An internal deletion in the cytoplasmic tail reverses the apical localization of human NGF receptor in transfected MDCK cells

A cDNA encoding the full-length 75-kD human nerve growth factor receptor was transfected into MDCK cells and its product was found to be expressed predominantly (80%) on the apical membrane, as a result of vectorial targeting from an intracellular site. Apical hNGFR bound NGF with low affinity and internalized it inefficiently (6% of surface bound NGF per hour). Several mutant hNGFRs were analyzed, after transfection in MDCK cells, for polarized surface expression, ligand binding, and endocytosis. Deletionof juxta-membrane attachment sites for a cluster of O-linked sugars did not alter apical localization. A mutant receptor lacking the entire cytoplasmic tail (except for the five proximal amino acids) was also expressed on the apical membrane, suggesting that information for apical sorting was contained in the ectoplasmic or transmembrane domains. However, a 58 amino acid deletion in the hNGFR tail that moved a cytoplasmic tyrosine (Tyr 308) closer to the membrane into a more charged environment resulted in a basolateral distribution of the mutant receptor and reversed vectorial (basolateral) targeting. The basolateral mutant receptor also internalized 125I-NGF rapidly (90% of surface bound NGF per hour), exhibited a larger intracellular fraction and displayed a considerably shortened half-life (approximately 3 h). We suggest that hNGFR with the internal cytoplasmic deletion expresses a basolateral targeting signal, related to endocytic signals, that is dominant over apical targeting information in the ecto/transmembrane domains. These results apparently contradict a current model that postulates that basolateral targeting is a default mechanism.     

Kinetics of spermatogenesis in megachiropteran bat, Rousettus leschenaulti (Desmarset): seminiferous epithelial cycle, frequency of stages, spermatogonial renewal and germ cell degeneration.

The paper describes in detail the cytomorphology of different types of germ cells, the 10 typical cellular associations or stages of the cycle of seminiferous epithelium (CSE), frequency of appearance of these stages, pattern of spermatogonial stem cell renewal and per cent degeneration of various germ cells in R. leschenaulti. Of the 14 steps of spermiogenesis (stained with PAS-haematoxylin) the first 10 were associated with the stages I-X, whereas, the remaining were found in association with one of the first six stages. The frequency of appearance of the various stages ranged from 3.84% (stage V) to 19.84% (stage I). These observations indicate that stage V is of shortest duration and stage I is of the longest duration in the bat. Five types of spermatogonia (A1, A2, A3, In and B) were identified based on their shape, size and nuclear morphology. Type A spermatogonia are oval with a large nucleus containing 1 or 2 nucleoli. The chromatin showed progressive condensation from A1 to A3 so that the latter appeared darkest among all the A type spermatogonia. The In type derived from A3 are smaller but appear darker than A3 due to heterochromatin crusts along the inner border of the nucleus. The B type spermatogonia derived from In are round and possess single nucleolus. The B type spermatogonia divided mitotically before entering meiosis or the actual production of the primary spermatocytes. The various spermatogonia divided mitotically at fixed stages of the cycle giving rise to their next generations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deletion of cytoplasmic sequences of the nerve growth factor receptor leads to loss of high affinity ligand binding

The nerve growth factor (NGF) receptor is a glycosylated transmembrane protein present on the cell surface as both high and low affinity forms, but biological responsiveness requires interactions of NGF with the high affinity site. We have tested the effects of mutations in the intracellular domain of the receptor upon its cell surface expression and equilibrium binding of 125I-NGF. Although mutant receptors lacking the entire cytoplasmic domain are processed and expressed at the cell surface and are capable of binding to NGF, the absence of cytoplasmic sequences leads to a loss of high affinity binding and to a lack of an appropriate cross-linking pattern as assessed by N-hydroxysuccinimidyl 4-azidobenzoate photoaffinity cross-linking. These results, taken together with the highly conserved nature of these cytoplasmic sequences, implies that the interaction of the receptor with an accessory molecule is necessary to form the high affinity receptor.     

Suppression of bioactive luteinizing hormone and testosterone by a progesterone antagonist ZK 98.734 in adult male common marmosets, Callithrix jacchus

The effects of a progesterone antagonist ZK 98.734 on release of bioactive luteinizing hormone (LH) and testosterone were studied in adult male common marmosets by using the following experimental protocols: (1) the blocking of the nocturnal rise in testosterone levels by ZK 98.734, (2) the pharmacodynamic effects of ZK 98.734 on testosterone and LH levels, (3) the reversal of ZK 98.734-induced decrease in testosterone by treatment with human chorionic gonadotropin (hCG), and (4) the blocking of estradiol-induced positive feedback release of LH by ZK 98.734. Sixteen adult male common marmosets were used for different experiments after resting them for at least 4 wk between experiments. Testosterone and bioactive LH levels were measured by specific radioimmunoassay and in vitro bioassay methods, respectively. Treatment (i.m.) of male common marmosets (n = 6/group) with ZK 98.734 (1 mg or 5 mg/day) at 1700 h for 3 consecutive days significantly (p less than 0.05) suppressed the nocturnal (2200 h) rise in testosterone levels. The effects of the two doses were not dose-related; however, the decrease on the first day of treatment was more pronounced with the 5-mg dose than with the 1-mg dose. Diurnal rhythms were restored during the post-treatment period. Similarly, treatment with ZK 98.734 (5 mg, n = 8/group) at 1000 h caused a decrease in testosterone and LH levels. The levels were significantly (p less than 0.05) lower at 3 and 6 h after treatment compared to pretreatment levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Degradation of carboxin and oxycarboxin in different soils

Summary Five different soils varying in physico-chemical properties were used for studying the persistence and degradation of carboxin and oxycarboxin. In one soil only both fungicides were degraded with accumulation of ammonium and nitrite. Under the conditions of forced circulation of air and continuous perfusion, oxycarboxin was found to be more susceptible to degradation than carboxin. Under simulated conditions of rice fields, conversion of carboxin to its sulphoxide and to a non-toxic derivative of oxycarboxin could only be seen in all the soils.The role of clay, humus and organic matter as protectants of fungicides against degradation indicated that the intermediary compound carboxin sulphoxide was strongly adsorbed probably on organic and inorganic colloids of most of the soils. Organic matter free soils delayed the degradation. Carboxin was rapidly converted to its sulphoxide on three forms of monoionic clays whereas oxycarboxin was transformed to an unidentified derivative.Part of Ph.D. thesis submitted to UAS, Bangalore-65.     

Roles of creatine in the regulation of cardiac protein synthesis

The observation that increased muscular activity leads to muscle hypertrophy is well known, but identification of the biochemical and physiological mechanisms by which this occurs remains an important problem. Experiments have been described (5, 6) which suggest that creatine, an end product of contraction, is involved in the control of contractile protein synthesis in differentiating skeletal muscle cells and may be the chemical signal coupling increased muscular activity and the increased muscular mass. During contraction, the creatine concentration in muscle transiently increases as creatine phosphate is hydrolyzed to regenerate ATP. In isometric contraction in skeletal muscle for example, Edwards and colleagues (3) have found that nearly all of the creatine phosphate is hydrolyzed. In this case, the creatine concentration is increased about twofold, and it is this transient change in creatine concentration which is postulated to lead to increased contractile protein synthesis. If creatine is found in several intracellular compartments, as suggested by Lee and Vissher (7), local changes in concentration may be greater then twofold. A specific effect on contractile protein synthesis seems reasonable in light of the work of Rabinowitz (13) and of Page et al. (11), among others, showing disproportionate accumulation of myofibrillar and mitochondrial proteins in response to work-induced hypertrophy and thyroxin-stimulated growth. Previous experiments (5, 6) have shown that skeletal muscles cells which have differentiated in vitro or in vivo synthesize myosin heavy-chain and actin, the major myofibrillar polypeptides, faster when supplied creatine in vitro. The stimulation is specific for contractile protein synthesis since neither the rate of myosin turnover nor the rates of synthesis of noncontractile protein and DNA are affected by creatine. The experiments reported in this communication were undertaken to test whether creatine selectively stimulates contractile protein synthesis in heart as it does in skeletal muscle.     

Assessment of genetic diversity and population structure in pomegranate (Punica granatum L.) using hypervariable SSR markers

Physiology and Molecular Biology of Plants - The present study investigates the genetic diversity and population structure among 42 diverse pomegranate genotypes using a set of twenty one class I...