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Biomedical subjects

A Jacob

Publications and source records attributed to A Jacob.

At least 19 recordsLinked to original sources
PubMed2000

Enzymatic activity of the Src homology 2 domain-containing inositol phosphatase is regulated by a plasma membrane location.

The negative regulatory role of the Src homology 2 domain-containing inositol 5-phosphatase (SHIP) has been invoked in a variety of receptor-mediated signaling pathways. In B lymphocytes, co-clustering of antigen receptor surface immunoglobulin with FcgammaRIIb promotes the negative effects of SHIP, but how SHIP activity is regulated is unknown. To explore this issue, we investigated the effect of SHIP phosphorylation, receptor tyrosine engagement by its Src homology 2 domain, and membrane recruitment of SHIP on its enzymatic activity. We examined two SHIP phosphorylation kinase candidates, Lyn and Syk, and observed that the Src protein-tyrosine kinase, Lyn is far superior to Syk in its ability to phosphorylate SHIP both in vitro and in vivo. However, we found a minimal effect of phosphorylation or receptor tyrosine engagement of SHIP on its enzymatic activity, whereas membrane localization of SHIP significantly reduced cellular phosphatidylinositol 3,4, 5-triphosphate levels. Based on our results, we propose that a membrane localization of SHIP is the crucial event in the induction of its phosphatase effects.

Animals↗
PubMed2000

Molecular characterization of a voltage-gated potassium channel expressed in rat testis.

Potassium (K(+)) channels are present in both mammalian testis and spermatozoa. Immunofluorescent detection of sperm-bound biotinylated charybdotoxin, an inhibitor of Ca(2+)-activated and of delayed rectifier K(+) channels, indicated that these ion channels are uniformly distributed over the surface of both heads and tails of unfixed rat epididymal spermatozoa. Reverse transcription-polymerase chain reaction (RT-PCR) analysis on rat testis RNA with PCR primers, based on known nucleotide sequences of different classes of K(+) channels, amplified sequences homologous to delayed rectifier K(+) channels. In-situ RT-PCR on rat testis sections showed that these K(+) channel transcripts are present in the cytoplasm of primary spermatocytes and post-meiotic elongating spermatids. Northern blot analysis of various rat tissues identified multiple K(+) channel transcripts, some of which were observed only in testis. An attempt to obtain a full length rat testis K(+) channel cDNA sequence gave an assembled sequence of 2693 base pairs with >90% homology to a delayed rectifier K(+) channel, Kv1.3. A method for rapid amplification of cDNA ends was employed to amplify the 5' sequences of the rat testis cDNA but a unique sequence could not be obtained. DNA sequencer traces suggest that multiple related K(+) channels which differed at their 5' ends were amplified in rat testis.

5' Untranslated Regions↗
PubMed1999

Single-or-two-stage laryngotracheal reconstruction; comparison of outcomes.

To compare single-stage laryngotracheal reconstruction (SSLTR) and reconstruction with tracheostomy and indwelling stent (two-stage LTR), a retrospective review was made of 69 patients undergoing laryngotracheal reconstruction for subglottic stenosis at Great Ormond Street Hospital for Sick Children. Pre-operative details recorded included grade and aetiology of subglottic stenosis, history of previous laryngeal surgery, sex of patient and age at reconstruction. As a measure of outcome, the total number of procedures including all endoscopy and further reconstruction was recorded as well as de-cannulation rate, and the need for more than one reconstruction. The patients undergoing two-stage reconstruction tended to have more severe stenosis (mean grade = 2.56) compared to the SSLTR group (mean grade = 2.14) and were more likely to have had previous laryngeal surgery. Inevitably, the outcome after reconstruction in the two-stage patients is therefore less favourable, and direct comparison of the two groups is not statistically valid. However, multiple regression analysis reveals that single-stage reconstruction does confer a significant independent advantage over the two-stage procedure in terms of average number of post reconstruction procedures (p = 0.006), and a significant advantage in de-cannulation rate (p = 0.03). No difference was noted in the requirement for further reconstruction between the two groups. Although a two-stage procedure is still required in certain cases such as those with very severe stenosis or respiratory insufficiency, the single-stage reconstruction is the procedure of choice for uncomplicated paediatric subglottic stenosis.

Child↗
PubMed1999

The HNF-3alpha transcription factor is a primary target for retinoic acid action.

We have previously demonstrated that gene expression of the hepatocyte nuclear factor 3alpha (HNF-3alpha) transcription factor is activated during retinoic-acid-induced differentiation of F9 embryonal carcinoma cells (A. Jacob et al. (1994). Nucleic Acids Res. 22, 2126-2133). We have extended these studies and now show that HNF-3alpha mRNA is induced approximately 6 h after addition of retinoic acid to the cells, peaks at 1 day postdifferentiation, and then declines to undetectable levels. Furthermore, HNF-3alpha induction occurs in the absence of de novo protein synthesis, suggesting that it is a primary target for retinoic acid action. In order to corroborate this hypothesis, we have mapped the cis-acting HNF-3alpha promoter site that mediates the retinoic acid response. DNA sequence analysis indicates that the HNF-3alpha promoter contains an authentic retinoic acid response element (RARE) of the DR5 class. As expected, this element is able to confer retinoic acid responsiveness to a heterologous promoter. In addition, the HNF-3alpha-specific RARE is able to interact with various retinoic acid receptor heterodimers of the RAR/RXR type. Since HNF-3alpha is induced early during mammalian neurogenesis, our data shed new light on the connection between retinoic-acid-mediated HNF-3alpha activation and establishment of the neuronal phenotype.

Animals↗
PubMed1999

Chronic suppurative otitis media: prevalence and practices among rural South Indian children.

In order to determine the prevalence of chronic suppurative otitis media (CSOM) in rural South Indian children, a cross-sectional survey was conducted among 914 children (484 boys and 430 girls) from four primary schools and 12 nurseries (balwadis; preschool), of adjacent villages of North Arcot District of Tamil Nadu state. The preschool children were aged 2-5 years, while the ages of the primary school children ranged from 6 to 10 years. The overall prevalence rate of CSOM was found to be 6%. The disease was equally prevalent in preschool children (5.7%) and primary school children (6.2%) (P = 0.94). Cholesteatomatous ear disease was observed in 1.2% of children, those of the older age group having a slightly higher prevalence rate (1.5%) than the younger age group (0.7%). Parental beliefs and existing practices with respect to the disease are also presented.

Attitude to Health↗
PubMed1999

FcgammaRIIb modulation of surface immunoglobulin-induced Akt activation in murine B cells.

We examined activation of the serine/threonine kinase Akt in the murine B cell line A20. Akt is activated in a phosphoinositide 3-kinase (PtdIns 3-kinase)-dependent manner upon stimulation of the antigen receptor, surface immunoglobulin (sIg). In contrast, Akt induction is reduced upon co-clustering of sIg with the B cell IgG receptor, FcgammaRIIb. Co-clustering of sIg-FcgammaRIIb transmits a dominant negative signal and is associated with reduced accumulation of the PtdIns 3-kinase product phosphatidylinositol 3,4,5-trisphosphate (PtdIns 3,4,5-P3), known to be a potent activator of Akt. PtdIns 3-kinase is activated to the same extent with and without FcgammaRIIb co-ligation, indicating conditions supporting the generation of PtdIns 3,4,5-P3. We hypothesized that the decreased Akt activity arises from the consumption of PtdIns 3,4,5-P3 by the inositol-5-phosphatase Src homology 2-containing inositol 5-phosphatase (SHIP), which has been shown by us to be tyrosine-phosphorylated and associated with FcgammaRIIb when the latter is co-ligated. In direct support of this hypothesis, we report here that Akt induction is greatly reduced in fibroblasts expressing catalytically active but not inactive SHIP. Likewise, the reduction in Akt activity upon sIg-FcgammaRIIb co-clustering is absent from avian B cells lacking expression of SHIP. These findings indicate that SHIP acts as a negative regulator of Akt activation.

Animals↗
PubMed1999

Numerical dose-compensated in vitro fertilization inseminations yield high fertilization and pregnancy rates.

OBJECTIVE: To evaluate in cases with morphologically abnormal sperm whether fertilization and pregnancy rates are increased by normalizing the number of sperm inseminated and whether biomarkers can identify cases of reduced or failed fertilization. DESIGN: Prospective studies of sperm morphology and function. SETTING: University hospital assisted human reproduction program. PATIENT(S): Partners of 308 women undergoing IVF. INTERVENTION(S): Motile sperm populations were assessed for sperm head morphology, for surface receptors for mannose and progesterone binding, and the ability to undergo a free mannose-induced acrosome reaction. Zinc in seminal plasma was determined by atomic absorption spectroscopy. MAIN OUTCOME MEASURE(S): Sperm morphology was associated with fertilization and clinical pregnancy rates. Biomarker analyses were correlated with fertilization rates using Kruskal-Wallis tests, chi2 tests, and Spearman rank order correlations. RESULT(S): Fertilization and pregnancy rates after numerical dose compensation inseminations were indistinguishable between men with differing percentages of normal sperm. Biomarker deficits were identified irrespective of sperm head morphology in 96% of cases of reduced or failed fertilization. CONCLUSION(S): Fertilization and pregnancy rates in cases of abnormal morphology are optimized by inseminating at least 25,000 sperm/mL with normal acrosomes. Reduced or failed fertilization can be predicted by testing for molecular deficits in mannose receptor expression and mannose-stimulated acrosome loss.

Biomarkers↗
PubMed1999

Standardization of creatine kinase-MB (CK-MB) mass assays: the use of recombinant CK-MB as a reference material.

BACKGROUND: The AACC assembled a committee to identify and validate a standard creatine kinase MB isoenzyme (CK-MB) material to improve the comparability of CK-MB mass assays. METHODS: Three protocols were used. In protocol I, various CK-MB materials prepared in different matrices were screened as candidate standards. In protocol II, participating manufacturers calibrated their systems with concentrates of human heart CK-MB and then tested 20 patient samples to evaluate calibration bias. In protocol III, participating manufacturers calibrated their immunoassay systems using recombinant CK-MB2 (rCK-MB2) diluted into their respective sample diluents and measured 50 samples. RESULTS: Candidate materials showed high recovery in stripped human serum, but bias improved only from 59% to 38%. These data led to the use of human heart CK-MB diluted in each manufacturer's sample diluent. This strategy reduced bias from 31% to 15%. Because human heart CK-MB is difficult to provide, a lyophilized source of CK-MB2 was identified. rCK-MB2 was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reversed-phase HPLC, intrinsic protein fluorescence, circular dichroism, agarose gel electrophoresis, immunoreactivity studies, high and low temperature stability, and reconstituted stability to be equivalent to human heart CK-MB. Calibration of immunoassay systems with rCK-MB2 added into each respective manufacturer's sample diluent showed a 13% between-manufacturer bias. CONCLUSION: Lyophilized rCK-MB2 was determined suitable for use as a reference material for CK-MB mass assays.

Calibration↗
PubMed1998

Mannose ligand receptor assay as a test to predict fertilization in vitro: a prospective study.

OBJECTIVE: To assess whether mannose receptor assays can predict fertilization outcome in vitro. DESIGN: A prospective, double-blind study of the mannose receptor properties of spermatozoa. SETTING: Assisted human reproduction program at a university hospital. PATIENT(S): Partners of 140 consecutive women undergoing their first in vitro fertilization cycle. INTERVENTION(S): Motile sperm populations were tested for surface receptors for mannose by measuring their ability to bind fluorescein-labeled mannosylated albumin and to undergo a free mannose-induced acrosome reaction as judged by Pisum, sativum agglutinin binding. MAIN OUTCOME MEASURE(S): Mannose receptor assay results were correlated with fertilization outcomes using several statistical tests, including the chi2 test, chi2 for proportions, t-tests, analysis of variance with Student-Newman-Keuls tests and correlational and receiver operating characteristic (ROC) curve analysis. RESULT(S): The fractional increment increase on incubation in the percent of sperm binding mannose ligand over an intact acrosome correlated with fertilization rates in vitro. Threshold values of mannose ligand binding and of mannose-induced acrosome reactions predictive of fertilization rates were identified by ROC curve analysis. Men were thus classified into one of four groups with differing fertilization rates in vitro. CONCLUSION(S): The increment increase in sperm surface mannose ligand binding by acrosome-intact sperm correctly predicts high and low fertilization rates in vitro and identifies cases where conventional insemination can result in failed fertilization.

Adult↗
PubMed1998

Release of clonal block in B cell chronic lymphocytic leukaemia by engagement of co-operative epitopes on CD40.

The clonal cells of patients with B-chronic lymphocytic leukaemia (B-CLL)--which essentially reside in a resting configuration--are characterised by a relative refractoriness to the normal signals for B cell growth and differentiation. Previously it has been shown that, using an in vitro culture system where CD40 is hyper-crosslinked by monoclonal antibody (mAb) held on CD32-transfected mouse L cells, the clonal block in B-CLL cells can be released with a resultant high rate of DNA synthesis ensuing. In the present study, we report that such release can be achieved purely with soluble reagents whereby co-operative epitopes on CD40 are targeted by the combined use of mAb and soluble recombinant CD40L. Substantial levels of DNA synthesis were induced under such conditions in 7/18 patients using CD40-targeted reagents alone and in 16/18 patients in the additional presence of interleukin 4. Possible extrapolation of these findings to novel therapeutic modalities could be envisaged.

Antibodies, Monoclonal↗
PubMed1998

Application of nested PCR and mass spectrometry for DNA-based virus detection: HBV-DNA detected in the majority of isolated anti-HBc positive sera.

DNA preparations from three different groups of serum samples were examined for HBV-DNA via a nested polymerase chain reaction assay (lower detection limit: 10 viral genomes in 100 microliters serum): Group I consisted of 11 uninfected control sera, group II consisted of sera obtained from 11 HBV infected patients and group III consisted of 21 isolated anti-HBc positive samples. The 21 samples from group III were HBV-DNA negative according to a conventional non-nested PCR assay and hybridization with a 32P-labelled probe. Using nested PCR and mass spectrometry, HBV-DNA was detected in none of group I and in all of group II samples. In 11 out of 21 (52%) of the isolated anti-HBc positive sera from group III, HBV-DNA was detected. No correlation was observed between HBV-DNA positivity and anti-HBc titers. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry provided a fast, sensitive and non-radioactive assay for the detection of PCR products without the need for gel electrophoresis or hybridization with labelled probes.

DNA, Viral↗
PubMed1998

Recovery of spermatogenesis following bone marrow transplantation.

Preparative regimens used in bone marrow transplantation usually result in impairment of male fertility. Although recovery of fertility is more likely after chemotherapy-based regimens, this is a rare occurrence after TBI containing regimens. We undertook a cross-sectional study of recovery of spermatogenesis in men who had undergone allogeneic and autologous marrow or peripheral stem cell transplant between 1984 and 1995. We identified six out of 42 patients eligible for analysis to have either recovered fertility or spermatogenesis. Seminal fluid analysis showed that four out of 25 patients recovered sperm counts. This has important implications for patient counselling, post-transplant follow-up and contraceptive advice.

Adult↗
PubMed1998

Human sperm non-nuclear progesterone receptor expression is a novel marker for fertilization outcome.

In a prospective, blind study, we have examined the relationship among the expression of human sperm surface progesterone receptors, the ability to undergo a mannose-stimulated acrosome reaction and the rate of fertilization in vitro. Individual aliquots of motile spermatozoa were surface-labelled with progesterone and/or mannose-fluoresceinated ligands. Spontaneous acrosome loss and the increase in acrosome reactions following exposure of spermatozoa to mannose ligands were assessed using rhodaminated Pisum sativum agglutinin. Progesterone fluoresceinated ligand binding was observed to occur in two patterns: (i) a uniform distribution of labelling over the acrosome cap (pattern II), and (ii) labelling limited to the equatorial and postacrosomal regions of the human sperm head (pattern III). A conversion of pattern II to pattern III binding was observed and was associated with the acrosome reaction. Pattern III binding was highly correlated with both fertilization potential and the ability to undergo a mannose-stimulated acrosome reaction (P < 0.001). In contrast, normal sperm mannose receptor expression was seen in five men whose abnormal progesterone receptor expression/function and inability to acrosome react after mannose treatment were correlated with their reduced fertility in vitro. In conclusion, surface progesterone receptor aggregation enhances the mannose ligand-stimulated acrosome reaction. Such detection of defective sperm surface progesterone receptor expression/function may be useful in the evaluation and management of male infertility.

Acrosome↗
PubMed1998

Bilateral simultaneous hearing preservation mastoidectomy in otogenic meningitis.

Bilateral simultaneous hearing conservation mastoidectomy has not been previously documented. We present our experience with this procedure. Three patients with pyogenic meningitis secondary to bilateral unsafe ears had hearing preservation surgeries of both ears in a single sitting. Although ear surgery carries risk of inner ear damage, in our series there was no deterioration of inner ear function in the 6 ears operated on. The air conduction threshold improved in 3 (50%) of the ears. When meningitis results from bilateral otogenic foci as in our series, simultaneous mastoidectomy in both ears may be the preferred method of treatment to eradicate the source of infection.

Adult↗
PubMed1998

Prevalence of primary glaucoma in an urban south Indian population.

Glaucoma is fast emerging as a major cause of blindness in India. In order to estimate the prevalence of primary open angle glaucoma (POAG) and primary angle closure glaucoma (PACG) in an urban South Indian population, we examined 972 individuals aged 30-60 years, chosen using a cluster sampling technique from 12 census blocks of Vellore town. They underwent a complete ocular examination, including applanation tonometry and gonioscopy, at the Medical College Hospital. Characteristic field defects on automated perimetry was a diagnostic requisite for POAG. Prevalence (95% CI) of POAG, PACG, and ocular hypertension were 4.1 (0.08-8.1), 43.2 (30.14-56.3), and 30.8 (19.8-41.9) per 1,000, respectively. All the PACG cases detected were of the chronic type. Hitherto unavailable community-based information on primary glaucoma in our study population indicates that PACG is about five times as common as POAG.

Adult↗
PubMed1998

Modified preparation and rapid quality control test for technetium-99m-tetrofosmin.

OBJECTIVE: The objectives of this study were to: modify the preparation of 99mTc-tetrofosmin by using twice the amount of 99mTcO4- recommended by the manufacturer; evaluate the use of miniaturized rapid paper chromatography (MRPC) for quality control (QC) testing; and determine the in vitro stability of the modified preparation using MRPC. METHODS: Two preparations of 99mTc-tetrofosmin were made: one with 4.4-8.8 GBq (120-240 mCi) and the other with 13.9-17.6 GBq (380-480 mCi) 99mTcO4-, referred to as regular and modified preparations, respectively. Routine QC tests were performed using MRPC and instant thin-layer chromatography/silica-gel (ITLC/SG) systems. The preparations were injected into 58 patients. Planar and SPECT images of stress and rest studies were obtained. The technical quality of the SPECT images was graded visually by four observers. Heart-to-lung and heart-to-background ratios were calculated from the planar images. RESULTS: The QC testing procedure took 4.18 +/- 0.15 min with MRPC and 54 +/- 5.3 min with ITLC/SG systems. The percent labeling efficiency, as determined by both techniques, ranged from 95.6 +/- 1.6 to 97.2% +/- 0.8%. Both preparations were stable up to 6 hr after reconstitution. There was no difference between the cardiac-to-lung and cardiac-to-background ratios of the two preparations. CONCLUSION: The results indicate that MRPC is a faster and effective chromatographic technique for routine QC testing of 99mTc-tetrofosmin. Doubling the amount of 99mTcO4- used in preparing 99mTc-tetrofosmin did not affect its in vitro stability, its efficacious use in patients or the technical quality of the images.

Chromatography, Paper↗