Evaluation of socio-economic factors causing discontinuation of epilepsy treatment resulting in seizure recurrence: a study in an urban epilepsy clinic in India.

BACKGROUND AND PURPOSE: The prevalence rate of epilepsy in India ranges between 4.15 and 7.03 per 1000 population. In the developing countries, the major problems of epilepsy are lying in the treatment gap and discontinuation of treatment due to various adverse socio-economic factors. The objective of this study was to evaluate the rate of discontinuation of epilepsy treatment and its related socio-economic factors responsible for discontinuation. MATERIAL AND METHODS: Among 1450 patients with epilepsy who were recurrently followed up at an intervals of 2 months from 05 January to 06 January; 620 patients discontinued their treatment. Among them 88.7% patient had breakthrough seizures for more than in two occasions. Socio-economic factors in respect to the treatment were evaluated during the follow-up period vis-a-vis income and expenditure, unemployment status, negative attitude towards medical treatment, non-availability of drugs locally, co-morbid psychiatric and other illnesses, polytherapy and socialillusional thoughts about epilepsy. RESULTS: Discontinuation of epilepsy treatment was detected in 42.75% (n = 620) of total patients resulting in recurrence of seizures. Reasons for discontinuation were multiple in most of the cases. The discontinued group had an average annual cost of treatment and income of Rs. 5500 ($110) and Rs. 12,800 ($256), respectively, amounting to 40% of their total income being expended for the cost of the treatment, while in continued group annual cost of treatment and income were Rs. 4500 ($ = 90) and Rs. 24,400 ($ = 580) respectively amounting to only 18% of the total income (p < 0.001) for the cost of treatment. Among the discontinued group, 90% of the patients reported the cost factors, 29.09% due to the unemployment, 20% from the frustration and despair, 20.09% due to non-availability of medicines locally, 17.27% spiritual illusional thoughts about epilepsy, 10% for marital disharmony were the causes for discontinuation of treatment. In the discontinued group, 10% got polytherapy against 9.03% in the continued group (p > 0.01), co-morbid psychiatric illnesses were observed in 4.54% against 3.25% in the continued group (p > 0.10). CONCLUSION: The study showed a significant number of patients (42.75%) discontinued epilepsy treatment within 1 year due to poor knowledge regarding the problem of discontinuation, cost and income disparity, unemployment, spiritual illusional thoughts about epilepsy, frustration and mental impairment, lack of uniform availability of drugs in local market. To tide these shortcomings, uniform availability of cheaper antiepileptic drugs with adequate information and communication regarding the disease and upliftment of socio-economic status are to be ensured.

Effects of 12-O-tetradecanoylphorbol-13-acetate on fibroblasts from individuals genetically predisposed to cancer.

The purpose of the present study was to determine whether skin fibroblasts from individuals, either with an inherited predisposition to cancer or with genetic disorders usually associated with a high risk of cancer, can be oncogenically transformed in vitro by a tumor promoter alone. The effects of chronic and limited applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) on several properties that are associated with transformation were examined using skin fibroblasts from individuals with polyposis coli, a familial cancer syndrome, xeroderma pigmentosum, Fanconi's anemia, and trisomy 21. The results of this study show that TPA treatment induces similar changes on cellular morphology, growth rate, saturation density, epidermal growth factor binding, and cytoskeleton in fibroblasts from both normal and genetically predisposed individuals. None of these cell lines, however, acquired anchorage-independent growth or unlimited growth potential in culture after chronic application of TPA. These observations suggest clearly that skin fibroblasts from individuals with either a genetic predisposition to or a high risk of cancer may not exist in a preneoplastic or "initiated" state susceptible to oncogenic transformation by TPA alone and that the mechanism of genetically determined cancer induction may be different from that of chemical carcinogenesis.

Altered biochemical properties of actin in normal skin fibroblasts from individuals predisposed to dominantly inherited cancers.

The data presented here show that normal skin fibroblasts from individuals with dominantly inherited retinoblastoma, polyposis coli, and nevoid basal cell carcinoma (predisposed cells), grown in the presence of [35S]methionine, contain more than 2.5-fold [35S]methionine-labeled actin as compared to normal fibroblasts from individuals without a prior history of predisposition to cancer (normal cells). The rate of incorporation of [35S]methionine into actin in predisposed cells is rapid and is not correlated with an increase in the total protein and actin contents of the cells, in the intracellular pool size of [35S]methionine, or in the synthesis of beta-actin-specific mRNA, as compared to normal cells. However, the half-life of actin in predisposed cells is less than 5 h, as compared to at least 48 h for normal cells. The significantly reduced half-life of actin and an increased incorporation of [35S]methionine specifically into actin in all predisposed cells studied may represent an inherited biochemical defect which leads to cytoskeletal disorganization previously observed in these cells. It can be speculated that the altered properties of actin in predisposed cells may be caused by the same genetic lesion(s) which is responsible for the induction of dominantly inherited cancers.

Transformation of normal rat kidney cells by v-K-ras enhances expression of transin 2 and an S-100-related calcium-binding protein.

v-K-ras transformants of normal rat kidney cells (KNRK) exhibit cell surface-related, transformation-specific properties, including cell-surface fibronectin depletion, induction of anchorage- and density-independent growth, and increased synthesis of transforming growth factors alpha and beta. To search for potential distal effectors of v-K-ras-mediated transformation, we prepared a rabbit antiserum directed against intact KNRK cells to immunoprecipitate and compare proteins from detergent lysates and conditioned media of labeled NRK, KNRK, B77-NRK (a v-src transformant) and ts-371-NRK cells (a Ki-MSV encoding a temperature-sensitive p21v-K-ras). Proteins with enhanced expression in both wild-type v-K-ras and v-src transformants included a cell-surface phosphoglycoprotein with apparent Mr of 79,000 (79K) modified from an 85K protein observed in NRK cell lysates, a cytoplasmic 47K and a 10K protein, and a 57K secreted glycoprotein. A KNRK-specific 21K membrane-associated protein and secreted 59K and 36K secreted glycoproteins were also detected. The expression of the 36K and 59K proteins best correlated with temperature-dependent activation of the ts-371-NRK p21v-K-ras. Immunoselection of recombinant clones from a KNRK-specific lambda gt11 cDNA library allowed identification of the 59K and 10K proteins as transin 2 and an S-100-related calcium-binding protein identified as p9Ka/42A but not previously associated with oncogenic transformation of rat cells. Transin 2 detection by a cell-derived antiserum may also suggest the presence of specific cell-surface binding sites for this enzyme.

Expression of antisense osteopontin RNA inhibits tumor promoter-induced neoplastic transformation of mouse JB6 epidermal cells.

Elevated expression of osteopontin (OPN), a secreted adhesive phosphoglycoprotein, is frequently associated with many transformed cell lines of epithelial and stromal origin. Moreover, several clonal lines of preneoplastic JB6 cells derived from Balb/c mouse epidermal cultures (Colburn et al., 1978, 1979), upon treatment with 12-O-tetradecanoyl phorbol-13-acetate (TPA), become irreversibly oncogenic and concomitantly synthesize OPN at elevated levels (Smith and Denhardt, 1989). In the present study we sought to determine whether OPN expression facilitates transformation of such preneoplastic (initiated) cells. We transfected TPA-promotable JB6 c141.5a cells with an expression vector containing mouse OPN cDNA in antisense orientation under transcriptional control of dexamethasone-inducible MMTV-LTR promoter. Four stably transfected clones, which expressed drastically reduced levels of OPN in the presence of both dexamethasone and TPA, were characterized. We found that (a) more than 20 copies of OPN antisense cDNA were stably incorporated into the genome of cells from two of these clones that were examined by Southern blot analysis; (b) dexamethasone-induced expression of antisense OPN RNA prevented augmented OPN expression at both mRNA and protein levels following TPA treatment; and (c) cells from all four clones failed to form colonies in soft agar medium containing both dexamethasone and TPA. Taken together, these data demonstrate that inhibition of elevated OPN expression blocks TPA-induced anchorage-independent growth of JB6 c141.5a cells, suggesting the possibility that OPN overproduction is causally related to transformation of preneoplastic cells.

Cells in vivo and in vitro from osteopetrotic mice homozygous for c-src disruption show suppression of synthesis of osteopontin, a multifunctional extracellular matrix protein.

Mice carrying homozygous disruption of the c-src proto-oncogene (Src-/-) develop osteopetrosis due to an impaired ability of osteoclasts to adhere to the bone surface and/or to form bone-resorbing ruffled border. It has also been reported that osteopontin (OPN), a secreted phosphoprotein, mediates osteoclast adherence to the bone matrix. We report here that cells from Src-/- mice, both in vitro and in vivo, express OPN mRNA and protein at a significantly reduced level as compared to cells from Src+/- and +/+ animals, suggesting a potential role for the proto-oncogene c-src in the regulation of OPN gene expression. Our data also show that OPN gene expression can be induced by treatment of SR-/- cells with epidermal growth factor (EGF) and 12-O-tetradecanoyl phorbol-13-acetate (TPA). Results obtained from studies using inhibitors of receptor tyrosine kinases (RTKs) and protein kinase C (PKC) suggest that PKC and RTK are positioned in a pathway with PKC as the downstream effector for the EGF-induced OPN gene expression in SRC-/- cells, and that pp60c-src and EGF may regulate OPN gene expression through a common signalling pathway. Furthermore, contrary to published reports, our study shows that EGF-mediated cell signalling does not require functional interaction between the EGF-receptor and pp60c-src.

Retinoic acid inhibits phospholipid turnover and protein kinase C activity in RA-sensitive but not in RA-resistant cells.

Treatment with 10(-5) M retinoic acid causes loss of anchorage-independent growth in src-transformed RR1022 cells but not in ras-transformed KNRK cells. In an effort to elucidate the mechanisms underlying this difference, we investigated the effect of RA on phospholipid turnover and PKC activity in these two cell lines. 10(-5) M RA treatment caused a drastic inhibition of 32P incorporation into PI and PA and a large increase in 32P incorporation into PC in RR1022 cells. Similar treatment of KNRK cells yielded no change in PC or PA labelling and a much smaller decrease in PI labelling. Furthermore, 10(-5) M RA treatment causes a large decrease in PKC activity in RR1022 cells (35% of control) but only a small decrease in KNRK cells (78% of control). We suggest that these effects are part of an altered signal transduction pathway which mediates the differential effects of RA on anchorage-independent growth in these two cell lines.

Epidermal and transforming growth factors modulate secretion of a 69 kDa phosphoprotein in normal rat kidney fibroblasts.

Our study shows that the secretion of a major non-glycosylated, phosphoprotein of 69 kDa (pp69) is a specific marker for non-transformed NRK-49F cells. Treatment of NRK-49F cells with EGF alone or with different combinations of EGF plus TGF-beta modulates the secretion of pp69, suggesting its relationship with cellular proliferation. Antibody raised against pp69 recognizes, in addition to pp69, another major phospho-protein of 62 kDa (pp62) secreted by RR1022 and spontaneously transformed NRK-49F cells. Immunoprecipitation of total cell lysates from both NRK-49F and RR1022 cells with anti-pp69 antibody detected only pp69. These observations suggest a precursor-product relationship between pp69 secreted by non-transformed NRK-49F cells and pp62 secreted by transformed cells.

Calcium-binding properties of osteopontin derived from non-osteogenic sources.

Osteopontin (OP), purified from rat bone, binds Ca2+ but whether different molecular forms of OPs derived from non-osteogenic sources and non-phosphorylated OP also possess this property remains to be determined. Furthermore, it is not known which specific site or sites of the molecule bind Ca2+. In the present study, following an established procedure, total proteins in the conditioned media from OP-synthesizing cell cultures were separated by SDS-PAGE, transferred to Immobilon-P membranes, and incubated with 45CaCl2, then Ca2+ ions bound to protein bands were analyzed by autoradiography. Purified OPs, and synthetic oligopeptides representing specific domains of the OP molecule were adsorbed on the membrane and processed as described above. Our results show that OPs synthesized by normal rat kidney cells, oncogenically transformed Rat-1 cells, OP purified from human milk, and non-phosphorylated OP secreted by 1 alpha, 25-dihydroxyvitamin D3-treated mouse epidermal JB6 cells all bind detectable levels of Ca2+ with specificity. We also show that a synthetic peptide representing the domain of OP which contains nine consecutive aspartic acid residues binds Ca2+ with specificity. It is probable, therefore, that a Ca(2+)-binding site resides in this region of the OP molecule. We conclude that Ca(2+)-binding is a general property of OP, irrespective of its molecular mass and origin, and the phosphate moieties of OP may not influence the conformation or accessibility of the Ca2+ affinity sites of the molecule.

Osteopontin: its transglutaminase-catalyzed posttranslational modifications and cross-linking to fibronectin.

Osteopontin (OP) is a component of extracellular, bone, and urinary stone matrices, but the mechanism by which it is stably incorporated into such matrices remains unknown. By SDS-PAGE analysis of [125I]OP, treated with a catalytic amount of TG, we first demonstrate both intra- and intermolecular covalent cross-linking of OP. Most importantly, the analysis of the products generated from reactions containing OP, Fn, and TG by SDS-PAGE, autoradiography, and Western blotting using either OP or Fn antibody, and quantitation of TG-catalyzed epsilon-(gamma-glutamyl)lysine isopeptide formation between OP and Fn demonstrate, for the first time, covalent cross-linking between these two proteins. Similar reactions in the presence of polyamine substrates of TG show OP-Fn intermolecular cross-linking via N,N-bis-(gamma-glutamyl)polyamine formation. Finally, immunoprecipitation of 125I-labeled NRK cell surface proteins with anti-OP and anti-Fn antibodies, SDS-PAGE analysis, and autoradiography provides critical evidence for nonreducible OP-Fn cross-linking in vivo. These results clearly suggest that TG-mediated cross-linking between OP and Fn represents one of the most likely mechanisms by which OP becomes covalently linked to bone matrix, urinary stone matrix, and to ECM.

Synthesis and biological activity of (2-hydroxy-1-naphthyl)-methylamino acetamido-epicillin and cephradine, and (2-hydroxy-1-naphthyl)-methylacetamido 6-APA.

Two new penicillins and a new cephalosporin have been synthesized by condensing 2-hydroxy-1-naphthaldehyde with epicillin, 6-aminopenicillanic acid and cephradine, and subsequently reducing the SCHIFF bases with NaBH4. The antimicrobial activities of these compounds are also described.

Trace metal levels of X-ray technicians' blood and hair.

Medical X-ray technicians are exposed to low-level ionizing radiation in their occupational field. There are very few data on low-dose radiation effects. The present study was designed to estimate few vital trace metals (Zn, Cu, Fe) in indicator tissues (blood and hair) of X-ray technicians and non-X-ray technicians (hospital employees were used as controls) by Atomic Absorption Spectrometry (AAS). This analysis noted a significant increase in Zn, Cu, and Fe concentrations in X-ray technicians' hair. But in blood, Zn and Cu were depleted, whereas Fe was increased. Such changes in trace metal concentrations among X-ray technicians were noted where occupational exposure to radiation was for longer than three years. Through composite risk analysis, by using Zn:Fe as an indicator, it was noted that blood gave a stronger indication than hair in analyzing and estimating risk.

Selective transcription of genomic sequences common to both N- and X-tropic endogenous retroviruses in BALB/c mouse tissues.

Total RNAs from four BALB/c mouse tissues, containing mostly non-dividing cells (liver and kidney) or variable proportions of dividing cells (uterus and embryo) were analysed for sequences complementary to 3H-DNA transcripts synthesized from BALB/c endogenous. N- and X-tropic retroviruses. Extensive transcription of virogene information was detected in the tissues examined, but such transcription was found to be mostly limited to the homologous regions of the two virus genomes. No additivity of hybridization values could be detected when RNAs from two different tissues were mixed, which suggests that BALB/c mouse liver, kidney, uterus and embryo transcribe a common set of nucleic acid sequences of the homologous regions of the N- and X-tropic viral genomes, in addition to other sequences of the same region that are specific for indiviual tissues.

Restriction of the rat-specific RNS sequences of Ki-MSV to the nucleus of dibutyryl cyclic AMP-treated K-A31 cells.

The treatment of oncogenically transformed cells in culture, with dibutyryl cyclic AMP (cAMP) has, in many cases, resulted in a general phenotypic change towards the normal state. A virus-specific gene product(s) is responsible for the transformation of cells by sarcoma viruses and it has been suggested that the src gene product may act through the alteration of cAMP levels. With these premises we have studied the effects of dibutyryl cAMP on cell growth and virus genome expression in a Kirsten sarcoma virus-transformed mouse cell line. Our results suggest that certain virus-specific RNA sequences are restricted to the nucleus of these cells after several days of growth in medium containing dibutyryl cAMP and that these sequences appear to be those coding for the sarcoma information.

Variations in hybridization of RNA from different mouse tissues and embryos to endogenous C-type virus DNA transcripts.

Several adult tissues, newborns, and embryos of uninfected BALB/c mice were analysed for RNA complementary to [3H]-DNA transcripts synthesized from an endogenous type-C virus of BALB/c 3T3. The technique of RNA:DNA hybridization was used and the extent of hybridization was measured by the use of a single-strand-specific nuclease (S-I), purified from Aspergillus oryzae. Virus-specific RNA was detected in all adult and embryonic tissues tested. However, the RNA extracted from tissues having higher proliferative activity, such as spleen, small intestine, uterus and embryos, hybridize the [3H]-DNA probe to a greater extent than the RNA from tissues with low proliferative activity, such as kidney and liver. These observations add further support to the view that the repression of the virus genome in normal cells is not complete, and suggest the existence of a correlation between a qualitative or quantitative change in the endogenous C-type virus genome transcription pattern and cell proliferation.

Nonrandom X-chromosome inactivation--an artifact of cell selection (mouse chimera).

The present study shows that a high degree of variability in the distribution of XX and XY cells exists among various tissues of artificially assembled or spontaneously occurring mouse chimeras. This variation from the expected equal distribution in various tissues of an animal may have resulted from random distribution, unequal segregation, or selective differences of such cells during development. This variation may also explain the observed intertissue and interanimal variations in the distribution of inactive paternal and maternal X-chromosomes in some mammalian females.

Altered processing of a major secreted phosphoprotein correlates with tumorigenicity in Rous sarcoma virus-transformed mammalian cells.

Anchorage-independent growth is highly correlated with neoplastic growth in vivo, and the retinoids (vitamin A and its analogs) inhibit this property in a wide variety of oncogenically transformed cells. We report here that retinoic acid-treated Rous sarcoma virus-transformed rat (RR1022) and vole (SR-1T) cells, which show reversible loss of anchorage-independent growth and assume nontransformed morphology, secrete a major 69-kilodalton phosphoprotein (pp69) instead of the 62-kilodalton phosphoprotein (pp62) secreted by their untreated counterparts. As determined by V8 protease mapping and by two-dimensional electrophoretic analysis, this 69-kilodalton polypeptide was indistinguishable from the pp69 released by nontransformed normal rat kidney cells. Neither retinoic acid-treated RR1022 cells nor normal rat kidney cells secreted pp62, and retinoic acid treatment did not have any significant effect on the synthesis, subcellular localization, or phosphokinase activity of pp60src. Furthermore, treatment with retinoic acid did not alter the synthesis of the transformation-specific 53-kilodalton phosphoprotein (p53) and secretion of the transforming growth factors in RR1022 cells. Our studies showed that there is a clear correlation between the release of pp69 or pp62 and the ability of cells to grow in vitro with or without anchorage. This may provide an important clue for elucidating specific biochemical events involved in anchorage regulation of growth.

Physiological properties and differential glycosylation of phosphorylated and nonphosphorylated forms of osteopontin secreted by normal rat kidney cells.

In a previous study we have shown that normal rat kidney (NRK) cells in vitro secrete a 69-kDa osteopontin in both phosphorylated (pp69) and nonphosphorylated (np69) forms. Only pp69 interacts with the cell surface and np69 forms a heat-dissociable complex with plasma fibronectin, suggesting functional modulation of osteopontin by phosphorylation. Using tunicamycin, an inhibitor of N-linked glycosylation, and peptide:N-glycosidase F, which removes N-linked oligosaccharide chains from glycoproteins, we show here that np69, but not pp69, contains N-linked carbohydrates. Our results also demonstrate that tunicamycin treatment does not inhibit the cell surface binding of pp69; however, np69 secreted by the treated cells fails to complex with plasma fibronectin, suggesting importantly, our data show that pp69 forms a heat-stable complex with cell surface fibronectin, suggesting that it is an integral component of the extracellular matrix of NRK cells. Finally, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of deglycosylated and in vitro translated osteopontin suggests that the acidic nature of osteopontin as well as its post-translational modifications play a role in the anomalous behavior of osteopontin in sodium dodecyl sulfate gels, observed in several laboratories. The data presented here provide evidence for possible functional roles of 69-kDa osteopontin and suggest that its physiological properties are regulated by post-translational modifications.

Callus culture and plantlet production in Carica papaya (Var. Honey Dew).

In order to develop techniques for efficient callus production and regeneration in Carica papaya (Var. Honey Dew), lamina, petiole, stem and root explants from in vitro plantlets were cultured in media supplemented with 2.0 mg/1 IBA and 0.5 mg/1 BAP. Use of in vitro-grown plantlets as an explant source helped to avoid contamination common in papaya tissue culture. Callusing was maximum in root explants cultured in a modified MS (half-strength) medium. Shoot reganeration was maxium in root-derived callus grown in full-strength modified MS medium supplemented with 0.5 mg/1 IBA and 1 to 2 mg/1 kinetin. A histological study indicated that shoot buds originated from peripheral cell layers of the callus. Each shoot regenerated from callus was subcultured using a multiplication medium. Root formation was induced in all shoots treated in half-strength of modified MS medium containing 2 mg/1 IBA and rooted shoots were transferred successfully to the field.