Plasticity in cultured carotid body chemoreceptors: Environmental modulation of GAP-43 and neurofilament

In this study we use dissociated cell cultures of the rat carotid body to investigate the adaptive capabilities of endogenous oxygen chemoreceptors, following chronic stimulation by various environmental factors. These oxygen chemoreceptors are catecholamine-containing glomus cells, which derive from the neural crest and resemble adrenal medullary chromaffin cells. Using double-label immunofluorescence, we found that chronic exposure of carotid body cultures to hypoxia (2% to 10% oxygen) caused a significant fraction of tyrosine hydroxylase-positive (TH+) glomus cells to acquire detectable immunoreactivity for growth-associated protein gap-43. The effect was dose-dependent and peaked around an oxygen tension of 6%, where approximately 30% of glomus cells were GAP-43 positive. Treatment with agents that elevate intracellular cyclic adenosine monophosphate (cAMP) (i.e., dibutyryl cAMP or forskolin) also markedly stimulated GAP-43 expression. Since hypoxia is known to increase cAMP levels in glomus cells, it is possible that the effect of hypoxia on GAP-43 expression was mediated, at least in part, by a cAMP-dependent pathway. Unlike hypoxia, however, cAMP analogs also stimulated neurofilament (NF 68 or NF 160 kD) expression and neurite outgrowth in glomus cells, and these properties were enhanced by retinoic acid. Nerve growth factor, which promotes neuronal differentiation in related crest-derived endocrine cells, and dibutyryl cGMP were ineffective. Thus, it appears that postnatal glomus cells are plastic and can express neuronal traits in vitro. However, since hypoxia stimulated GAP-43 expression, without promoting neurite outgrowth, it appears that the two processes can be uncoupled. We suggest that stimulation of GAP-43 by hypoxia may be important for other physiological processes, e.g., enhancing neurotransmitter release or sensitization of G-protein–coupled receptor transduction. © 1995 John Wiley & Sons, Inc.     

Using haplotypes to monitor the migration of fall armyworm (Lepidoptera: Noctuidae) corn-strain populations from Texas and Florida

Fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), infestations in most of North America north of Mexico arise from annual migrations of populations that overwinter in southern Texas and Florida. A comparison of the cytochrome oxidase I haplotype profiles within the fall armyworm corn-strain, the subgroup that preferentially infests corn (Zea mays L.) and sorghum (Sorghum vulgare Pers.), identified significant differences in the proportions of certain haplotypes between the Texas and Florida populations. These proportional differences were preserved as the populations migrated, providing a molecular metric by which the source of a migrant population could be identified. The migratory pattern derived from this method for several southeastern states was shown to be consistent with predictions based on analysis of historical agricultural and fall armyworm infestation data. These results demonstrate the utility of haplotype proportions to monitor fall armyworm migration, and they also introduce a potential method to predict the severity of cotton crop infestations in the short term.     

The measurement of membrane potential during photosynthesis and during respiration in intact cells of Rhodopseudomonas capsulata by both electrochromism and by permeant ion redistribution.

Adenosine deaminase was purified 3038-fold to apparent homogeneity from human leukaemic granulocytes by adenosine affinity chromatography. The purified enzyme has a specific activity of 486 mumol/min per mg of protein at 35 degrees C. It exhibits a single band when subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, non-denaturing polyacrylamide-gel electrophoresis and isoelectric focusing. The pI is 4.4. The enzyme is a monomeric protein of molecular weight 44000. Both electrophoretic behaviour and molecular weight differ from those of the low-molecular-weight adenosine deaminase purified from human erythrocytes. Its amino acid composition is reported. Tests with periodic acid-Schiff reagent for associated carbohydrate are negative. Of the large group of physiological compounds tested as potential effectors, none has a significant effect. The enzyme is specific for adenosine and deoxyadenosine, with Km values of 48 microM and 34 microM respectively. There are no significant differences in enzyme function on the two substrates. erythro-9-(2-Hydroxy non-3-yl) adenine is a competitive inhibitor, with Ki 15 nM. Deoxycoformycin inhibits deamination of both adenosine and deoxyadenosine, with an apparent Ki of 60-90 pM. A specific antibody was developed against the purified enzyme, and a sensitive radioimmunoassay for adenosine deaminase protein is described.     

A comparative binding of platinum anti-tumour compounds to plasma proteins in the rat (in vivo) and mouse (in vitro).

Plasma protein binding of 195mPt-labelled cisplatin, carboplatin and iproplatin has been studied in vivo in rat and in vitro in mouse, using both electrophoresis and trichloroacetic acid precipitation. After intravenous injection plasma clearance rates were biphasic for all 3 compounds, (t1/2 alpha, 13-17 min) but cisplatin was retained thereafter longer than the others. By 5 min, gel electrophoresis showed protein labelling with all 3 drugs but none involved low mol.wt. proteins (< 16 kDa). At 2 h a notable proportion of the protein bound platinum was associated with the latter components. There was a general resemblance between the distribution patterns of cisplatin and carboplatin whereas iproplatin showed a persistent retention of the label with time to higher mol. wt. proteins. From in vitro incubation with mouse plasma, rates of interaction respectively were cisplatin t1/2 alpha, 35 min, beta 8 h, carboplatin t1/2, 44 h and iproplatin t1/2, 104 h. By electrophoresis the protein bound fraction pattern (1 h) was again similar for cisplatin and carboplatin with virtually no binding to low mol. wt. proteins. After 24 h these were now involved to a high degree (40%). Iproplatin showed relatively marked binding to proteins of higher mol. wt. but no transfer with time to the low mol. wt. protein zone. A possible explanation is the need for in vivo metabolism for this compound as manifest in the rat. It is suggested that the significance of interaction with low mol. wt. proteins merits further investigation in relation to the antitumour and toxicological actions of these drugs.     

Insertion of a MalE β-Galactosidase Fusion Protein into the Envelope of Escherichia coli Disrupts Biogenesis of Outer Membrane Proteins and Processing of Inner Membrane Proteins

The synthesis of a membrane-bound MalE β-galactosidase hybrid protein, when induced by growth of Escherichia coli on maltose, leads to inhibition of cell division and eventually a reduced rate of mass increase. In addition, the relative rate of synthesis of outer membrane proteins, but not that of inner membrane proteins, was reduced by about 50%. Kinetic experiments demonstrated that this reduction coincided with the period of maximum synthesis of the hybrid protein (and another maltose-inducible protein, LamB). The accumulation of this abnormal protein in the envelope therefore appeared specifically to inhibit the synthesis, the assembly of outer membrane proteins, or both, indicating that the hybrid protein blocks some export site or causes the sequestration of some limiting factor(s) involved in the export process. Since the MalE protein is normally located in the periplasm, the results also suggest that the synthesis of periplasmic and outer membrane proteins may involve some steps in common. The reduced rate of synthesis of outer membrane proteins was also accompanied by the accumulation in the envelope of at least one outer membrane protein and at least two inner membrane proteins as higher-molecular-weight forms, indicating that processing (removal of the N-terminal signal sequence) was also disrupted by the presence of the hybrid protein. These results may indicate that the assembly of these membrane proteins is blocked at a relatively late step rather than at the level of primary recognition of some site by the signal sequence. In addition, the results suggest that some step common to the biogenesis of quite different kinds of envelope protein is blocked by the presence of the hybrid protein.     

Conformational dynamics of the molecular chaperone Hsp90 in complexes with a co-chaperone and anticancer drugs

The molecular chaperone Hsp90 is essential for the correct folding, maturation and activation of a diverse array of client proteins, including several key constituents of oncogenic processes. Hsp90 has become a focus of cancer research, since it represents a target for direct prophylaxis against multistep malignancy. Hydrogen-exchange mass spectrometry was used to study the structural and conformational changes undergone by full-length human Hsp90beta in solution upon binding of the kinase-specific co-chaperone Cdc37 and two Hsp90 ATPase inhibitors: Radicicol and the first-generation anticancer drug DMAG. Changes in hydrogen exchange pattern in the complexes in regions of Hsp90 remote to the ligand-binding site were observed indicating long-range effects. In particular, the interface between the N-terminal domain and middle domains exhibited significant differences between the apo and complexed forms. For the inhibitors, differences in the interface between the middle domain and the C-terminal domain were also observed. These data provide important insight into the structure of the biologically active form of the protein.     

Dynamic expression of ancient and novel molluscan shell genes during ecological transitions

Background  

The Mollusca constitute one of the most morphologically and ecologically diverse metazoan phyla, occupying a wide range of marine, terrestrial and freshwater habitats. The evolutionary success of the molluscs can in part be attributed to the evolvability of the external shell. Typically, the shell first forms during embryonic and larval development, changing dramatically in shape, colour and mineralogical composition as development and maturation proceeds. Major developmental transitions in shell morphology often correlate with ecological transitions (e.g. from a planktonic to benthic existence at metamorphosis). While the genes involved in molluscan biomineralisation are beginning to be identified, there is little understanding of how these are developmentally regulated, or if the same genes are operational at different stages of the mollusc's life.