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R Latif

Publications and source records attributed to R Latif.

13 recordsLinked to original sources
PubMed2004

Monomerization as a prerequisite for intramolecular cleavage and shedding of the thyrotropin receptor.

The TSH receptor (TSHR) undergoes intramolecular cleavage of the ectodomain yielding a two-subunit structure on the cell surface. Subsequently, the TSHR ectodomains (the alpha- or A-subunits) are shed from the cell surface. In this study we first confirmed TSHR alpha-subunit shedding from tagged-TSHR transfected Chinese hamster ovary cells. We found that TSH exacerbated this phenomenon of TSHR subunit shedding. The 125I-TSH cross-linking technique has been suggested as useful in the assessment of dynamic changes in TSHR processing. In our hands this technique did not detect any enhancement of cleavage by TSH. However, we found that the cross-linking method had an inherent insensitivity for studying receptor dynamics as exhibited by its inability to detect even major degrees of TSHR down-regulation. We, therefore, used a cell-based, double-antibody, flow cytometric immunoassay to quantitate TSHR cleavage in real time. We then found that different lines of Chinese hamster ovary TSHR cells, when treated with TSH, showed a time- and dose-dependent increase in TSHR cleavage in addition to ectodomain shedding. We previously reported that monoclonal TSHR stimulating antibody (MS-1) did not always act like TSH. In particular, MS-1 did not enhance TSHR cleavage. However, when we used the Fab fragment of MS-1, we were able to induce cleavage in a similar time frame to TSH. These results suggested that the intact bivalent antibody immobilized the TSHRs in their multimeric state and inhibited intramolecular cleavage. In support of these observations, fluorescence recovery after photo bleaching measurements demonstrated a greater increase in TSHR mobility with MS-1 Fab fragments than with the intact MS-1 IgG. In conclusion, these data indicated that monomer formation from multimeric TSHRs might be an important requirement for TSHR cleavage and TSHR ectodomain shedding.

Animals↗
PubMed2003

Localization and regulation of thyrotropin receptors within lipid rafts.

The TSH receptor (TSHR) is a prototypic G protein-coupled receptor with a large extracellular domain. We have previously demonstrated homophilic interactions of TSHRs and their existence as constitutive oligomers. However, we have also shown that TSH itself promotes the formation of receptor monomers. We hypothesized, therefore, that TSHR monomers induced by TSH ligand may move into lipid rafts before effective TSH-induced signaling by bringing the cognate signaling molecules resident in such rafts together with the TSHRs. Thus, we aimed to determine whether the TSHRs would partition into these lipid rafts. The B subunit of cholera toxin (CTxB) binds to lipid raft-enriched GM1 ganglioside and has been widely exploited to visualize lipid rafts. Using such a method, we demonstrated the presence of these GM1-enriched lipid microdomains in Chinese hamster ovary cells by using CTxB labeled with a red dye (Alexa 594). To provide evidence for the presence of TSHRs in lipid rafts, we stained Chinese hamster ovary cells expressing TSHRGFP with labeled CTxB. Our results demonstrated that the TSHRGFP complexes localized to GM1-enriched lipid raft microdomains as evidenced by colocalization of the green fluorescent protein tag with the labeled CTxB. Hence, we concluded that a significant proportion of TSHRs were constitutively associated with lipid rafts. Furthermore, upon activation of these stained raft-receptor complexes with increasing concentrations of TSH, we observed that the raft-receptor complexes decreased significantly. The relevance of such receptor movement out of the rafts suggested that these may be the receptors critical in the initiation of signal transduction

Animals↗
PubMed2003

Functional assessment of the thyrotropin receptor-beta subunit.

Posttranslational processing of the TSH receptor (TSHR) involves proteolysis of a single chain holoreceptor into TSHR-alpha (or A) and TSHR-beta (or B) subunits, which remain associated via disulfide bonds and which may then form oligomers. As both uncleaved and cleavage-derived forms of this receptor have been reported to bind TSH and transduce signals, reasons for this cleavage into alpha- and beta-subunits have remained enigmatic. Recently we suggested that TSHR cleavage was related to receptor oligomerization and now we have asked if cleavage influenced the binding of G proteins to this receptor. Furthermore, as TSHR-alpha subunits are subject to shedding from the cell surface membrane, we have examined whether the remaining TSHR-beta subunits could mediate signaling themselves, either constitutively and /or ligand-induced. We found that only the cleaved form of the TSHR in transfected Chinese hamster ovary cells was able to bind Gsalpha protein, suggesting that cleavage of the native TSH receptor was associated with receptor activation. We also found that independently expressed TSHR-beta subunits on stable cell lines were unable to mediate either constitutive or TSH-induced signaling, as monitored by their inability to induce cAMP accumulation. These data suggested that receptor cleavage was intimately associated with receptor activation in the wild-type TSH receptor and that the residual TSHR-beta subunits left on the thyroid cell membrane, after TSHR cleavage and subsequent TSHR-alpha shedding, were essentially silent and did not participate in signal transduction.

Animals↗
PubMed2001

Oligomerization of the human thyrotropin receptor: fluorescent protein-tagged hTSHR reveals post-translational complexes.

To examine thyrotropin (TSH) receptor homophilic interactions we fused the human TSH receptor (hTSHR) carboxyl terminus to green fluorescent protein (GFP) and the corresponding chimeric cDNA was expressed in Chinese hamster ovary cells. Fluorescent TSH receptors on the plasma membrane were functional as assessed by TSH-induced cAMP synthesis. The binding of TSH, as well as TSHR autoantibodies, induced time- and dose-dependent receptor capping. Fluorescence resonance energy transfer between receptors differentially tagged with GFP variants (RFP and YFP) provided evidence for the close proximity of individual receptor molecules. This was consistent with previous studies demonstrating the presence of TSHR dimers and oligomers in thyroid tissue. Co-immunoprecipitation of GFP-tagged and Myc-tagged receptor complexes was performed using doubly transfected cells with Myc antibody. Western blotting of the immunoprecipitated complex revealed the absence of noncleaved TSH holoreceptors. This further suggested that cleavage of the holoreceptor into its two-subunit structure, comprising disulfide-linked TSHR-alpha and TSHR-beta subunits, was required for the formation of TSHR dimers and higher order complexes.

Animals↗
PubMed2001

Reversal of the CD4(+)/CD8(+) T-cell ratio in lymph node cells upon in vitro mitogenic stimulation by highly purified, water-soluble S3-S4 dimer of pertussis toxin.

Pertussis toxin (PT), a holomer consisting of a catalytic S1 subunit and a B oligomer composed of S2-S4 and S3-S4 dimers, held together by the S5 subunit, exerts profound effects on immune cells, including T-cell mitogenicity. While the mitogenic activity of PT was shown to reside fully within the B oligomer, it could not be assigned to any particular B-oligomer component. In this study, we purified the S3-S4 dimer to homogeneity under conditions propitious to maintenance of the native conformation. In contrast to previous reports which suggested that both S3-S4 and S2-S4 dimers are necessary for mitogenic activity, our preparation of the highly purified S3-S4 dimer was as strongly mitogenic as the B oligomer, suggesting that the S3-S4 dimer accounts for the mitogenic activity of the B oligomer. Moreover, in vitro stimulation of naive lymphocytes by the S3-S4 dimer resulted in reversal of the normal CD4(+)/CD8(+) T-cell ratio from approximately 2:1 to 1:2. The reversal of the CD4(+)/CD8(+) T-cell ratio is unlikely to be due to preferential apoptosis-necrosis of CD4(+) T cells, as indicated by fluorescence-activated cell sorter analysis of annexin-stained T-cell subsets, or to preferential stimulation of CD8(+) T cells. The mechanism underlying the reversal requires further investigation. Nevertheless, the data presented indicate that the S3-S4 dimer may have potential use in the context of diseases amenable to immunological modulation.

Animals↗
PubMed2000

Fluorescent probes: looking backward and looking forward.

Probe technology has been advancing very rapidly but to study molecular events in real time, there has to be a discrete choice in the use of "probes" and the "labels" that they carry. In this minireview, we shed light on the use of fluorescent probes, especially the use of green fluorescent protein (GFP) and its variants as tools to cell biologists studying protein secretion and trafficking. The use of these GFP variants has further widened the application of Fluorescence Resonance Energy Transfer (FRET) in analyzing protein-protein interaction.

Endocrinology↗
PubMed1992

A dipstick immunobinding enzyme-linked immunosorbent assay for serodiagnosis of hepatitis B and delta virus infections.

A simple, specific and economical dipstick immunobinding enzyme-linked immunosorbent assay (DIA) for detecting hepatitis B surface antigen (HBsAg) and antibodies to hepatitis delta virus (anti-HDV), utilizing cellulose nitrate membrane is described. Screening of 815 serum specimens for HBsAg by DIA and micro ELISA revealed a positivity of 22.69% and 22.94% respectively. In the detection of antibodies to delta antigen, DIA was compared with an indirect immunofluorescence technique using A3 cell line as antigen substrate and a commercial macro ELISA. Of the 143 HBsAg positive sera tested for anti-HDV, 59 (41.25%) were positive by both immunofluorescence and macro ELISA and 61 (42.65%) by DIA. While the positive and negative predictive values of DIA for HBsAg were 100% and 99.6%, for anti-HDV by DIA these were 96.7% and 100% respectively. Based on the simplicity of performance and the economical nature of the test system, DIA is recommended as a diagnostic tool for field surveys and small laboratories in developing countries.

Enzyme-Linked Immunosorbent Assay↗
PubMed1989

Non-organ specific & organ-specific auto-antibodies in hepatitis-B virus associated glomerulonephropathies.

Eighty eight patients of glomerulonephropathies (HBsAg positive 67; HBsAg negative 21) and 88 matched and healthy controls were screened for non-organ specific autoantibodies-ANA, AMA, ASMA and APCA by indirect immunofluorescent technique. The 2.3 per cent positivity in the test group and the 8 per cent positivity in the control group did not suggest the involvement of hepatitis-B virus (HBV), as an influencing or associated agent. When 48 patients with glomerulonephropathies and 23 controls were screened for liver cell membrane (LMA) and renal cell membrane antibodies (RMA) by indirect immunofluorescent technique using isolated rat hepatocytes and renal cells, 79.2 per cent LMA positivity was seen in the HBsAg positive group and 41.7 per cent in the negative group and RMA positivity was 58 per cent in the positive group and 25 per cent in the negative group. Simultaneous positivity for both LMA and RMA was recorded in 50 per cent of the HBsAg positive patients and 15.7 per cent of the negative ones. The results suggest the possibility of an organ specific autoimmune trigger more frequently in HBV associated glomerulonephropathy.

Antibody Specificity↗
PubMed1983

Ceftriaxone diffusion into cerebrospinal fluid of children with meningitis.

We evaluated the diffusion of ceftriaxone into the cerebrospinal fluid of 27 infants and children with meningitis who were receiving conventional antimicrobic therapy. Ceftriaxone was administered as a single 75 mg/kg dose and was given early or late or both in the course of the illness. Three hours after a dose, the mean cerebrospinal fluid ceftriaxone level was 5.7 micrograms/ml in patients studied early in the course of meningitis and 2.1 micrograms/ml in patients studied later in the illness. Six hours after a dose, the mean cerebrospinal fluid ceftriaxone levels early and late in meningitis were 7.2 and 2.5 micrograms/ml, respectively. The diffusion did not correlate with the leukocyte count or the protein or glucose content of the cerebrospinal fluid. Serial, simultaneous cerebrospinal fluid and plasma ceftriaxone levels were also determined in three additional patients with ventriculo-peritoneal shunt infections and external ventriculostomy drainage. The cerebrospinal fluid ceftriaxone levels in these patients ranged from 0.7 to 8.3 micrograms/ml. Our data indicate that ceftriaxone diffuses sufficiently and consistently into the cerebrospinal fluid to warrant its assessment in the treatment of meningitis.

Adolescent↗
PubMed1981

Pharmacokinetic and clinical evaluation of moxalactam in infants and children.

37 patients ranging in age from 9 months to 14 years, with various infections, were treated with moxalactam. The pharmacokinetics of the drug were studied in 18 patients. The mean plasma concentration 1 h after a 25 mg/kg intravenous dose was 41.4 micrograms/ml +/- 15.6 SD, the half-life was 1.5 h +/- 0.4 SD and the mean Vd was 550 ml/kg +/- 239 SD. The total body clearance of moxalactam was 4.1 ml/min/kg +/- 1.5 SD and the mean renal clearance 4.5 ml/min/kg +/- 2.8 SD. Between 52 and 107% of the administered dose was recovered in the urine within 8 h after administration. The pharmacokinetics after the first and multiple doses of moxalactam were similar, indicating no accumulation of the drug with repeated administration. The clinical response was adequate in 35 of 37 patients. Moxalactam was well tolerated and only minor and transient hematological abnormalities were observed.

Adolescent↗
PubMedDate not supplied

Comparison of a miniaturized culture test and sheep blood agar for the recovery of Group A streptococci from throat cultures.

A miniaturized culture test (strep test) was compared with conventional sheep blood agar (SBA) for the detection of Group A streptococci (GAS) from throat swabs. A total of 355 throat cultures were obtained in duplicate from symptomatic children and assessed by the two methods after overnight incubation. GAS were detected in 109 specimens by the strep test, in 100 specimens by SBA and in 118 specimens by both methods. There was no significant difference between the recovery rate of GAS by the strep test (92.4%) and SBA (84.7%) (0.1 greater than P greater than 0.05). The strep test was 85.2% specific for detection of GAS. The rates of false positive and false negative reactions with the strep test were 24.3 and 4.3%, respectively. Beta-hemolytic streptococci other than Group A, Staphylococcus aureus and alpha-hemolytic streptococci produced false positive reactions. The viability of GAS on the strep test was also tested. GAS were recovered from 27 of 28 (96.4%) strep tests at 24 hours and 25 of 27 (92.6%) tests at 72 hours after inoculation. These results indicate that strep test is as sensitive as the conventional SBA method in recovering GAS. The strep test is simple to use, is easy to interpret and has a long shelf life. Because of the high rate of false positive results (24.3%), this test may not be the best alternative method to SBA for use in office practice.

Agar↗