of HMG nonhistone proteins of the rat liver.

HMG proteins were extracted with 5% PCA or 0.35 M NaCl from whole tissue, nuclei or chromatin of the liver of young (19 weeks) and old (118 weeks) male rats. They were resolved on acetic acid-urea polyacrylamide gel. The electrophoretic patterns of the major HMG proteins 1, 2, 14 and 17 of both ages are similar. The in vitro synthesis of HMG 1 and 2 decreases, but that of HMG 14 and 17 increases considerably in the liver of old rats. The synthesis of different HMG proteins is modulated differentially by spermine, butyrate, dexamethasone and 3-aminobenzamide in the liver of young and old rats. These findings suggest that HMG proteins contribute to alterations in the organization of chromatin and expression of genes during aging.

Aging↗