Encapsulating the right chemistry.
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Biotechnology↗
Biomedical subjects
B Trivedi
Publications and source records attributed to B Trivedi.
Real-time patch-cram detection of intracellular cGMP reveals long-term suppression of responses to NO and muscarinic agonists.
Cyclic GMP (cGMP) is a crucial intracellular messenger in neuronal, muscle, and endocrine cells. The intracellular concentration of cGMP is regulated by various neurotransmitters, including acetylcholine (ACh) and nitric oxide (NO). While much is known about the biochemical steps leading to cGMP synthesis, little is known about cGMP kinetics in intact cells. Here, we use "patch-cramming," in which an excised, inside-out membrane patch containing cyclic nucleotide-gated ion channels is used as a biosensor, to obtain the first real-time measurements of cGMP in intact cells. Patch-cramming experiments on neuroblastoma cells show that both muscarinic agonists and NO rapidly elevate cGMP. NO elicits cGMP responses repeatedly without decrement, whereas responses to muscarinic agonists exhibit a profound and prolonged desensitization. Remarkably, muscarinic agonists also cause long-term (>30 min) suppression (LTS) of cGMP responses elicited by NO. Biochemical measurements reveal that rat sympathetic neurons also exhibit LTS of cGMP, suggesting that LTS is a widespread mechanism that may contribute to synaptic plasticity.
Animals↗
Modeling the oddities of biology.
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Animals↗
Modulation of rod photoreceptor cyclic nucleotide-gated channels by tyrosine phosphorylation.
Cyclic nucleotide-gated (CNG) channels in vertebrate photoreceptors are crucial for transducing light-induced changes in cGMP concentration into electrical signals. In this study, we show that both native and exogenously expressed CNG channels from rods are modulated by tyrosine phosphorylation. The cGMP sensitivity of CNG channels, composed of rod alpha-subunits expressed in Xenopus oocytes, gradually increases after excision of inside-out patches from the oocyte membrane. This increase in sensitivity is inhibited by a protein tyrosine phosphatase (PTP) inhibitor and is unaffected by three different Ser/Thr phosphatase inhibitors. Moreover, it is suppressed or reversed by application of ATP but not by a nonhydrolyzable ATP analog. Application of protein tyrosine kinase (PTK) inhibitors causes an increase in cGMP sensitivity, but only in the presence of ATP. Taken together, these results suggest that CNG channels expressed in oocytes are associated with active PTK(s) and PTP(s) that regulate their cGMP sensitivity by changing phosphorylation state. The cGMP sensitivity of native CNG channels from salamander rod outer segments also increases and decreases after incubation with inhibitors of PTP(s) and PTK(s), respectively. These results suggest that rod CNG channels are modulated by tyrosine phosphorylation, which may function as a novel mechanism for regulating the sensitivity of rods to light.
Adenosine Triphosphate↗
Viral hepatitis B with pleural complication.
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Adult↗
Non-islet cell tumour induced hypoglycaemia with acromegaloid facial and acral swelling.
The syndrome of non-islet cell tumour hypoglycaemia (NICTH) has been linked with the synthesis and secretion of 'big' IGF-II. We report a patient with a large pelvic clear cell sarcoma who developed recurrent severe hypoglycaemia and in addition presented with severe soft tissue facial swelling, skin tags and nuchal hyperpigmentation. After resection of the tumour serum 'big' IGF-II returned to normal and the acromegaloid skin changes remitted.
Acromegaly↗
The diastrophic dysplasia gene encodes a novel sulfate transporter: positional cloning by fine-structure linkage disequilibrium mapping.
Diastrophic dysplasia (DTD) is a well-characterized autosomal recessive osteochondrodysplasia with clinical features including dwarfism, spinal deformation, and specific joint abnormalities. The disease occurs in most populations, but is particularly prevalent in Finland owing to an apparent founder effect. DTD maps to distal chromosome 5q and, based on linkage disequilibrium studies in the Finnish population, we had previously predicted that the DTD gene should lie about 64 kb away from the CSF1R locus. Here, we report the positional cloning of the DTD gene by fine-structure linkage disequilibrium mapping. The gene lies in the predicted location, approximately 70 kb proximal to CSF1R, and encodes a novel sulfate transporter. Impaired function of its product is likely to lead to undersulfation of proteoglycans in cartilage matrix and thereby to cause the clinical phenotype of the disease. These results demonstrate the power of linkage disequilibrium mapping in isolated populations for positional cloning.
Amino Acid Sequence↗
Serum "big insulin-like growth factor II" from patients with tumor hypoglycemia lacks normal E-domain O-linked glycosylation, a possible determinant of normal propeptide processing.
The insulin-like growth factor II (IGF-II) gene is overexpressed in many mesenchymal tumors and can lead to non-islet-cell tumor hypoglycemia (NICTH). ProIGF-II consists of the 67 aa of IGF-II with a carboxyl 89-aa extension, the E domain. A derivative of proIGF-II containing only the first 21 aa of the E domain [proIGF-II-(E1-21)] has been isolated by others from normal serum and has O-linked glycosylation. We found that the "big IGF-II" of normal serum, as detected by an RIA directed against residues 1-21 of the E domain of proIGF-II, was reduced in size by treatment with neuraminidase and O-glycosidase. The big IGF-II, which is greatly increased in NICTH sera, was unaffected by neuraminidase and O-glycosidase treatment. We have also shown that big IGF-II from normal serum is retained by jacalin lectin columns and that big IGF-II from NICTH serum was not retained, indicating that it lacked O-glycosylation. Normal O-linked glycosylation may be required for proper peptidase processing of proIGF-II. The lack of normal O-linked glycosylation by tumors may explain the predominance of big IGF-II in NICTH sera. In normal serum, most of the IGF-II is present in a 150-kDa ternary complex with IGF-II binding protein (IGFBP) 3 and alpha subunit. In NICTH serum, however, the complexes carrying big IGF-II are < 50 kDa. We investigated whether big IGF-II of NICTH was responsible for this abnormality. Tumor big IGF-II and IGF-II were equally effective in forming the 150-kDa complex with purified IGFBP-3 and 125I-labeled alpha subunit. Both 125I-labeled IGF-II and 125I-labeled proIGF-II-(E1-21), when incubated with normal serum, formed the 150-kDa complex as detected by Superose 12 exclusion chromatography. We conclude that the nonglycosylated big IGF-II of NICTH serum can form normal complexes with serum IGFBPs. The defective binding in NICTH is attributable to defective IGFBP-3 binding.
Adult↗
Prevalence of microalbuminuria in diabetic subjects.
Total 193 diabetic patients were investigated to assess the prevalence of microalbuminuria. Urinary albumin excretion rate (UAER) was measured by radioimmunoassay (RIA) on 3 hours urine samples. The prevalence of microalbuminuria (UAER) > 15 micrograms/min was 41%. Microalbuminuria was commonly observed in patients having diabetes for more than 5 years. A significant correlation was found between duration of diabetes and microalbuminuria (p < 0.01). Glycemic control (fasting and postprandial blood sugar) did not show any correlation with UAER, whereas blood urea (r.39, p < 0.01), creatinine (r.26, p < 0.05) and chloride (r.24, p < 0.05) were positively correlated. A significant correlation was found between raised blood pressure and UAER (p < 0.01).
Adult↗
Measurement of derivatives of proinsulin-like growth factor-II in serum by a radioimmunoassay directed against the E-domain in normal subjects and patients with nonislet cell tumor hypoglycemia.
We describe a modified RIA using a rabbit polyclonal antiserum directed against the first 21 amino acids of the E-domain (E-21) of proinsulin-like growth factor-II (pro-IGF-II). For standardization, we purified big IGF-II from patients with nonislet cell tumor hypoglycemia (NICTH). Under the conditions of our assay there was no significant interference from IGF-binding proteins. The big IGF-II present in the serum of a patient with NICTH displaced [125I]E-(1-21) from antibody parallel to our big IGF-II standard. We found a progressive rise in E-21 immunoactivity (IA) during childhood, with somewhat higher values in girls than in boys. In normal adults the mean E-21 IA level was 138 +/- 49 (+/- SD) micrograms/L. Women with twin pregnancies had higher E-21 IA than women with single pregnancies (302 +/- 66 compared with 120 +/- 18 micrograms/L). We found a marked elevation of E-21 IA in patients with NICTH due to sarcomas (n = 3), hepatoma (n = 2), adrenal carcinoma (n = 1), and carcinoma of the lung (n = 1). No elevation of E-21 IA was present in the serum of a hypoglycemic patient with a hypernephroma or another patient with carcinoma of the lung. Marked elevation of E-21 IA was observed in the serum of patients with renal failure receiving chronic hemodialysis. We conclude that this assay will prove useful in the diagnosis of NICTH in patients who are not azotemic and the investigation of the role of the kidney in clearing products of pro-IGF-II processing.
Adolescent↗
Heterogeneity of serum peptides with immunoactivity detected by a radioimmunoassay for proinsulin-like growth factor-II E domain: description of a free E domain peptide in serum.
We have reported that normal human sera contain immunoactivity (IA) detected by a RIA directed against the first 21 amino acids of the E domain of proinsulin-like growth factor-II (pro-IGF-II). Marked elevations of E-21 IA were found in the serum of patients with nonislet cell hypoglycemia (NICTH) and patients with renal failure receiving chronic hemodialysis. In this paper we describe some of the properties of the E-21 IA of normal and abnormal sera. The E-21 IA eluted from a calibrated acid Sephadex G-50 column as two major peaks. In normal serum the first major peak had a mol wt (Mr) between 14,000-15,000, and the second peak had a Mr between 5,000-6,000. When E-21 IA from serum of a patient with NICTH was similarly studied, most of the IA was present as a Mr 11,000 peak and only a small amount was present as a 5,000-6,000 Mr peak. In contrast, most of the E-21 IA present in the sera of patients on hemodialysis was present in the smaller molecular form, which eluted from a reverse phase column as a single component. This small Mr peak lacked determinants for the IGF-II monoclonal antibody (Amano), for pooled serum IGF-binding proteins, and for the IGF-I receptors on human placental membranes. We suggest that the 15- and 11-kilodalton peaks represent the glycated and unglycated forms of pro-IGF-II (E-21) reported by others. The glycated form appears to predominate in normal serum, whereas the nonglycated form predominates in the serum of patients with NICTH and renal failure. The 5,000-6,000 Mr E-21 IA probably represents a fragment of the free E domain of pro-IGF-II. Its size is consistent with cleavage of the free E domain between Arg46 and Arg47. The accumulation of this E-21 IA in renal failure is evidence that the kidney has a major role in the clearance of this fragment, which is not accomplished by the membranes used in hemodialysis.
Aged↗
Clinical aspects of GH binding proteins.
The high affinity specific binding of 125I-hGH in human serum is attributable to the GH binding protein, which is derived from the extracellular domain of the receptor. Most measurements have been made by gel filtration methods and expressed with a normal human reference serum (RSGHB). The RSGHB is virtually unmeasurable in the cord sera of premature infants and is higher in cord serum of term infants. RSGHB rises progressively in childhood and reaches adult levels after age 20 years. GH deficiency or excess exerts little effect on RSGHB. RSGHB in GH resistance of the Laron-type is virtually undetectable and such measurements are confirmatory of the diagnosis. In African pygmies, the RSGHB fails to rise in late childhood and correlates with decreased IGF-I concentrations and reduced growth velocity at that time. Studies of abnormalities of the GH-BP in nutritional and other medical conditions are in their infancy, but will be greatly aided by simpler assay methods.
Aging↗
Whole body nitrogen kinetics and their relationship to growth in short children treated with recombinant human growth hormone.
We studied the effects of growth hormone on retention of 15N-labeled amino acids in 34 short, prepubertal, growth hormone-sufficient children and three growth hormone-deficient subjects. All 34 non-growth hormone-deficient children had apparently normal circulating growth hormone molecules and no mutations were detected in the growth hormone or IGF-I genes of any subjects. Fibroblasts from 34 children responded normally when challenged with recombinant human IGF-I. During the last 72 h of a 4-d challenge with recombinant human growth hormone (16 micrograms/kg body wt), retention of a mixed 15N-amino acid dose varied between 5.7 and 50.5%. Whole body protein synthesis, breakdown, and net anabolism calculated from the 15N kinetics were all increased by the acute growth hormone challenge. However, no routine clinical feature or laboratory determination correlated with the nitrogen retention response. After subsequent treatment (75 micrograms/kg three times a week) with recombinant human growth hormone for 1 y, there was a significant increase in height velocity, but this increase was not related significantly to pretreatment variables other than inversely to pretreatment height velocity. There was a significant (p = 0.03) correlation between the change in height velocity Z score and the degree of nitrogen retention to acute challenge with growth hormone, but this correlation was too weak (r = 0.37) to be of practical value in predicting the treatment growth response in an individual child.
Adolescent↗
Hypersomatotropism in pregnant women, as measured by a human liver radioreceptor assay.
Radioreceptor assays (RRAs) provide useful information about the bioactivity of peptide hormones. We have developed a RRA for human GH using membranes prepared from human liver. The assay has 1% cross-reactivity with human PRL, 0.11% cross-reactivity with human chorionic somatomammotropin, and negligible cross-reactivity with bovine and rat GH. The assay has distinct advantages over the IM-9 lymphocyte RRA in having reduced nonspecific interference from serum proteins and providing a uniform source of receptors which can be stored for long periods at -70 C. The coefficient of variation of the assay is 13%. In studies of 55 sera from short children with normal or elevated serum GH concentrations the mean ratio of RRA GH to immunoradiometric assayed GH was 1.28. This assay provided important information about the total GH activity in pregnancy. There was a progressive rise in RRA GH during pregnancy, reaching 64.7 +/- 2.5 (+/- SE) micrograms/L at term. This level is 2-3 times higher than suggested by earlier estimates made with monoclonal antibody RIAs for pituitary GH. At term the contributions to total GH activity of serum are less than 3% from the pituitary, 12% from human chorionic somatomammotropin reacting with the GH receptor, and 85% from placental GH. Despite this great increase in receptor-reactive GH, somatomedin activity by bioassay is reduced, and IGF-I, determined by RIA after acid-gel filtration, is minimally elevated.
Adult↗
Embryotoxicity and fetotoxicity of orally administered hexavalent chromium in mice.
The embryotoxic and fetotoxic potential of hexavalent chromium (Cr+6) in mice was investigated by administering 250, 500, and 1000 ppm of potassium dichromate daily through drinking water during the entire gestation period. An increase in embryonic deaths was observed; however, in the mothers treated with the highest dose, there was complete absence of implantation sites. No major abnormality was observed in the fetuses except that Cr+6 exposure increased the incidences and types of external and skeletal malformations. It is concluded that oral exposure to Cr+6 causes dose-dependent embryolethal effects in mice.
Abnormalities, Drug-Induced↗
Reduced 125I-hGH binding by serum of dwarf pigs but not by serum of dwarfed poodles.
1. Normal and growth-deficient poodle and swine strains were characterized for serum growth hormone-binding protein (GH-BP) content as well as other growth-related hormones, and the relationship between these factors and body size was examined. 2. GH-BPs were found in all strains of pigs and poodles. Concentrations of GH-BPs (as expressed by specific bindings) did not vary among the poodle breeds, but did correlate with body size in pigs. 3. Insulin-like growth factors (IGFs) I and II were decreased 71 and 44% respectively in miniature compared to standard size poodles. 4. Only the Yucatan micro pig strain had reduced serum IGF-I concentrations compared to normal controls. 5. Growth hormone concentrations however were normal to elevated in all micro and miniature pig strains. 6. Serum triiodothyronine concentrations were reduced in Yucatan mini and micro pigs in spite of normal circulating levels of thyroxine. 7. Body size reductions in the swine and dog strains are probably attributable to different primary defects of various growth related hormones or hormone receptors. 8. Each species breed therefore could serve as a model for a different human growth-deficient condition.
Animals↗
Calcium compartmentation in isolated renal tubules in suspension.
Substantial increases of total cell Ca2+ have been observed in suspensions of isolated rabbit proximal tubules subjected to hypoxic injury or treated with exogenous ATP followed by apparent recovery with reoxygenation of the hypoxic tubules or continued incubation of ATP-treated tubules. Ca2+ compartmentation studies using digitonin and metabolic inhibitors were done to clarify the basis for these changes. Digitonin, 40-90 micrograms/mg tubule protein, rapidly permeabilized the tubule cells and did not impair mitochondrial Ca2+ sequestration. Most of the increases of tubule cell Ca2+ produced by hypoxia and ATP were accounted for by pools which could be rapidly removed by exposure of tubules to EGTA and the uncoupler carbonyl cyanide m-chlorophenyl hydrazone without concomitant use of digitonin, suggesting that the changes of Ca2+ predominantly reflect sequestration by mitochondria in severely damaged cells or mitochondria already released to the medium from them. The time course of uptake followed by spontaneous release of mitochondrial Ca2+ from tubule cells deliberately permeabilized with digitonin, then incubated for prolonged periods, indicated that the decreases of tubule cell Ca2+ during reoxygenation of hypoxic suspensions and prolonged incubation of ATP-treated tubules were likely to be attributable to loss of Ca2+ from free mitochondria and those in damaged cells rather than to extrusion by intact cells.
Adenosine Triphosphate↗