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Biomedical subjects

R Joseph

Publications and source records attributed to R Joseph.

At least 73 records · Page 4Linked to original sources

p23 transplantation antigen mRNA is differentially expressed in human fetal brain.

As gene expression governs development, we attempted to isolate differentially expressed genes in fetal and adult human brain. RNA samples extracted from adult and 18-24-week-old fetal human brain were reverse transcribed, amplified using twenty combinations of 3'-anchored primers and degenerate 5'-primers, and the resulting cDNA fragments separated on denaturing polyacrylamide gels. Thereafter, 45 (H1-H45) differentially displayed cDNA bands were extracted from the gels, amplified by polymerase chain reaction, and used as probes to detect their mRNA by northern blotting. One of these fragments, H8, confirmed on northern blotting to be highly expressed in fetal brain, was cloned and sequenced. This fragment was homologous to wild type p23 human transplantation antigen. This is phylogenetically a well conserved gene and appears to play an important role in cell growth. Even a single point mutation in the mouse gene results in cell destruction secondary to a cytotoxic T-lymphocyte response. Therefore, our finding that normal human fetal brain expresses high levels of wild type p23 transplantation antigen may have importance in maintaining cell growth during human brain development.

Antigens, Neoplasm↗

The pruritogenic and inflammatory effects of prostanoids in the conjunctiva.

The therapeutic utility of cyclooxygenase (CO) inhibitors, such as ketorolac, in reducing the inflammatory events associated with allergic conjunctivitis is not unexpected since prostanoids (PG) elicit conjunctival redness (PGD2, PGE2, PGF2 alpha), edema (PGD2, TxA2), eosinophil infiltration (PGD2, PGJ2) and mucous cell discharge (PGD2, PGJ2, TxA2). Recently, topically administered ketorolac has also been reported to alleviate the itching associated with allergic conjunctivitis. This was viewed as intriguing since CO inhibitors are not regarded as useful for treating itching dermatoses and PGs do not elicit itching when applied to the skin. In order to investigate the antipruritic activity of ketorolac, we developed a model for reproducibly measuring ocular surface itch responses. The model involves itch-scratch responses to pruritogens applied locally to the ocular surface. Painful and foreign body stimuli do not produce an itch-scratch response. Unlike reported skin studies, PGE2 was a potent itch-producing substances in the conjunctiva. PGD2 was weakly pruritogenic but PGF2 alpha and the TxA2-mimetic U-46619 were inactive. The PG precursor arachidonic acid was also a potent pruritogen and its effects were inhibited by ketorolac pretreatment. Ketorolac also dose-dependently inhibited the itching associated with experimental allergic conjunctivitis. It appears that PGs are potent itch-producing substances in the conjunctiva and the anti-itch efficacy of ketorolac in allergic conjunctivitis appears to involve inhibition of conjunctival PG biosynthesis from arachidonic acid.

Animals↗

Prospects for the secondary prevention of colorectal cancer: screening by flexible sigmoidoscopy?

It may be useful to draw an analogy between the proposed screening programme for colorectal cancer and the cervical cancer screening programme. Both tumours show a spectrum of histological abnormalities consistent with a premalignant phase. The natural history of these premalignant lesions is poorly understood and although some will progress, if untreated, to invasive disease, most will not. Light microscopy cannot confidently distinguish which cases will progress and which will regress, and clinicians are therefore obliged to treat all. This will result in the destruction of many lesions of uncertain malignant potential. The secondary prevention of cervical cancer, although therapeutically efficacious, is inefficient. A lack of understanding of the natural history of intraepithelial neoplasia has frustrated attempts to develop rational referral criteria, and it is only now that the appropriate trials are being undertaken. The development of outpatient investigative and therapeutic procedures has resulted in many more women being referred for investigation and treatment, with predictable pressure on other services offered by gynaecologists, but no demonstrable saving of life. Similar uncertainties surround a screening programme for colorectal cancer. The principal concerns are not about the efficacy of polypectomy in interrupting the polyp cancer sequence, although uncertainties about the frequency with which cancer arises de novo do require that the effectiveness of this intervention is formally tested. Our major concerns are with compliance, and the management of the individual who tests positive--that is, who is found to have a distal polyp. Technological advances and operator enthusiasm may, as has happened with the cervical screening programme, lead to a relaxation in the indications for further investigation and treatment. Such a development would affect resources substantially if a population screening programme were in place. Nevertheless, there are grounds for believing that a screening programme for colorectal cancer, using sigmoidoscopy, might be successful in certain age groups if compliance was satisfactory. The scale of benefits may be comparable with those achieved by the breast screening programme. Our limited cost analysis, which relates to only to specific items of clinical activity, suggest that the mean cost for each case of cancer prevented will be about 8000 pounds sterling. These conclusions suggest that screening by flexible sigmoidoscopy merits serious consideration. It is also imperative, however, that consideration should be given to resolving some of the uncertainties about the clinical management and surveillance of those found to have distal polyps.

Age Factors↗

Towards improved perinatal care--perinatal audit.

Perinatal audit is a measure of quality of care given in pregnancy and it gives an idea as to how the resources need to be allocated for better outcome. The perinatal mortality data in the National University Hospital over a 7-year period (1986-1992) were compiled and compared with that of the year 1982. The perinatal mortality rate (PNMR) of 14.6/1000 in 1982 declined to 8.9/1000 for the period 1986 to 1992 and the reduction was noticeable in all ethnic groups, particularly in the Malays. When lethal congenital malformations (LCMs) were excluded, the PNMR decreased to 5.7/1000. Such reduction is due to easy availability and acceptance of antenatal care, improvement in antenatal and intrapartum fetal surveillance and advances in neonatal care. Neonatal audit was extended beyond the first 7 days of birth which showed that the majority (65%) of deaths occurred in the first week and 15% occurred after the first month. The fear that intensive neonatal care serves to postpone death is not entirely substantiated. There was nearly a ten-fold rise in PNMR between the non-low birthweight and low birthweight groups. The important causes of perinatal mortality during the review period were LCMs (35.7%), complications of prematurity (17.9%) and asphyxia (15.3%). No cause was identifiable in 28.5%. Detailed analysis revealed that the standard of care could have been improved in a third of the cases (83/235) which could have led to further reduction of perinatal mortality rate.

Asphyxia Neonatorum↗

The normal preterm foetal heart rate pattern.

A longitudinal study was carried out on 38 women with low risk pregnancies. These women had cardiotocography at 27-28 wk initially, at fortnightly intervals thereafter until 36 wk and at weekly intervals thereafter until delivery. All cardiotocographs were analyzed by one investigator who was not aware of the individual clinical situation. Of the 232 cardiotocographs, 12 (0.5%) of poor quality were excluded from analyses. The mean base-line heart rate decreased from 142.5 (SD 6.03) beats per min at 27-30 wk to 138.2 (SD 7.4) at term. Analysis of variance for repeated measures showed that the decrease in foetal heart rate with gestation was statistically significant (P < 0.001). The number of accelerations increased with gestation (P = 0.002). There were no significant changes in variability and decelerations with increasing gestation.

Female↗

Molecular cloning of a novel mRNA (neuronatin) that is highly expressed in neonatal mammalian brain.

As differential gene expression governs the progression of development into senescence, we attempted to define the genes that are selectively expressed during postnatal brain development. A cDNA fragment selectively expressed in neonatal rat brain was identified by differential display and used to screen a cDNA library prepared from the same mRNA sample. The full length cDNA, neuronatin, was 1195bp long and coded for a novel protein of 81 amino acids. The cDNA detected an mRNA species of similar size that was highly expressed in rat neonatal and human fetal brain. The deduced protein exhibited a hydrophobic N-terminal and hydrophilic C-terminal, suggesting that it is membrane bound and might function in signal transduction. The selective expression of this novel mRNA in late fetal and early postnatal brain development, and loss of expression in adulthood and senescence, suggests that downregulation of neuronatin may be involved in terminal brain differentiation.

Age Factors↗

The identification of nuclear and mitochondrial genes by sequencing randomly chosen clones from a marsupial mammary gland cDNA library.

To increase the number of genes that can be mapped to the genome of the tammar wallaby (Macropus eugenii), we sequenced 100 randomly chosen clones from a mammary gland cDNA library. Provisional identifications were made of seven nuclear genes and one mitochondrial gene encoding two caseins, beta-galactosidase, acetyl-coenzyme A synthetase, lipoprotein lipase, inorganic pyrophosphatase, an ATP-dependent RNA helicase, and cytochrome c oxidase I. Highly conserved genes, such as that encoding acetyl-coenzyme A synthetase, were easily identified even from cross-kingdom matches. Genes which are highly divergent, however, such as those encoding the mature casein peptides, could not be aligned with homologues in the databases. Even in an organ where there is high mRNA species redundancy, the sequence characterization of expressed sequence tags provides a rapid means of gene identification for mapping purposes.

Amino Acid Sequence↗

Double staining in situ study of mRNAs encoding milk proteins in the mammary gland of the tammar wallaby (Macropus eugenii).

Oligonucleotides, differentially tagged with fluorochromes, were used to determine whether the distribution of mRNAs encoding the major milk proteins is heterogeneous within the mammary gland of the tammar wallaby (Macropus eugenii). This method also allowed direct visualization of two species of mRNA within the same cell. Sections of early and late lactating glands of tammar wallabies were hybridized with oligonucleotides labelled with fluorescein isothiocyanate or rhodamine isothiocyanate either alone or in combination. The results support the hypothesis that milk secretion is an all-or-none process with all epithelial cells in a given alveolus producing the same suite of milk proteins. In tammar wallabies, a gene encoding a protein specific to the latter phase of lactation appears to be expressed in those cells already secreting the other major milk proteins.

Animals↗

Occurrence and de novo biosynthesis of follicle stimulating hormone (FSH) in benign and malignant conditions of human breast.

We report the occurrence as well as biosynthesis of a pituitary hormone, follicle stimulating hormone (FSH) in human breast. Using immunoperoxidase localization technique, both FSH and beta-FSH were localized in cytoplasm of epithelial cells but not in stromal cells. Immunostaining was more intense in benign and malignant specimens as compared to normal. In vitro radiolabelled precursor experiments with breast tissue explants indicate de novo synthesis of FSH. Human milk had higher concentrations of FSH as compared to serum. In gonads, FSH is involved in the cellular growth, differentiation and function. The presence of higher levels of FSH in benign mammary tumors and breast cancer when compared to normal breast supports the suggestion that FSH might have a role in the process of breast malignant transformation.

Breast↗

Neuronal beta-amyloid precursor protein gene expression: regulation by aurintricarboxylic acid.

beta-Amyloid precursor protein (beta-APP) and its derivative, amyloid beta-protein (beta-A4), may cause death of differentiated neurons and aurintricarboxylic acid (ATA), a metabolic inhibitor, improves neuronal survival. Therefore, we studied the effect of ATA on neuronal beta-APP gene expression. ATA decreased beta-APP mRNA levels by increasing its degradation, without changing the rate of transcription. ATA decreased both steady state and interleukin-1 (IL1)-induced increase in beta-APP mRNA levels. These effects of ATA were associated with rounding of cells suggestive of decreased cell adhesion or neurite retraction that was completely reversible when ATA was removed. However, beta-APP mRNA levels continued to remain suppressed in neurons that were actively regrowing neurites following discontinuation of ATA. In studies carried out upto 24 h, ATA did not damage cells as determined by Trypan blue exclusion, lactate dehydrogenase (LDH)-release and transmission electron microscopy. The findings suggest that constitutive or steady state levels of beta-APP mRNA may not be essential for the survival and growth of neurons and that ATA suppresses beta-APP expression without causing cell damage. These observations may be a basis for studying whether ATA or a related compound could beneficially regulate beta-APP levels in vivo.

Amyloid beta-Protein Precursor↗

A novel member of the lipocalin superfamily: tammar wallaby late-lactation protein.

The finding that tammar wallaby late-lactation protein is linked to beta-lactoglobulin prompted a search of current GenPeptide and NBRF-PIR protein databases for sequence similarities to late-lactation protein. Similarities were found to von Ebner's gland protein and other members of the lipocalin superfamily of proteins. A conservative replacement of Trp with Tyr suggests that late-lactation protein may represent an unusual member of this protein superfamily.

Amino Acid Sequence↗

Neuronal death, cytoplasmic calcium and internucleosomal DNA fragmentation: evidence for DNA fragments being released from cells.

Neuronal death, secondary to endogenous agents such as glutamate, may involve changes in cytoplasmic calcium. Besides its well recognized role as a second messenger mediating cellular response, calcium is necessary for the activation of endonuclease(s), resulting in DNA fragmentation and cell death. Therefore, we investigated the relationship between changes in cytoplasmic calcium, DNA fragmentation and neuronal death, using PC12 and NCB-20 cell lines. The calcium ionophore, A23187, caused a dose-dependent increase in cytoplasmic calcium, loss of cell viability, increased lactate dehydrogenase (LDH)-release, and DNA fragmentation. DNA fragments, typical of internucleosomal digestion of genomic DNA, characteristic of endonuclease(s) activation, were consistently detected in the incubating medium. Release of DNA fragments into the medium was seen with A23187 in concentrations as low as 10 nM, and within an hour of treatment. Furthermore, calcium added to preparations of PC12 nuclei also produced DNA fragmentation, although, less pronounced than when intact cells were treated with A23187. The findings indicate that A23187-induced neuronal death involves the activation of endonuclease(s). The role of cytoplasmic calcium in this process is supported by evidence that A23187 selectively mobilizes cytoplasmic calcium, and that calcium can directly activate endonuclease(s) in nuclear preparations.

Animals↗

Free thyroxine as a supplement to thyrotropin in cord screening for hypothyroidism.

The low specificity of cord serum T4 levels for detecting hypothyroidism, the need to reduce recall rates and the availability of a reliable and quick assay system led to this study designed to determine the advantages of using cord serum free thyroxine levels over total thyroxine levels as a supplement to cord thyroid stimulating hormone (TSH) determination. Sixty-eight out of 75 newborns with a cord TSH > 23 mU/l had both free thyroxine (fT4) and T4 levels measured in the cord serum. All were recalled within the first month of life for reevaluation of their thyroid status. In the majority of cases (46), the fT4 and T4 values corresponded. Hypothyroidism was diagnosed in eight cases; both fT4 and T4 were below the mean in five cases and above the mean in one case; only T4 was below the mean in one and in the remaining one, only fT4 was below the mean. Recall rates were 0.9% with TSH alone and 0.7% when either a fT4 or T4 level that was less than 1 sd above the mean was used as a supplement to TSH. In this cohort, using the fT4 levels instead of the T4 levels brought no change to the specificity or the sensitivity of the screening. The diagnostic sensitivity would have dropped from 100% to 75% if either T4 or fT4 values below the mean were used as a cut-off point for recall.

Congenital Hypothyroidism↗

Gonadal function in prepubertal boys following treatment for Hodgkin's disease.

PURPOSE: Gonadal functions were evaluated in 26 male patients with Hodgkin's disease (HD), who were in continuous unmaintained remission following combination chemotherapy consisting of COPP/MOPP. MATERIALS AND METHODS: These patients had received chemotherapy during the prepubertal phase. The median duration after termination of chemotherapy was 72 months. RESULTS: Semen analysis of 18 patients showed azoospermia. Hormonal analysis showed elevated mean levels follicle-stimulating hormone (FSH) and inhibin as compared to age-matched controls, whereas luteinizing hormone levels were only marginally elevated. CONCLUSIONS: These results suggest that COPP/MOPP causes severe damage to germinal epithelium even when given during prepubertal age. Sertoli cells, which are responsible for secretion of inhibin, are resistant to these cytotoxic agents. Our data emphasize the lack of gross dysfunction of Leydig cells. It is possible that an alternative chemotherapy protocol (ABVD) may be used in young patients to minimize the gonadal damage.

Adolescent↗

On the significance of different aequorin loading techniques on intracellular aequorin discharge, baseline calcium, platelet aggregation and aequorin-indicated Ca(2+)-transients.

The study compares the decay of intracellular luminescence activity (Lmax), the levels of basal [Ca2+]i in resting platelets, and agonist-induced peak [Ca2+]i-signals in platelets loaded with aequorin using the EGTA-, DMSO- and hypoosmotic shock treatment (HOST)-techniques. The highest load of intracellular aequorin with almost unchanged luminescence activity during 4 h was achieved with HOST. Lmax decreased linearly in EGTA- and HOST-platelets, but the decay rate and the levels of basal [Ca2+]i were significantly lower in HOST-platelets. Platelet aggregation and aequorin-indicated [Ca2+]i-rise induced by thrombin and collagen were similar in EGTA- and HOST-platelets. In HOST-platelets, ADP-induced platelet aggregation was always accompanied by aequorin-signals, while at a similar time point, aequorin-signals were absent in 3 of 5 cases in EGTA-platelets. The initial aequorin loading was highest in DMSO-platelets, but Lmax described an exponential decay, which was most pronounced when DMSO-platelets were maintained in Ca(2+)-free buffer (R2 = 0.86). Agonist-induced platelet aggregation was significantly reduced in DMSO-platelets: thrombin-stimulation was accompanied by a significantly lower and delayed [Ca2+]i-rise and no aequorin-signal was obtained in response to ADP in 3 of 5 cases. The study shows that in addition of being a rapid loading-technique, the criteria of high intracellular aequorin load with low luminescence consumption, low basal [Ca2+]i and completely preserved platelet functions are most convincingly met by the HOST-method.

Aequorin↗

Amyloid beta-protein fragment 25-35 causes activation of cytoplasmic calcium in neurons.

The cellular mechanism by which beta-amyloid has its effect on neurons is unknown. Based on observations that endogenous neurotoxins, such as glutamate and platelet activating factor (PAF), cause activation of cytoplasmic calcium, we tested if this was true with beta-amyloid. Using nerve growth factor-treated PC12 cells, we noted that the active beta-amyloid fragment, containing residues 25 to 35, caused a specific and dose-dependent increase in intracellular calcium due to an influx of extracellular calcium.

Aequorin↗

Further studies on platelet-mediated neurotoxicity.

The mechanism of ischemic neuronal injury is not fully resolved. The present view is that vascular occlusion per se does not fully account for the extent of neurological dysfunction. We hypothesized that platelet secretory products might contribute to ischemic neuronal injury in the central nervous system (CNS) (Joseph et al., Stroke, 20 (1989) 38-44 and 1316-1319). Our preliminary studies using organotypic rat spinal cord cultures exposed to human platelet and its secretory products, revealed that platelet product(s) had neurotoxicity. Further studies, using the same methods, were conducted here, with the addition of several refinements such as use of gel-filtered platelets (as opposed to washed platelets), adding additional relevant controls including platelet membranes, red blood cells and washed rat platelets. The results confirmed our initial finding that an agent(s) in platelet secretion is neurotoxic. Subsequently, we identified serotonin (5HT), a major platelet product, as having toxic effects on neurons. This toxicity of 5HT appeared to be blocked by ketanserin, a 5HT2 receptor antagonist. Judging by the concentrations of 5HT that demonstrated neurotoxicity in these in vitro studies, it appears that products secreted from activated platelets could have pathological significance in vivo.

Animals↗

Serotonin may have neurotoxic properties.

Serotonin (5HT), a major platelet secretory product, has been shown to suppress CNS function in vivo. As part of an ongoing project to study interactions between neuron and platelet, we used organotypic explant cultures of rat spinal cord to study if 5HT had a morphologically demonstrable neurotoxic effect. The results suggest that serotonin may be neurotoxic, and that this effect may be prevented by ketanserin, a specific 5HT2 antagonist. Related experiments, using acetylcholinesterase (AChE) enzyme activity as a biochemical parameter, indicate that 5HT hastens the decline of enzyme activity. The concentrations of 5HT at which neurotoxicity was demonstrated were comparable to the calculated 5HT concentration potentially present in the vicinity of an acute cerebral thrombus. These findings could provide new insight into the mechanism of ischemic neuronal injury.

Acetylcholinesterase↗