Biomedical subjects

R Fotedar

Publications and source records attributed to R Fotedar.

At least 19 recordsLinked to original sources

PubMed2007

PCR detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in stool samples from Sydney, Australia.

This study investigated the presence of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in stool samples from a patient population in Sydney, Australia. Stool samples were tested by microscopy and PCR. Five patients were found with E. histolytica infections, while E. dispar and E. moshkovskii were observed in 63 (70.8%) and 55 (61.8%) patients, respectively, by PCR. This is the first study in Australia using molecular techniques to determine the presence of E. histolytica, E. dispar, and E. moshkovskii.

Adult↗

PubMed2005

Comparison of phospholipase and proteinase activity in Candida albicans and C. dubliniensis.

Although the production of virulence enzymes by Candida albicans has been extensively explored, little attention has been given to the virulence factors of C. dubliniensis. In the present study, an attempt was made to investigate phospholipase activity (Pz value) and secretory aspartyl proteinase production of C. dubliniensis and compare it with C. albicans. None of the 87 C. dubliniensis isolates tested, produced phosholipases whereas, in contrast all the 52 (100%) C. albicans isolates tested demonstrated varying degree of phospholipase activity (Pz value: 0.37-0.74), with 35 (67.3%) of them eliciting a higher phospholipase activity (Pz values between 0.37 and 0.50). Only 32% of the C. dubliniensis isolates exhibited moderate activity (score of 1+) of secretory aspartyl proteinase whereas a vast majority (68%) of them were non-proteolytic. On the contrary, a strong proteinase activity (score of 2+) was observed for 79% of C. albicans while the remaining 21% isolates showed moderate proteinase activity (score of 1+). As phospholipases and aspartyl proteinases of C. albicans are considered important virulence factors, the absence or lowered expression of these enzymes in C. dubliniensis may indicate the less virulent nature of this novel yeast species when compared with C. albicans.

Aspartic Acid Endopeptidases↗

PubMed2004

Prevalence of Candida dubliniensis among germ tube-positive yeasts recovered from the respiratory specimens in HIV-negative patients.

The present investigation was conducted to identify Candida dubliniensis, from respiratory specimens, recovered from HIV-negative patients. Over a 7-month period, 75 germ tube and chlamydospore-positive yeasts were screened for C. dubliniensis, using a variety of phenotypic characteristics. Their identification was based on sugar assimilation reactions using API 20 C Aux. A total of seven (9%) isolates recovered from sputum, bronchial lavage and nasopharyngeal aspirate were identified as C. dubliniensis. All the isolates were susceptible to amphotericin B. One isolate each showed resistance to fluconazole and ketoconazole, and two were resistant to itraconazole. A significantly high percentage (43%) of C. dubliniensis showed resistance to flucytosine.

Adult↗

PubMed2003

Identification of chlamydospore-negative Candida albicans using CHROMagar Candida medium.

This study was undertaken to evaluate the utility of CHROMagar Candida medium for the identification of chlamydospore-negative Candida albicans. A total of 60 isolates including 45 chlamydospore-negative C. albicans, 10 chlamydospore-positive C. albicans (positive controls) and five non-C. albicans (negative controls) were investigated. On the basis of germ tube test, assimilation of trehalose (Tre), glucosamine (GlcN), N-acetyl glucosamine (GlcNAc), secretory aspartyl production and serotyping, the 45 chlamydospore-negative C. albicans isolates were assigned to the reported three groups. Eighteen isolates showing positive germ tube test, negative for the assimilation of Tre, GlcN/GlcNAc, strong producers of proteinase (2+) and assigned to serotype B belonged to group I. Whereas, the isolates in group II and group III showed common characteristics including assimilation of Tre, GlcN/GlcNAc, moderate production of proteinase (1+) and were serotype A, except for the fact that group II isolates were germ tube positive and group III isolates were negative. Using CHROMagar Candida medium, all the 45 chlamydospore-negative and 10 positive control isolates were accurately identified on the basis of characteristic green color at 37 degrees C for 48 h of incubation. On the other hand at an optimum incubation temperature of 37 degrees C none of the non-C. albicans (negative controls) showed characteristic green color thus yielding a 100% sensitivity and specificity. Isolates in group-I showed a slow growth rate and no visible growth was observed at 24 h, whereas, groups II, III and the control isolates showed visible growth at 24 h. Besides differences in growth rates, these isolates also varied in their characteristic colony color which gradually changed over a period of time. The results of this study clearly suggest that CHROMagar Candida medium is not only a simple, reliable and cost effective method for the identification of chlamydospore-negative atypical C. albicans, but can also be used to differentiate various groups of chlamydospore-negative C. albicans.

Adult↗

PubMed2003

Candida dubliniensis at a university hospital in Saudi Arabia.

Candida dubliniensis is a newly described yeast species that is a close phylogenetic relative of C. albicans. Although it has been reported from different parts of the world, no detailed investigation of this species has been done in Saudi Arabia. The purpose of the present study was to identify C. dubliniensis isolates recovered from clinical specimens at a tertiary-care hospital in Riyadh, Saudi Arabia, and to determine the drug susceptibility profiles of those isolates. Over a period of 8 months, 823 germ tube- and chlamydospore-positive yeasts identified as C. albicans and recovered from different clinical specimens were screened for their ability to grow at 45 degrees C on Sabouraud dextrose agar. Isolates which failed to grow at 45 degrees C were presumptively identified as C. dubliniensis. The species identities were further confirmed by the production of pseudohyphae and chlamydospores on Staib agar and their inability to assimilate D-xylose and alpha-methyl-D-glucoside by using the API 20C AUX system. A total of 27 (3.3%) isolates were identified as C. dubliniensis. They were all recovered from 23 human immunodeficiency virus-negative patients. The prevalence of C. dublinensis in bronchoalveolar lavage (33.3%), oral (16.7%), and blood (16.7%) specimens was high. In addition, 33 isolates previously identified as C. albicans and preserved among our stock blood culture isolates were also recruited for the study. Of these, 5 isolates were found to be C. dubliniensis, thus making the total number of isolates identified as this species 32. Antifungal susceptibility testing of the C. dubliniensis isolates showed 100% sensitivity to amphotericin B, 97% sensitivity to each of fluconazole and ketoconazole, and 87.5% sensitivity to itraconazole. However, in contrast to other studies, the majority of the isolates (65.6%) showed high levels of resistance to flucytosine (MIC > 64 microg/ml). Further studies are warranted to investigate the cause of this unusually high rate of resistance to flucytosine of the C. dubliniensis isolates in this region.

Adolescent↗

PubMed2002

Recovery and studies on chlamydospore-negative Candida albicans isolated from clinical specimens.

During a period of 31 months, we isolated 3525 strains of Candida albicans from different patient specimens. Twenty-five of these (0.71%), obtained from female patients, displayed morphological and biochemical characteristics different from those seen in typical C. albicans. The failure to produce chlamydospores in cornmeal agar was the common denominator in this group. The strains were categorized into three groups: Group I contained 13 isolates that produced germ tubes but were unable to assimilate trehalose (TRE), glucosamine (GLN) and N-acetylglucosamine (NAG); Group II contained four isolates that were germ-tube positive and able to assimilate TRE, GLN and NAG; and Group III contained eight isolates that were germ-tube negative and able to assimilate TRE, GLN and NAG. These isolates were further studied to determine their biotypes, serotypes, extracellular proteinase production and antifungal susceptibility. Group I isolates were of serotype B, whereas Groups II and III were serotype A. All isolates produced high to moderate amounts of extracellular proteinase. Six group I isolates were resistant to 5-fluorocytosine, whereas all groups II and III isolates were susceptible to this drug. Five of the 12 isolates of group II and III were resistant to fluconazole, itraconazole and ketoconazole.

Acetylglucosamine↗

PubMed2001

Vector potential of houseflies (Musca domestica) in the transmission of Vibrio cholerae in India.

It is well known that diarrhoeal infections due to Vibrio cholerae are spread through fecal-oral route of transmission. In the present study an attempt was made to isolate and identify V. cholerae from houseflies, Musca domestica collected from a low socioeconomic area in Delhi, India, where an outbreak of cholera was encountered. Of the ten fly pools examined, six (60%) were positive for V. cholerae. Of these six pools, three (50%) showed V. cholerae Ogawa T2 El Tor and one (17.5%) V. cholerae non-O1. Two isolates could not be typed. During the outbreak period, V. cholerae Ogawa T2 El Tor was isolated from stools of patients suffering from diarrhoea. These findings suggest that houseflies act as mechanical vectors of V. cholerae biotype El Tor and may help in their dissemination. The present study highlights the recovery of V. cholerae El Tor from M. domestica which, to the authors knowledge, has not been reported previously.

Agglutination Tests↗

PubMed2001

The large subunit of replication factor C promotes cell survival after DNA damage in an LxCxE motif- and Rb-dependent manner.

Retinoblastoma (Rb) protein promotes cell survival after DNA damage. We show here that the LxCxE binding site in Rb mediates both cell survival and cell-cycle arrest after DNA damage. Replication factor C (RF-C) complex plays an important role in DNA replication. We describe a novel function of the large subunit of RF-C in promoting cell survival after DNA damage. RF-Cp145 contains an LxCxE motif, and mutation of this motif abolishes the protective effect of RF-Cp145. The inability of wild-type RF-Cp145 to promote cell survival in Rb-null cells is rescued by Rb but not by Rb mutants defective in binding LxCxE proteins. RF-C thus enhances cell survival after DNA damage in an Rb-dependent manner.

Amino Acid Motifs↗

PubMed2000

The functions of the cdk-cyclin kinase inhibitor p21WAF1.

p21WAF1 plays a critical role in regulating cell growth and the cell response to DNA damage. The primary targets of p21WAF1 (hereafter referred to as p21) are the cdk-cyclins which regulate the progression of eukaryotic cells through the cell cycle, and proliferating cell nuclear antigen (PCNA), an accessory protein of DNA polymerase delta. p21 forms complexes with a class of cdk-cyclins to inhibit their kinase activity and with PCNA to inhibit DNA synthesis. These distinct properties map to the N-terminal and the C-terminal regions of p21, respectively. Cell cycle arrest in G-1 (G-1 checkpoint) following DNA damage is mediated by p53 and is deficient in p21 null cells. p53 thus upregulates p21 expression in response to DNA damage, which in turn inhibits cdk2-associated kinase activity. Retinoblastoma protein is regulated by cdk-cyclin kinases, and acts as a downstream target of p21 in DNA damage-induced G-1 arrest. Furthermore, accumulating evidence indicates that p21 may play a role in maintaining G-2 arrest after DNA damage. Transcriptional control of p21 by factors other than p53 is critical for growth arrest and for cell differentiation in many instances.

Animals↗

PubMed1999

Growth inhibition by CDK-cyclin and PCNA binding domains of p21 occurs by distinct mechanisms and is regulated by ubiquitin-proteasome pathway.

The CDK inhibitor, p21WAF1/Cip1 blocks cell cycle progression. In vitro, the N-terminus of p21 binds and inhibits CDK-cyclin kinase activity, whereas the C-terminus binds and inhibits PCNA (proliferating cell nuclear antigen) function. PCNA is essential for processivity of both DNA polymerase delta and epsilon. We have performed a detailed analysis of growth inhibition by the N- and C-terminal regions of p21, and determined whether the N- and C-terminal regions mediate this effect by different mechanisms. Expression of either the N- or the C-terminal region of p21 inhibits DNA synthesis and cell growth, but not as efficiently as full length p21. The effectiveness of the two p21 domains is dependent on their stability which is determined by the ubiquitin-proteasome pathway. The stabilization of the N- and C-terminal region of p21 increases their effectiveness as inhibitors of DNA synthesis to levels comparable to full length p21. Inhibition of DNA synthesis by the N-terminal region of p21 involves suppression of E2F activity. In contrast, inhibition by the C-terminal region of p21 is not accompanied by suppression of E2F activity, but is mediated via PCNA binding. The C-terminal region of p21 therefore inhibits cell growth by a mechanism distinct from that of the N-terminal region containing the CDK-cyclin inhibitory domain.

3T3 Cells↗

PubMed1999

Effect of p21waf1/cip1 transgene on radiation induced apoptosis in T cells.

The cyclin kinase inhibitor p21WAF1/Cip1 is upregulated by the tumor suppressor p53. While p21 is central for the G-1 arrest mediated by p53, it is still unclear if p21 also functions as a downstream effector of p53 dependent apoptosis. Apoptosis induced by DNA damage but not dexamethasone is p53 dependent in thymocytes. To investigate the physiological role of p21 in apoptosis, we have generated transgenic mice in which the p21 transgene is targeted for restricted expression in the T cell lineage. Thymocytes from p21 transgenic mice were hypersensitive to cell death induced by DNA damaging agents such as ionizing radiation and UV, but not be dexamethasone. Irradiated p21 transgenic thymocytes had approximately twofold more apoptotic cells as compared to irradiated age matched littermate control mice. Radiation induced death is comparable in thymocytes from p21 + Bcl2 + double transgenic mice and age matched littermate controls, indicating that the Bcl2 transgene rescues the radiation hypersensitivity imposed by p21. However, thymocytes from p53-/- mice even when they expressed the p21 transgene, were resistant to death induced by radiation. Together these results show that thymocytes from p21 transgenic mice are hypersensitive to radiation induced programmed cell death and suggest that the radiation hypersensitivity of p21 transgenic thymocytes involves p53 dependent pathway and signals in addition to p21.

Animals↗

PubMed1999

Growth inhibition by CDK-cyclin and PCNA binding domains of p21 occurs by distinct mechanisms and is regulated by ubiquitin-proteasome pathway.

The CDK inhibitor, p21(WAF1/Cip1) blocks cell cycle progression. In vitro, the N-terminus of p21 binds and inhibits CDK-cyclin kinase activity, whereas the C-terminus binds and inhibits PCNA (proliferating cell nuclear antigen) function. PCNA is essential for processivity of both DNA polymerase delta and epsilon. We have performed a detailed analysis of growth inhibition by the N- and C-terminal regions of p21, and determined whether the N- and C-terminal regions mediate this effect by different mechanisms. Expression of either the N- or the C-terminal region of p21 inhibits DNA synthesis and cell growth, but not as efficiently as full length p21. The effectiveness of the two p21 domains is dependent on their stability which is determined by the ubiquitin-proteasome pathway. The stabilization of the N- and C-terminal region of p21 increases their effectiveness as inhibitors of DNA synthesis to levels comparable to full length p21. Inhibition of DNA synthesis by the N-terminal region of p21 involves suppression of E2F activity. In contrast, inhibition by the C-terminal region of p21 is not accompanied by suppression of E2F activity, but is mediated via PCNA binding. The C-terminal region of p21 therefore inhibits cell growth by a mechanism distinct from that of the N-terminal region containing the CDK-cyclin inhibitory domain.

3T3 Cells↗

PubMed1998

Stepwise reconstitution of interphase microtubule dynamics in permeabilized cells and comparison to dynamic mechanisms in intact cells.

Microtubules in permeabilized cells are devoid of dynamic activity and are insensitive to depolymerizing drugs such as nocodazole. Using this model system we have established conditions for stepwise reconstitution of microtubule dynamics in permeabilized interphase cells when supplemented with various cell extracts. When permeabilized cells are supplemented with mammalian cell extracts in the presence of protein phosphatase inhibitors, microtubules become sensitive to nocodazole. Depolymerization induced by nocodazole proceeds from microtubule plus ends, whereas microtubule minus ends remain inactive. Such nocodazole-sensitive microtubules do not exhibit subunit turnover. By contrast, when permeabilized cells are supplemented with Xenopus egg extracts, microtubules actively turn over. This involves continuous creation of free microtubule minus ends through microtubule fragmentation. Newly created minus ends apparently serve as sites of microtubule depolymerization, while net microtubule polymerization occurs at microtubule plus ends. We provide evidence that similar microtubule fragmentation and minus end-directed disassembly occur at the whole-cell level in intact cells. These data suggest that microtubule dynamics resembling dynamics observed in vivo can be reconstituted in permeabilized cells. This model system should provide means for in vitro assays to identify molecules important in regulating microtubule dynamics. Furthermore, our data support recent work suggesting that microtubule treadmilling is an important mechanism of microtubule turnover.

3T3 Cells↗

PubMed1998

Proteolytic cleavage of retinoblastoma protein upon DNA damage and Fas-mediated apoptosis.

Proteolytic cleavage of key cellular proteins by caspases (ICE, CPP32, and Ich-1/Nedd2) may be crucial to the apoptotic process. The retinoblastoma tumor suppressor gene is a negative regulator of cell growth and the retinoblastoma protein (pRb) exhibits anti-apoptotic function. We show that pRb is cleaved during apoptosis induced by either UV irradiation or anti-Fas antibody. Our studies implicate CPP32-like activity in the proteolytic cleavage of pRb. The kinetics of proteolytic cleavage of pRb during apoptosis differ from that observed for other cellular proteins, suggesting that the specific cleavage of pRb during apoptosis may be an important event.

Antibodies↗

PubMed1997

Association of cyclin A and cdk2 with SV40 DNA in replication initiation complexes is cell cycle dependent.

The cell cycle is driven by the sequential activation of a family of cyclin-dependent kinases (CDK) in association with cyclins. In mammalian cells the timing of activation of cyclin A-associated kinase activity coincides with the onset of DNA synthesis in S-phase. Using in vitro replication of SV40 origin-containing DNA as a model system, we have analyzed the proteins associated with DNA during initiation of DNA replication in S-phase cell extracts. This analysis reveals that, in addition to replication initiation proteins, cyclin A and cdk2 are also specifically associated with DNA. The association of cyclin A and cdk2 with DNA during initiation is cell cycle regulated and occurs specifically in the presence of SV40 origin-containing plasmid and SV40 T antigen (the viral replication initiator protein). The interactions among proteins involved in initiation play an important role in DNA replication. We therefore investigated the ability of cyclin A and cdk2 to associate with replication initiation proteins. Under replication initiation conditions, cyclin A and cdk2 from S-phase extracts specifically associate with SV40 T antigen. Further, the interaction of cyclin A-cdk2 with SV40 T antigen is mediated via cyclin A, and purified recombinant cyclin A associates directly with SV40 T antigen. Taken together, our results suggest that cyclin A and cdk2 are components of the SV40 replication initiation complex, and that protein-protein interactions between cyclin A-cdk2 and T antigen may facilitate the association of cyclin A-cdk2 with the complex.

Animals↗

PubMed1997

Prevalence of Candida tropicalis in clinical specimens from patients with variable clinical syndromes over a 5-year period.

In this study, we have examined our records for the isolation of Candida tropicalis from clinical specimens of patients with heterogeneous clinical presentations during the past 5 years. We have found that this species ranks third among all yeasts in frequency of isolation from clinical specimens and that the trend of recovery from the specimens is rising over the years. The isolation rate of C. tropicalis was highest from urine specimens (36%) followed by respiratory specimens (22%). The frequency of isolation of C. tropicalis from vaginal specimens was relatively high (14%), however the trend was declining over the years. In general, the high recovery of Candida tropicalis from clinical specimens of patients with variable disease supports the views of this organism being a major pathogen.

Candida↗

PubMed1996

Role for cyclin A-dependent kinase in DNA replication in human S phase cell extracts.

Cell cycle progression is regulated by cyclin-dependent kinases. Using in vitro replication of SV40 origin containing DNA as a model system, we have performed a detailed analysis of the dependence on cyclin-associated kinases of mammalian DNA replication. Complete immunodepletion of cyclin A from human S phase cell extracts decreases replication, and replication activity of cyclin A-depleted S phase extracts can subsequently be restored by the addition of purified CDK2-cyclin A kinase. Addition of cyclin A alone reconstitutes both kinase activity and DNA replication, whereas addition of cyclin E or cyclin B reconstitutes neither. We therefore conclude that reconstitution of DNA replication specifically correlates with an increase in kinase activity. By comparison, depletion of cyclin E from S phase cell extracts does not have any significant inhibitory effect on DNA replication. Moreover, specific p21(Waf1) mutants that bind to CDK2-cyclin and inhibit both cyclin A and cyclin E kinase activities, but do not bind to proliferating cell nuclear antigen, inhibit DNA replication to the same extent as cyclin A depletion. Together, these results show that the kinase activity associated with cyclin A, but not with cyclin E, is primarily responsible for activating SV40 plasmid replication in mammalian S phase cell extracts. Finally, we present evidence that the cyclin-dependent kinase does not influence the assembly of initiation complexes but acts at a stage prior to elongation.

Cyclin-Dependent Kinase Inhibitor p21↗

PubMed1996

A conserved domain of the large subunit of replication factor C binds PCNA and acts like a dominant negative inhibitor of DNA replication in mammalian cells.

Replication factor C (RF-C), a complex of five polypeptides, is essential for cell-free SV40 origin-dependent DNA replication and viability in yeast. The cDNA encoding the large subunit of human RF-C (RF-Cp145) was cloned in a Southwestern screen. Using deletion mutants of RF-Cp145 we have mapped the DNA binding domain of RF-Cp145 to amino acid residues 369-480. This domain is conserved among both prokaryotic DNA ligases and eukaryotic poly(ADP-ribose) polymerases and is absent in other subunits of RF-C. The PCNA binding domain maps to amino acid residues 481-728 and is conserved in all five subunits of RF-C. The PCNA binding domain of RF-Cp145 inhibits several functions of RF-C, such as: (i) in vitro DNA replication of SV40 origin-containing DNA; (ii) RF-C-dependent loading of PCNA onto DNA; and (iii) RF-C-dependent DNA elongation. The PCNA binding domain of RF-Cp145 localizes to the nucleus and inhibits DNA synthesis in transfected mammalian cells. In contrast, the DNA binding domain of RF-Cp145 does not inhibit DNA synthesis in vitro or in vivo. We therefore conclude that amino acid residues 481-728 of human RF-Cp145 are critical and act as a dominant negative mutant of RF-C function in DNA replication in vivo.

Amino Acid Sequence↗