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Biomedical subjects

S Pandey

Publications and source records attributed to S Pandey.

At least 73 records · Page 4Linked to original sources
PubMed1997

Identification of a novel 97 kDa endonuclease capable of internucleosomal DNA cleavage.

The two steps of DNA digestion seen in apoptotic cells were recreated in nuclei isolated from 5123tc rat hepatoma cells. The initial DNA cleavage, into high molecular weight fragments (300-50 kb), was stimulated by magnesium ions alone, whereas the second step required both calcium and magnesium ions and produced the ladder of oligonucleosomes. Endonucleolytic activities involved in both steps of DNA cleavage could be separated under appropriate conditions since the magnesium-modulated activity was tightly bound to the chromatin whereas the calcium/magnesium-dependent internucleosomal cleaving activity was easily extractable with a low ionic strength buffer. This calcium/ magnesium-dependent activity was attributed to a novel 97 kDa endonuclease which was also activated by manganese and cobalt and inhibited by millimolar concentrations of zinc, consistent with the properties ascribed to the apoptotic endonuclease. Furthermore, this activity became resistant to extraction with a low salt buffer in nuclei of apoptotic cells. Isoelectrofocusing revealed that the p97 protein existed in multiple forms of different isoelectric points (pI range 4.6-5.0), indicative of its postranslational modification. The p97 enzyme was present constitutively in a variety of cultured cells and rat tissues. It was active over a broad range of pH (6-9), but it was inactivated by reducing agents. In vitro, it displayed both endo- and exonucleolytic activities, and it was capable of both single- and double-stranded DNA cleavage. Rabbit polyclonal anti-p97 antibodies were generated and used to further distinguish this protein from other known cellular nucleases, namely, DNases I and II.

Animals↗
PubMed1997

Absorption of ciprofloxacin and norfloxacin when administered as niosome-encapsulated inclusion complexes.

An attempt has been made to design suitable niosome-encapsulated drug delivery system for ciprofloxacin and norfloxacin. Encapsulation of ciprofloxacin and norfloxacin in niosomes was investigated and the nasal and intestinal absorption of the products studied. More than 80% of the drugs were successfully encapsulated to give products with sustained release characteristics. Encapsulation in niosomes also improved the stability of the antibacterial compounds. Although the systemic availability of these niosome-encapsulated antibacterial compounds was not increased after nasal administration, intestinal absorption was significantly higher in comparison with that of plain inclusion complexes.

Absorption↗
PubMed1997

Spectrochemical investigations of fluorescence quenching agents. Part 5. Effect of surfactants of the ability of nitromethane to selectively quench fluorescence emission of alternant PAHs.

Applicability of the nitromethane selective quenching rule for discriminating between alternant vs. nonalternant polycyclic aromatic hydrocarbons (PAHs) is examined for 18 representative PAH solutes dissolved in micellar cetyltrimethylammonium chloride (CTACl), micellar dodecyltrimethylammonium bromide (DTAB), micellar Brij-35 and micellar sodium octanoate (SO) solvent media. Experimental results show that nitromethane quenched fluorescence emission of only the 10 alternant PAHs in the two cationic (CTACl and DTAB) and nonionic Brij-35 surfactant solvent media as expected. Emission intensities of nonalternant PAHs, except for the few exceptions noted previously, were unaffected by nitromethane addition. Unexpected quenching behavior was observed, however, in the case of nonalternant PAHs dissolved in micellar sodium octanoate solvent media. Nitromethane quenched fluorescence emission of all nonalternant PAHs studied in the SO solvent media, which is contrary to the selective quenching rule.

Fluorescent Dyes↗
PubMed1997

Antitumor and antifertility activities of plumbagin controlled release formulations.

With a view to increase efficiency and reduce toxicity of Plumbagin, an attempt was made to formulate plumbagin as a controlled release preparation using various carriers and test for their antitumor and antifertility activities. Niosomes and albumin microspheres were used as carriers. In vitro data showed promising results for these formulations thus they were taken up for in vivo assessment. Given at a dose of 5 mg/kg, ip the albumin microspheres showed promising antitumor and antifertility activity when compared to the niosomes on control. Animal survival data also indicated slight improvement in survival rate and thus antitumoral activity. Also, an interesting point was that the antifertility activity was affected through an antiovulatory action as seen from histopathological studies.

Animals↗
PubMed1996

Understanding the relationship between neighborhood poverty and specific types of child maltreatment.

In this paper we examined the relationship between neighborhood poverty and three different types of child maltreatment; neglect, physical abuse, and sexual abuse. We explore both rates of reporting and rates of substantiated reports across low, moderate, and high poverty areas. We use 1990 Census data and Child Protective Services (CPS) data to study this relationship. We find that neighborhood poverty is positively associated with all three forms of child maltreatment, but to different degrees. Of the three types of child maltreatment, child neglect is most powerfully associated with neighborhood poverty status.

Adolescent↗
PubMed1996

Bifunctionality of the AcMNPV homologous region sequence (hr1): enhancer and ori functions have different sequence requirements.

The Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) homologous region sequence hr1 is a putative origin of replication (ori) sequence and can also function as a transcriptional enhancer for delayed-early genes. We demonstrate that this 750-bp sequence, carrying five 28-bp core palin-dromes, enhances expression from the very late polyhedrin promoter up to 11-fold in a classical enhancer fashion in transient expression assays. Enhancement is at the level of transcription, as evident from RNase protection assay analysis. It is mediated by an alpha-amanitin-insensitive RNA polymerase from the authentic polyhedrin promoter transcription start site and follows the temporal activation profile characteristic of the polyhedrin promoter. Three lines of evidence conclusively demonstrated that hr1 acts typically as an enhancer of polyhedrin gene transcription independent of its role as an ori: (i) linearized hr1-reporter plasmids, incapable of replicating in the host cell, could enhance transcription from the promoter; (ii) reporter plasmid copy number was not affected by the presence of aphidicolin during transfection; (iii) reporter plasmid DNA recovered from Sf9 cells was sensitive to Dpn I confirming its unreplicated state in the transfection regime followed by us. Molecular dissection of the hr1 sequence elements revealed that a core palindrome alone can function as an ori sequence whereas a palindrome along with flanking sequences is essential for the enhancer activity. Enhancement of luciferase expression from the polyhedrin promoter is a function of the number of core palindromes and flanking sequences. Our results demonstrate that hr1, which has several motifs for enhancer binding proteins and transcription factors, has a dual role associated with both DNA replication and transcriptional enhancement.

Animals↗
PubMed1996

Active cell death induced by the anti-estrogens tamoxifen and ICI 164 384 in human mammary carcinoma cells (MCF-7) in culture: the role of autophagy.

Active cell death in hormone-dependent cells was studied using cultured human mammary carcinoma cells (MCF-7) treated with the anti-estrogens (AEs) tamoxifen (TAM), 4-hydroxy-tamoxifen (OH-TAM) or ICI 164 384 (10(-8)-10(-5) M) as a model. The following results were obtained. (i) In untreated MCF-7 cells a wave of replication occurred in the first 5 days of culture. All three AEs caused a dose-dependent inhibition of cell replication. (ii) TAM and OH-TAM at 10(-5) M, but not ICI 164 384, caused lytic cell death (necrosis) within 24 h, which was not inhibited by estradiol (10(-9)-10(-6)M). (iii) Lower concentrations of TAM or OH-TAM (up to 10(-6) M) or ICI 164 384 induced a more gradual appearance of cell death beginning at day 3. This type of cell death was inhibited by estradiol (10(-9) M), indicating its active nature. (iv) Nuclei showed two distinct patterns of alteration: (a) apoptosis-like condensation and fragmentation of chromatin to crescent masses abutting the nuclear envelope; (b) condensation of the chromatin to a single, pyknotic mass in the center of the nucleus, detached from the nuclear envelope. Quantitative histological evaluation revealed the predominance of pyknosis. (v) Biochemical DNA analysis revealed that only a relatively small amount of the total DNA was finally degraded into low molecular weight fragments (20 kb and less). (vi) Active cell death, with both apoptotic and pyknotic nuclear morphology, was associated with extensive formation of autophagic vacuoles (AV).3-Methyladenine, a known inhibitor of AV formation, partially prevented cell death as detected by nuclear changes. (vii) ICI 164 384 was about 10 times more effective than TAM or OH-TAM at inhibiting DNA synthesis, but had equal potency in inducing active cell death. It is concluded that AEs have anti-proliferative and anti-survival effects on MCF-7 human mammary cancer cells in culture. These two effects are under separate control because they differ by kinetics, dose dependence and sensitivity to the various AEs. Active cell death in MCF-7 cells seems to be initiated by autophagy, in contrast to concepts of apoptosis, and thus corresponds to autophagic/ lysosomal or type II death as previously defined. This may be important because of biochemical and molecular differences between these various subtypes of active cell death.

Autophagy↗
PubMed1996

Status of free radicals and their scavenging enzymes in pregnancy induced hypertension (PIH).

The levels of lipid peroxidation (malonaldialdehyde), one of the consequence of free radical damage, and the antioxidant enzymes superoxide dismutase and catalase were estimated in the blood samples of fourteen normal and thirteen pregnancy induced hypertensive patients. A marked increase in malonaldialdehyde (p < 0.001) with concomitant decrease in superoxide dismutase (p < 0.001), catalase (p < 0.001) activities were observed in PIH as compared to normal pregnancy, thereby indicating the involvement of free radicals in PIH.

Adult↗
PubMed1996

A study of free radicals and scavenging enzyme in tonsillitis.

The present study is a comparison of malonaldialdehyde (MDA) level and superoxide dismutase (SOD) activity between controls and in tonsillitis patients of different degree before (pre) and after (post) surgery (tonsillectomy). The SOD activity increases in pretonsillectomy cases according to severity of disease and there is a rise in MDA level whereas after tonsillectomy although the increase in SOD is marginal but MDA declines sharply as compared to pretonsillectomy patients indicating that the SOD exerts its protective effect after surgery.

Free Radicals↗
PubMed1995

Cells en route to apoptosis are characterized by the upregulation of c-fos, c-myc, c-jun, cdc2, and RB phosphorylation, resembling events of early cell-cycle traverse.

Density-arrested quiescent murine Balb/c-3T3 cells are dependent upon growth factors for their survival. Withdrawal of serum from their medium induces rapid cell death, the mechanism of which is not yet fully understood. We have studied the effect of serum deprivation on density-inhibited quiescent Swiss 3T3 cells and found that they undergo rapid cell death upon total withdrawal of serum. The nature of this cell death is similar to apoptosis, as shown by cellular and nuclear morphology and DNA fragmentation into oligonucleosomal fragments. Investigating the regulation of early cell-cycle genes during this process, we found that c-myc, c-jun, c-fos, and cdc2 protein presence is induced after serum deprivation, when the phosphorylated form of the RB protein also appears. The upregulation of these genes' protein products is coupled with the appearance of PCNA, a proliferation-specific nuclear antigen, as well as significant incorporation of BrdU, which may reflect DNA repair activity; in situ analysis shows that BrdU-positive cells are also positive for DNA fragmentation. These results suggest that en route to apoptosis, cells undergo events typical of early cell-cycle traverse by expressing early G1 genes and may even experience the late G1/S phase boundary, as shown by the presence of PCNA. However, the demonstrated ability of these cells to traverse the G1 phase of the cell cycle seems to be an abortive event, since they die shortly afterwards.

3T3 Cells↗
PubMed1995

Down-regulation of statin, a nonproliferation-specific nuclear protein, and up-regulation of c-myc after initiation of programmed cell death in mouse fibroblasts.

Deprivation of growth factors has been shown to induce programmed cell death in many cell types, including mouse 3T3 fibroblasts. Programmed cell death (apoptosis) is an active process of self-destruction which is thought to require the expression of unique genes. Recently, the expression of cell cycle genes such as c-fos and c-myc, and re-entrance to cell cycle traverse, are thought to be necessary to induce programmed cell death. Previous work in this laboratory has shown that statin is a nonproliferation-specific nuclear protein present in the nuclei of young quiescent or senescent human fibroblasts, as well as in growth-arrested mouse 3T3 fibroblasts; we have reported that statin disappears rapidly after the blockage of growth arrest is removed and cells are allowed to resume cell cycle traverse. In this report we address the question of whether cells induced to enter the programmed cell death process also lose the expression of statin. We studied density-arrested quiescent mouse 3T3 cells, which undergo rapid cell death by apoptosis upon serum deprivation. Our results suggest that c-myc expression is induced, as previously reported in other systems of apoptotic death. Interestingly, we also find that statin indeed disappears after the induction of programmed cell death is initiated. These results further support the notion that when apoptosis is induced, cells behave as though released from replication arrest, and experience some part of the G1 phase of the cell cycle. The difference between this event and normal cell cycle traverse is that this experience of the G1 phase in the apoptotic process is an abortive one, with the end result of cell demise.

3T3 Cells↗
PubMed1995

Expression of proliferation-specific genes in the mucosa adjacent to colon carcinoma.

BACKGROUND: One of the main concerns in colon carcinoma therapy is local recurrence of the malignancy at the site of resection. Previous studies have shown that morphologically normal-appearing mucosa adjacent to colon carcinoma is different from mucosa distant from carcinoma. Mucosa adjacent to a carcinoma is characterized by crypt lengthening, cell hypertrophy, and change in production of mucopolysaccharides from sulfomucin in normal mucosa to sialomucins in carcinomas and adjacent mucosa. Recently there have been reports suggesting that there is an upward extension of the proliferative compartment in colonic crypts of this adjacent mucosa. METHODS: Immunoblot analysis using antibodies to retinoblastoma, statin, c-Fos, c-Jun, and Cdc-2 proteins was used for our study on the expression of early cell cycle genes in carcinoma and its adjacent mucosa. In all, 15 tissue samples obtained from patients with colon carcinoma were analyzed. Tissue specimens were collected and immediately dissected as tumor, 0 to 1, 1 to 2, 2 to 3, 3 to 4, and 4 to 5 cm from the primary lesion. Dissected pieces were homogenized separately and subjected to immunoblot analysis. RESULTS: We found upregulation of c-Fos, c-Jun, and Cdc-2 expression in carcinoma and adjacent mucosa up to 4 cm from the edge of the carcinoma. The phosphorylated form of retinoblastoma is present in the carcinoma as well as in adjacent mucosa up to 4 cm from the margin of the carcinoma. Furthermore, we observed that the level of statin, a nonproliferation-specific nuclear protein, is very low in the primary lesion and in adjacent mucosa up to 3 cm. CONCLUSIONS: These results indicate that adjacent tissue up to 3 to 4 cm from the carcinoma has elevated levels of expression for cell cycle traverse-associated genes and down-regulation of nonproliferation-specific gene expressions such as statin. This imbalance indicates that within 3 to 4 cm from the edge of the carcinoma, colonic epithelial cells are already abnormal and may be in the hyperproliferative and preneoplastic state, susceptible to further steps leading to eventual malignant transformation.

Carcinoma↗
PubMed1995

Alternative splicing in human retinal mRNA transcripts of an opsin-related protein.

An opsin-related gene encodes a putative RPE-retinal G-protein-coupled receptor (RGR) that is most homologous to the visual pigments and invertebrate retinochrome. A splice variant of human RGR mRNA can be demonstrated by the sequence of isolated cDNA clones and by the amplification and analysis of human retinal mRNA. The shortened transcript contains a deletion of 114 nucleotides that correspond exactly to the sequence of exon 6 in the human rgr gene. The predicted RGR variant lacks the putative sixth transmembrane domain and has a calculated molecular weight of 27,726. Variable amounts of a 28-kDa protein were found in the retinas of some individuals by immunoblot assay. Since a similar shortened RGR transcript was not detected in bovine retina or RPE, the RGR variant is not essential for vertebrate vision. Analysis of the structure of the rgr gene and of the sequences of cDNA clones indicates that the truncated mRNA may be produced through alternative splicing of pre-mRNA from which a cassette exon is removed and the predicted RGR variant is radically altered in primary structure.

Base Sequence↗
PubMed1995

Degradation of chromatin in apoptotic cells.

We present here a model for the degradation of chromatin in cells undergoing apoptosis. This model rationalises all aspects of the fragmentation process that have been described to date, explaining not only the patterns of degradation seen within individual cells, but also the variability in extent of degradation seen in different cells. Although DNA fragmentation in apoptosis was initially considered to be solely internucleosomal, it is now apparent that the process is much more complex and most, if not all, cells also produce much larger DNA fragments. However, in the same way that internucleosomal DNA fragmentation is a reflection of chromatin structure, the generation of these larger fragments is a reflection of chromatin structure, too. By comparing the ionic requirements for the complete pattern of chromatin degradation in nuclei with those required for apoptosis, it is apparent that the whole process may be catalysed by two pools of Mg-activated\Ca-modulated DNase I-like enzyme activities.

Journal Article↗
PubMed1994

Control of fibroblast senescence and activation of programmed cell death.

We have characterized a nuclear phosphoprotein of 57 kda, statin, found only in nonproliferating cells of both quiescent and senescent natures. Emerging results suggest that statin may function as a sequester to block the early G1 phase phosphorylation for the RB protein. A second protein, terminin, undergoes senescence-specific posttranslational modification from 90 to 60 kda, and further death-specific conversion from 60 to 30 kda. We also found that apoptotic mouse 3T3 fibroblasts express c-fos, c-myc, c-jun, and cdc2, as well as the upregulation of RB phosphorylation and BrdU incorporation, just before final DNA fragmentation and death. It seems that en route to death, cells re-enter the cell-cycle transverse and experience early G1 and part of S Phase; however, this cycling event is an abortive one. In contrast, senescent fibroblasts are resistant to the initiation of the death program, since they are unable to enter cell cycle traverse. Long-term serial passaging of normal human fibroblasts may be inadvertently selecting those, while termed as senescent, are also specialized survivors, and thus a good culture model to study both the control of permanent departure from cell cycle traverse and the mechanism underlying the survival or antideath cellular program.

Animals↗
PubMed1994

Commitment to cell death is signaled by the appearance of a terminin protein of 30 kDa.

Programmed cell death appears to be regulated by a specific molecular program, often dependent upon the activation of unique genes. We have identified terminin in the 60-kDa form as the unique subspecies present only in senescent human diploid fibroblasts. In this report, the biochemical properties of terminin during the process of induced cell death in Swiss 3T3 cells is investigated by total removal of serum, which subsequently activates apoptosis as determined by DNA degradation. Evidence presented here indicates that a specific proteolytic cleavage of terminin proteins occurs during apoptosis, leading to the appearance of a protein species with a molecular mass of 30 kDa (Tp-30) in serum-deprived mouse 3T3 cells. The appearance of Tp-30 can be modulated up to 24 h after serum deprivation by pretreatment with cycloheximide or returning to serum-containing conditions. These studies thus define the association of apoptosis-specific proteolysis of terminin and the appearance of Tp-30 with cell death in mouse 3T3 fibroblasts and can serve as a marker for cells on their way to apoptosis.

3T3 Cells↗