In vivo relevance for photoprotection by the vitamin D rapid response pathway.

Vitamin D is produced by exposure of 7-dehydrocholesterol in the skin to UV irradiation (UVR) and further converted in the skin to the biologically active metabolite, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and other compounds. UVR also results in DNA damage producing cyclobutane pyrimidine dimers (CPD). We previously reported that 1,25(OH)(2)D(3) at picomolar concentrations, protects human skin cells from UVR-induced apoptosis, and decreases CPD in surviving cells. 1,25(OH)(2)D(3) has been shown to generate biological responses via two pathways-the classical steroid receptor/genomic pathway or a rapid, non-genomic pathway mediated by a putative membrane receptor. Whether the rapid response pathway is physiologically relevant is unclear. A cis-locked, rapid-acting agonist 1,25(OH)(2)lumisterol(3) (JN), entirely mimicked the actions of 1,25(OH)(2)D(3) to reduce fibroblast and keratinocyte loss and CPD damage after UVR. The effects of 1,25(OH)(2)D(3) were abolished by a rapid-acting antagonist, but not by a genomic antagonist. Skh:hr1 mice exposed to three times the minimal erythemal dose of solar-simulated UVR and treated topically with 1,25(OH)(2)D(3) or JN immediately after UVR showed reduction in UVR-induced UVR-induced sunburn cells (p<0.01 and <0.05, respectively), CPD (p<0.01 for both) and immunosuppression (p<0.001 for both) compared with vehicle-treated mice. These results show for the first time an in vivo biological response mediated by a rapid-acting analog of the vitamin D system. The data support the hypothesis that 1,25(OH)(2)D(3) exerts its photoprotective effects via the rapid pathway and raise the possibility that other D compounds produced in skin may contribute to the photoprotective effects.

Purification and characterization of Escherichia coli xanthine-guanine phosphoribosyltransferase produced by plasmid pSV2gpt.

The enzyme xanthine-guanine phosphoribosyltransferase from Escherichia coli cells harboring the plasmid pSV2gpt has been purified 30-fold to near homogeneity by single-step GMP-agarose affinity chromatography. It has a Km value of 2.5, 42 and 182 microM for the substrates guanine, xanthine and hypoxanthine, respectively, with guanine being the most preferred substrate. The enzyme exhibits a Km value of 38.5 microM for PRib-PP with guanine as second substrate and of 100 microM when xanthine is used as the second substrate. It is markedly inhibited by 6-thioguanine, GMP and to a lesser extent by some other purine analogues. Thioguanine has been found to be the most potent inhibitor. The subunit molecular weight of xanthine-guanine phosphoribosyltransferase was determined to be 19 000. The in situ activity assay on a nondenaturing polyacrylamide gel electrophoresis gel has indicated that a second E. coli phosphoribosyltransferase preferentially uses hypoxanthine as opposed to guanine as a substrate, and it does not use xanthine.

Skin cancer prevention: a possible role of 1,25dihydroxyvitamin D3 and its analogs.

We previously reported that the natural hormone 1,25dihydroxyvitamin D3 (1,25(OH)(2)D(3)) protects human skin cells from ultraviolet radiation (UVR)-induced apoptosis. UVR-induced pre-mutagenic cyclobutane pyrimidine dimers are diminished in number from 0.5h after cessation of UVR in all skin cell types, by treatment with three different Vitamin D compounds: by 1,25(OH)(2)D(3), by the rapid acting, low calcemic analog, 1alpha,25(OH)(2)lumisterol(3) (JN) and by the low calcemic but transcriptionally active hybrid analog 1alpha-hydroxymethyl-16-ene-24,24-difluoro-25-hydroxy-26,27-bis-homovitamin D3 QW-1624F2-2 (QW), which may explain the enhanced cell survival. The rapid response antagonist analog 1beta,25(OH)(2)D(3) (HL) abolished the photoprotective effects of 1,25(OH)(2)D(3) whilst a genomic antagonist, (23S)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647), had no effect. UVR increased p53 expression in human skin cells, whilst concurrent treatment with 1,25(OH)(2)D(3) further enhanced this effect several fold, at 3 and 6h after UVR. Combined with previously reported lower nitrite levels with 1,25(OH)(2)D(3), this increased p53 expression may favor DNA repair over apoptosis. We now report that topical application of 1,25(OH)(2)D(3) or QW also suppressed solar simulated UV (SSUVR-induced pyrimidine dimers in the epidermis of irradiated hairless Skh:HR1 mice, measured 24h after irradiation. Furthermore, UVR-induced immunosuppression in the mice was markedly reduced by topical application of either 1,25(OH)(2)D(3) or QW. These preliminary results show, for the first time, a protective effect of Vitamin D compounds against DNA photodamage in vivo.

Design and development of a web-based application for diabetes patient data management.

A web-based database management system developed for collecting, managing and analysing information of diabetes patients is described here. It is a searchable, client-server, relational database application, developed on the Windows platform using Oracle, Active Server Pages (ASP), Visual Basic Script (VB Script) and Java Script. The software is menu-driven and allows authorized healthcare providers to access, enter, update and analyse patient information. Graphical representation of data can be generated by the system using bar charts and pie charts. An interactive web interface allows users to query the database and generate reports. Alpha- and beta-testing of the system was carried out and the system at present holds records of 500 diabetes patients and is found useful in diagnosis and treatment. In addition to providing patient data on a continuous basis in a simple format, the system is used in population and comparative analysis. It has proved to be of significant advantage to the healthcare provider as compared to the paper-based system.

1,25-Dihydroxyvitamin D and three low-calcemic analogs decrease UV-induced DNA damage via the rapid response pathway.

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] is anti-apoptotic in human keratinocytes, melanocytes and fibroblasts after ultraviolet (UV)-exposure. To date, there is no published data on the effects of 1,25(OH)(2)D(3) or its analogs on DNA damage in irradiated skin cells. In these skin cells, 24h pre-treatment with 1,25(OH)(2)D(3) dose-dependently (10(-12) to 10(-8)M) decreased CPD damage by up to 60%. This photoprotective effect was also seen if the 1,25(OH)(2)D(3) was added immediately after irradiation and was mimicked by QW-1624F2-2 (QW), a low-calcemic 1beta-hydroxymethyl-3-epi-16-ene-24,24-difluoro-26,27-bis homo hybrid analog. The well-studied low calcemic, rapid acting agonist analogs 1alpha,25(OH)(2)lumisterol(3) (JN) and 1alpha,25(OH)(2)-7-dehydrocholesterol (JM) also protected skin cells from UV-induced cell loss and CPD damage to an extent comparable with that of 1,25(OH)(2)D(3). In contrast, the rapid response antagonist analog 1beta,25(OH)(2)D(3) (HL) completely abolished the photoprotective effects (reduced cell loss and reduced CPD damage) produced by treatment with 1,25(OH)(2)D(3), JN, JM and QW. Evidence for involvement of the nitric oxide pathway in the protection from CPD damage by 1,25(OH)(2)D(3) was obtained. These data provide further evidence for a role of the vitamin D pathway in the intrinsic skin defenses against UV damage. The data also support the hypothesis that the photoprotective effects of 1,25(OH)(2)D(3) are mediated via the rapid response pathway(s).

Japanese encephalitis in Sangli district, Maharashtra.

During the months June to December, 1997, 52 cases of suspected viral encephalitis were admitted at the Government Hospital, Sangli. These cases were from the congested areas of Sangli and the adjoining villages. All age groups and both genders were affected. IgM antibodies to Japanese encephalitis (JE) virus were detected in the sera of five of the 52 cases. Perhaps JE virus has established itself in a new locality in Maharashtra and could cause serious public health problems.

Drimarene brilliant blue--a better pre-stain in protein separation and visualization.

Prestaining of human serum proteins with a new reactive dye Drimarene Brilliant Blue (DBB), was standardized employing 940 separations and examining 30 variables. Under the critical condition, the serum and the soluble dye (0.1 g/100 ml in working Tris-glycine buffer, pH, 8.3), was mixed in equal proportion, conjugate warmed at 40 degrees C for 2 hr and a 30 microliter of the sample electrophoresed by disc electrophoresis. The method when compared with prestaining by Remazol Brilliant Blue (RBB) and postelectrophoretic staining by Amido Black (AB) in 50 normal sera, revealed that the discs stained with DBB were intense and well defined and appeared in 2 hr on a sparkingly clear gel. Quality of resolution was better than RBB and AB. Protein bands eluted from the DBB prestained gels retained their immunoreactivity. The dye-protein complex of albumin and transferrin produced high-titre monospecific antisera in rabbits.

Elevated polymorphonuclear phagocytic function in thalassemia patients by chemiluminescence.

Twenty five patients with beta thalassemia major, with no evidence of infection were evaluated for their polymorphonuclear cell (PMN) metabolic function and serum opsonic activity by chemiluminescence assay. These were divided into Group I of normal adults (n = 21), Group II thalassemia major < 5 years (n = 9) and Group III thalassemia major > 5 years (n = 16). The ability of the chemiluminescence assay (CL) to reflect opsonic and phagocytic dysfunction suggested its potential application in the evaluation of phagocytic function. The peak count of Group I was (1.07 +/- 0.24 x 10(-5)), Group II (1.60 +/- 0.83 x 10(-5)) and Group III was (2.71 +/- 0.98 x 10(-5)) respectively in the presence of autologous sera. The peak count compared between Group I and III was found to be statistically significant (p < 0.05). The peak count of Group I and II when compared showed a trend in the increase activity not statistically significant. The polymorph function of all the groups were compared with autologous serum as well as normal serum. There was no increase in polymorph function of Group III in the presence of thalassemia serum, nor any decrease in the polymorph function of thalassemia patients of Group II and III. This concluded that polymorphs of thalassemia patients are active in the presence of autologous as well as normal serum. The increased activity of thalassemia polymorphs may be due to antigenic stimulation which may be due to multiple transfusion and not due to circulating iron load.

Increased prevalence of metabolic syndrome in non-obese asian Indian-an urban-rural comparison.

BACKGROUND: In the present study we evaluated the association of insulin resistance (IR) with different components of Metabolic Syndrome (MS) in an Asian Indian population, and performed a comparative study between urban and rural populations of India. METHODS: A Total of 267 urban men and women aged 25-70 years participated in this study. RESULTS were compared with rural data from a previously published study. Fasting serum insulin, uric acid, and lipid profile were measured along with fasting and 2 hour plasma glucose. Association of MS and IR was studied by using univariate regression analysis. RESULTS: Prevalence of MS was significantly higher in the urban population compared to that of the rural population (35.2% vs 20.6%, chi(2) = 23.2, p < 0.001). Calculated insulin resistence (HOMA-IR) was common in MS group of both populations. Percentage prevalence of IR was high and almost the same in both population (42%). Percentage prevalence of abdominal obesity and hypertriglyceridemia was significantly higher in the urban population compared to the rural population. Linear regression analysis of IR significantly correlated with different components of MS of both the population. CONCLUSIONS: The significant finding of the present study was that the rural population exhibited a high prevalence of MS and IR, though nonobese. IR correlated with components of MS not only in the urban but also in the rural population. To reduce the incidence of Type 2 Diabetes (T2DM) and cardiovascular disease (CVD) in our populations, early identification of populations at risk based on prevalence of MS and IR will become of prime importance.

Insulin sensitivity as a risk factor for common carotid intima media thickness (IMT): its relation to atherosclerosis.

INTRODUCTION: The relationship between insulin resistance and atherosclerosis (ATH) in non-diabetic hypertensive patients from the Asian Indian population remains poorly understood. To resolve this issue, the present study was designed to analyze whether insulin sensitivity in a non-diabetic individual is related to the development of ATH.(by using IMT as an index) and whether this relationship is dependent on the presence of other cardiovascular disease (CVD) risk factors such as dyslipidemia and hypertension. METHODOLOGY: This study included 68 healthy controls with no diabetes and hypertension and 41 hypertensive patients who underwent four-point oral glucose tolerance test (OGTT) and intravenous glucose tolerance test (IVGTT). A biochemical profile, beta mode ultrasonography for intima media thickness of carotid artery, and ECG determination was carried out. RESULTS: Hypertensive patients in our study exhibited significantly increased abdominal obesity. Blood pressure, fasting and 2 hr plasma glucose (4.62 +/- 0.08 and 5.55 +/- 0.17 mmol/l), and triglyceride (1.47 +/- 0.067 mmol/l) levels were compared to those of control subjects (p < 0.05). The fasting insulin levels and HOMA-IR were also significantly increased and Composite Insulin Sensitivity Index (CISI) reduced compared to controls with p < 0.01. Intima media thickness of the left (0.08 +/- 0.01) and right (0.069 +/- 0.008) CA were both significantly increased in hypertensives (p < 0.01). Correlation analysis showed that IMT of the left carotid artery was significantly associated with triglyceride levels (r = 0.813, p < 0.05) but not with insulin measures such as HOMA-IR and CISI. CONCLUSION: Hyperinsulinemia was observed in our non-diabetic hypertensive patients, but no association was found between IMT and insulin resistance. That IMT of hypertensives was associated with triglyceride levels suggests that high levels of insulin may be related to the development of ATH indirectly through its effects on lipid metabolism in our population.

Integration of a dominant selectable marker into human chromosomes and transfer of marked chromosomes to mouse cells by microcell fusion.

A method for the production of stable mouse-human cell hybrids containing a single human chromosome is described. As a first step in this method, a cloned selectable marker, the E. coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, was transferred to human cells to generate cell lines each carrying Ecogpt integrated into a different site. Human chromosomes marked with Ecogpt were transferred further into mouse cells by microcell fusion. Monochromosomal hybrids, in which the human chromosome is maintained by selection, have been produced for chromosomes 2, 5, 16, and a rearranged chromosome involving a translocation between chromosomes 1 and 2. In addition to these monochromosomal hybrids, we have also obtained monochromosomal hybrids for human chromosomes 6, 12, and 17 by selection for the loss of marked chromosome from the microcell hybrids each containing two human chromosomes. Although the human chromosome present in these hybrids cannot be maintained by selection, 80-90% of cells retained the transferred chromosome on continuous growth for 15 days. Monochromosomal hybrids would provide biological materials to construct genetic maps of human chromosomes. In addition, chromosomes marked with dominant selectable markers can be transferred further to any cell line of interest in inter- or intra-species combination.

Conformational transitions of an unmodified tRNA: implications for RNA folding.

Unmodified tRNAs are powerful systems to study the effects of posttranscriptional modifications and site-directed mutations on both the structure and function of these ribonucleic acids. To define the general limitations of synthetic constructs as models for native tRNAs, it is necessary to elucidate the conformational states of unmodified tRNAs as a function of solution conditions. Here we report the conformational properties of unmodified yeast tRNAPhe as a function of ionic strength, [Mg2+], and temperature using a combination of spectroscopic measurements along with chemical and enzymatic probes. We find that in low [Na+] buffer at low temperature, native yeast tRNAPhe adopts tertiary structure in the absence of Mg2+. By contrast, tertiary folding of unmodified yeast tRNAPhe has an absolute requirement for Mg2+. Below the melting temperature of the cloverleaf, unmodified yeast tRNAPhe exists in a Mg2+-dependent equilibrium between secondary and tertiary structure. Taken together, our findings suggest that although the tertiary structures of tRNAs are broadly comparable, the intrinsic stability of the tertiary fold, the conformational properties of intermediate states, and the stability of intermediate states can differ significantly between tRNA sequences. Thus, the use of unmodified tRNAs as models for native constructs can have significant limitations. Broad conclusions regarding "tRNA folding" as a whole must be viewed cautiously, particularly in cases where structural changes occur, such as during protein synthesis.

Attitudes and treatment outcome of breast conservation therapy for stage I & II breast cancer using peroperative iridium-192 implant boost to the tumour bed.

Breast conservation therapy for early breast cancer is an established but grossly under-utilized treatment option in India for various reasons. Breast conservation therapy was offered to 200 suitable breast cancer patients between June 1993 and June 1998. Fifty-one patients (25%) opted for breast conservation and the remaining preferred mastectomy. In patients agreeing to conservation therapy, surgery was performed first along with peroperative implantation of iridium-192 to deliver a boost. Whole breast irradiation of 45 Gy was delivered 3-4 weeks after the boost. Cosmesis was assessed at the end of 6 months from completion of therapy. The main reason for refusal of breast conservation therapy was fear of recurrence in the remaining breast (60%). There were no locoregional failures in our study at a median follow up of 42 months; one patient experienced a systemic relapse. Cosmesis was good to excellent in 80% of patients. Breast conservation therapy using peroperative iridium-192 implant provides excellent locoregional disease control and cosmesis. The results of our study indicate that patient preference for mastectomy is an important reason for the under-utilization of breast conservation therapy in India.