Expression of the gap junction protein connexin43 in embryonic chick lens: Molecular cloning,ultrastructural localization,and post-translational phosphorylation

Summary Lens epithelial cells are physiologically coupled to each other and to the lens fibers by an extensive network of intercellular gap junctions. In the rat, the epithelial-epithelial junctions appear to contain connexin43, a member of the connexin family of gap junction proteins. Limitations on the use of rodent lenses for the study of gap junction formation and regulation led us to examine the expression of connexin43 in embryonic chick lenses. We report here that chick connexin43 is remarkably similar to its rat counterpart in primary amino acid sequence and in several key structural features as deduced by molecular cDNA cloning. The cross-reactivity of an anti-rat connexin43 serum with chick connexin43 permitted definitive immunocytochemical localization of chick connexin43 to lens epithelial gap junctional plaques and examination of the biosynthesis of connexin43 by metabolic radiolabeling and immunoprecipitation. We show that chick lens cells synthesize connexin43 as a single, 42-kD species that is efficiently posttranslationally converted to a 45-kD form. Metabolic labeling of connexin43 with³²P-orthophosphate combined with dephosphorylation experiments reveals that this shift in apparent molecular weight is due solely to phosphorylation. These results indicate that embryonic chick lens is an appropriate system for the study of connexin43 biosynthesis and demonstrate for the first time that connexin43 is a phosphoprotein.     

Chronic Electroconvulsive Seizures Down–Regulate Expression of the Immediate-Early Genes c-fos and c-jun in Rat Cerebral Cortex

Acute seizures and other stimuli that increase neuronal activity cause a rapid induction of the immediate-early genes c-fos and c-jun, also referred to as nuclear proto-oncogenes, in the nervous system. In the present study, rats were administered one or more electroconvulsive seizures (ECS) and the responsiveness of c-fos and c-jun to an acute, "test" seizure was examined. Four hours after a single ECS, the induction of c-fos mRNA by a test seizure was blocked, in agreement with earlier findings, but by 18 h the levels of c-fos mRNA could be reinduced by the test seizure, suggesting that 1 day is sufficient to "reset" the responsiveness of this system. However, it was found that chronic, daily ECS treatments resulted in a time-dependent decrease in the expression of c-fos mRNA in response to a test seizure administered 18 h after the last daily ECS; this effect was maximal after 8-10 days of treatment, at which time the induction of c-fos mRNA by the test seizure was blocked dramatically. Chronic ECS also blocked the induction of c-jun in response to an acute, test seizure. The effect of chronic ECS on levels of Fos protein was also investigated. It was found that basal levels of Fos protein were reduced after chronic (10 days) ECS and were not induced by a test seizure. Because levels of Fos protein remain elevated 4 h after a single seizure this finding suggests that the mechanisms by which acute (4 h) and chronic (8-10 days) ECS block the induction of c-fos may differ.     

Human monocytes selectively bind to cells expressing the tumorigenic phenotype

Summary The characteristics of the binding of human monocytes to tumor cells were studied by a newly developed microassay. First, we determined the kinetics and optimal conditions of the binding. Monocytes recognized and bound to tumor cells very rapidly within 10–20 min of cellular interaction. Binding was also more efficient at 37°C suggesting that active metabolism of monocytes is required. Second, we determined that selective binding of monocytes to cells with tumorigenic phenotypes occurs. For this purpose, lymphocytic leukemia cell lines versus normal lymphocytes, and tumorigenic versus nontumorigenic hybrids from the same parental lines were compared as the targets of the binding assay. In both cases, neoplastic cells were selectively bound by monocytes. Although tumor cells were bound rapidly and selectively by monocytes, initial recognition and binding did not necessarily lead to subsequent tumor cell lysis. This is based on the observation that some tumorigenic parental and hybrid lines were avidly bound by monocytes yet not subsequently killed in a cytotoxicity assay.This work was supported in part by a grant from the National Institutes of Health CA42992 and a grant from the Kleberg foundation Abbreviations used: [¹²⁵I]IdUrd [¹²⁵I]iododeoxyuridine; rIFN-, recombinant human interferon ; IL-1, interleukin 1; rTNF, recombinant human tumor necrosis factor     

Effects of seven different mutations in the pho1 gene on enzymatic activity, glycosylation and secretion of acid phosphatase in Schizosaccharomyces pombe

Summary Structural gene mutants of the cell-surface glycoprotein acid phosphatase of Schizosaccharomyces pombe were analysed to define structural determinants that are responsible for enzymatic activity, N-glycosylation and secretion. All seven defined mutations cause a single amino acid substitution in the mature acid phosphatase protein and destroy the enzymatic activity. The mutational lesions are distributed throughout the pho1 gene. A ser to phe substitution at position 349 abolishes enzymatic activity only and does not affect glycosylation and secretion. Two mutations create a new N-glycosylation site by substitution of pro at position 56 by phe and ser, respectively. This new site is apparently used in the mutants. Their core-glycosylated acid phosphatase is slightly larger than that of the wild type. Overglycosylation seems not to affect secretion. Four different mutations (a gly to asp substitution at position 281 and ser to phe substitutions at positions 150, 271 and 277) cause intracellular accumulation of enzymatically inactive core-glycosylated acid phosphatase precursor. These mutational lesions apparently block transport of acid phosphatase from the endoplasmic reticulum to the Golgi apparatus.     

Identification of fungi for sweet whey permeate utilization and eicosapentaenoic acid production

Summary Eighteen selected organisms of theEumycota division of the fungi kingdom were examined for eicosapentaenoic acid production and utilization of sweet whey permeate. The organisms belong to the subdivisionsMastigomycotina, Zygomycotina, Ascomycotina andDeuteromycotina. Seven organisms were initially identified as lactose utilizers (the predominant sugar in sweet whey permeate_ and eicosapentaenoic acid (EPA) producers. Utilization of lactose was demonstrated and EPA production was confirmed for four organisms, all of the subdivisionMastigomycotina. Growth studies showed thatP. ultimum had the best potential for future work.     

Plant growth analysis: an evaluation of experimental design and computational methods

Journal of Experimental Botany, Vol. 47, No. 302, pp. 1343–1351,September 1996 On page 1345, equation (2) should have read:      

Intracellular Calcium Dynamics and Cellular Energetics in Ischemic NG108-15 Cells Studied by Concurrent 31P/19F and 23Na Double-Quantum Filtered NMR Spectroscopy

Abstract: The role of voltage-sensitive Ca²⁺ channels in mediating Ca²⁺ influx during ischemia was investigated in NG108-15 cells, a neuronal cell line that does not express glutamate-sensitive receptor-mediated Ca²⁺ channels. Concurrent ³¹P/¹⁹F and ²³Na double-quantum filtered (DQF) NMR spectra were used to monitor cellular energy status, intracellular [Ca²⁺] ([Ca²⁺]_i), and intracellular Na⁺ content in cells loaded with the calcium indicator 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5FBAPTA) during ischemia and reperfusion. Cells loaded with 5FBAPTA were indistinguishable from unloaded cells except for small immediate decreases in levels of phosphocreatine (PCr) and ATP. Ischemia induced a steady decrease in intracellular pH and PCr and ATP levels, and a steady increase in intracellular Na⁺ content; however, a substantial increase in [Ca²⁺]_i (about threefold) was seen only following marked impairment of cellular energy status, when PCr was undetectable and ATP content was reduced to 55% of control levels. A depolarization-induced increase in [Ca²⁺]_i could be completely blocked by 1 µM nifedipine, whereas up to 20 µM nifedipine had no effect on the increase in [Ca²⁺]_i seen during ischemia. These data demonstrate that voltage-gated Ca²⁺ channels do not mediate significant Ca²⁺ flux during ischemia in this cell line and suggest an important role for Ca²⁺_i stores, the Na⁺/Ca²⁺ antiporter, or other processes linked to cellular energy status in the increase in cytosolic Ca²⁺ level during ischemia.     

EPISTASIS AS A SOURCE OF INCREASED ADDITIVE GENETIC VARIANCE AT POPULATION BOTTLENECKS

The role of epistasis in evolution and speciation has remained controversial. We use a new parameterization of physiological epistasis to examine the effects of epistasis on levels of additive genetic variance during a population bottleneck. We found that all forms of epistasis increase average additive genetic variance in finite populations derived from initial populations with intermediate allele frequencies. Average additive variance continues to increase over many generations, especially at larger population sizes (N = 32 to 64). Additive-by-additive epistasis is the most potent source of additive genetic variance in this situation, whereas dominance-by-dominance epistasis contributes smaller amounts of additive genetic variance. With additive-by-dominance epistasis, additive genetic variance decreases at a relatively high rate immediately after a population bottleneck, rebounding to higher levels after several generations. Empirical examples of epistasis for murine adult body weight based on measured genotypes are provided illustrating the varying effects of epistasis on additive genetic variance during population bottlenecks.     

Perinatal Steroid Treatments Alter Alloparental and Affiliative Behavior in Prairie Voles

This experiment was designed to examine the hypothesis that perinatal manipulation of gonadal or adrenal steroids can alter the subsequent expression of juvenile parental (alloparenting) and affiliative behavior in prairie voles (Microtus ochrogaster). Corticosterone (PRECORT), testosterone (PRE-TP), or oil injections (PRESES) were given on Prenatal Days 12–20 or on Postnatal Days 1–6 (CORT6, TP6, or SES6, respectively). Alloparenting was reduced significantly in females in the CORT6 group and in males in the TP6 group. Sibling affiliative preferences were increased significantly in PRE-TP females and stranger preferences were increased in TP6 and CORT6 females. The results suggest timing is a critical factor determining whether hormones have a facilitative or inhibitory effect on alloparental and affiliative behavior in prairie voles. In this species, corticosterone and testosterone have similar organizational effects on affiliative behavior in females. Alloparental behavior is inhibited by postnatal corticosterone administration in females and by postnatal testosterone administration in males, whereas prenatal steroid administration had no significant effect on alloparenting in either gender.