Improved production and stability ofE. coli recombinants expressing transketolase for large scale biotransformation

Summary TheE.coli tkt gene has been subcloned into high copy number vectors. In fed batch fermentations up to 4gL^–1 of soluble intracellular transketolase was produced representing 43% of the total cell protein. Increased plasmid stability during fed-batch fermentations was obtained by using kanamycin resistant pBGS vectors rather than the ampicillin resistant pUC vectors. Plasmid stability was maintained throughout growth in a complex medium without any selective pressure by incorporating thecer region fromColE1 into the expression construct.     

Amphiphysin II (SH3P9; BIN1), a Member of the Amphiphysin/Rvs Family, Is Concentrated in the Cortical Cytomatrix of Axon Initial Segments and Nodes of Ranvier in Brain and around T Tubules in Skeletal Muscle

Amphiphysin (amphiphysin I), a dominant autoantigen in paraneoplastic Stiff-man syndrome, is a neuronal protein highly concentrated in nerve terminals, where it has a putative role in endocytosis. The yeast homologue of amphiphysin, Rvs167, has pleiotropic functions, including a role in endocytosis and in actin dynamics, suggesting that amphiphysin may also be implicated in the function of the presynaptic actin cytoskeleton. We report here the characterization of a second mammalian amphiphysin gene, amphiphysin II (SH3P9; BIN1), which encodes products primarily expressed in skeletal muscle and brain, as differentially spliced isoforms. In skeletal muscle, amphiphysin II is concentrated around T tubules, while in brain it is concentrated in the cytomatrix beneath the plasmamembrane of axon initial segments and nodes of Ranvier. In both these locations, amphiphysin II is colocalized with splice variants of ankyrin3 (ankyrin_G), a component of the actin cytomatrix. In the same regions, the presence of clathrin has been reported. These findings support the hypothesis that, even in mammalian cells, amphiphysin/Rvs family members have a role both in endocytosis and in actin function and suggest that distinct amphiphysin isoforms contribute to define distinct domains of the cortical cytoplasm. Since amphiphysin II (BIN1) was reported to interact with Myc, it may also be implicated in a signaling pathway linking the cortical cytoplasm to nuclear function.     

Characterization of Myelin Basic Protein Charge Microheterogeneity in Developing Mouse Brain and in the Transgenic Shiverer Mutant

Abstract: Myelin basic protein (MBP) is a highly heterogeneous family of membrane proteins consisting of several isoforms resulting from alternative splicing and charge isomers arising from posttranslational modifications. Although well characterized in the bovine and human species, those in the mouse are not. With the availability of a number of transgenic and knockout mice, the need to understand the chemical nature of the MBPs has become very important. To isolate and characterize the MBP species in murine brain, two methods were adapted for use with the small amounts of MBP available from mice. The first was a scaled-down version of the preparative CM-52 chromatographic system commonly used to isolate MBP charge isomers; the second was an alkaline-urea slab gel technique that required five times less material than the conventional tube gel system and, from these gels, western blots were readily obtained. Murine MBP was resolved into two populations of charge isomers: the 18.5- and 14-kDa isoforms. Isolation and characterization of these charge isomers or components permitted us to assign possible posttranslational modifications to some of them. Component 1 (C-1), the most cationic isomer, had a molecular weight of 14,140.38 ± 0.79. C-2 consisted of two 14-kDa species, 14,136.37 ± 0.74 and 14,204.45 ± 0.70. Two variants, 14,215.57 ± 0.94 and 18,413.57 ± 0.76, constituted C-3. C-4, C-5, and C-8 (the least cationic isomer) each consisted of both 14- and 18.5-kDa isoforms. During myelinogenesis, the 18.5-kDa isoform appeared first (day 4); the 14-kDa isoform appeared at day 16 and subsequently became the dominant isoform. The transgenic shiverer mutant synthesized mainly the 18.5-kDa isoform, but none of the 14-kDa isoform, similar to the 4-day-old mouse. We concluded that the trangenic shiverer was able to initiate myelinogenesis with the 18.5-kDa isoform, but was unable to complete myelinogenesis because of the absence of the 14-kDa isoform.     

Comparison of Type 2 Inositol 1,4,5-Trisphosphate Receptor Distribution and Subcellular Ca2+ Release Sites that Support Ca2+ Waves in Cultured Astrocytes

Abstract: We have examined the mechanisms that underlie Ca²⁺ wave propagation in cultured cortical astrocytes. Norepinephrine evoked Ca²⁺ waves in astrocytes that began at discrete initiation loci and propagated throughout the cell by regenerative amplification at a number of cellular sites, as shown by very high Ca²⁺ release rates at these regions. We have hypothesized previously that domains displaying elevated Ca²⁺ release kinetics in astrocytes may correspond to sites of high inositol 1,4,5-trisphosphate receptor (InsP₃R) density. To examine this possibility, we compared the distribution pattern of endoplasmic reticulum (ER) and InsP₃Rs with Ca²⁺ release kinetics in subcellular regions during propagation of norepinephrine-evoked waves. 3,3'-Dihexyloxacarbocyanine iodide staining revealed that the ER in astrocytes exists as a meshwork of membranes extending throughout the cells, including fine processes. A specific antibody directed against type 2 InsP₃Rs (InsP₃R2) detected a 260-kDa band in western blotting of astrocyte membranes. Immunocytochemistry using this antibody stained the entire ER system in a punctate, variegated manner. When Ca²⁺ responses and InsP₃R2 immunofluorescence were compared in the same cell, domains of elevated Ca²⁺ response kinetics (high amplitude and rapid rate of rise) showed significant positive correlation with high local intensity of InsP₃R2 staining. It appears, therefore, that specializations in the ER responsible for discrete local Ca²⁺ release sites that support regenerative wave propagation include increased levels of InsP₃R2 expression.     

Temperature preference in two populations of juvenile coho salmon,oncorhynchus kisutch

Synopsis Two groups of coho salmon,Oncorhynchus kisutch, were raised under identical regimes to test the hypothesis that the group from a stream with lower and less variable temperatures would have a lower and less variable preferred temperature than would the group from a stream with warmer and more variable temperatures. The preferred (modal) temperatures in an electronic shuttlebox of coho salmon young from a relatively cool, groundwater-fed stream were slightly lower and less variable than those of young from a warmer and more heterothermal stream (mean = 9.6° C, range: 6–16° C vs. mean = 11.6° C, range: 7–21° C). However, there was a great deal of variation within and among individual fish. While some genetic variation in thermal preference may exist, the species seems best characterized as tolerant of relatively large temperature fluctuations.     

Development of cerebellar astroglia: Transitions in form and cytoskeletal content

The forms, disposition, and cytoskeletal contents of astroglia in immature mouse cerebellum were studied by immunocytochemical staining with antisera against two intermediate filament proteins, vimentin (Vim) (58,000 daltons) and glial filament protein (GF) (51,000 daltons). From embryonic (E) Day 15 to postnatal (P) Day 2, Vim is expressed in cells throughout the cerebellar anlage, including radial glia and Bergmann fibers, cells with amorphous shapes and 2–3 processes, and thick longitudinal elements oriented parallel to axons within axon tracts. GF is not expressed during the first few postnatal days, but by P7, there is a dramatic increase in GF-positive astrocyte-like cells in the putative white matter that are more densely stained and more crowded than at any other age. Between P7 and P14 all astrocytes throughout the cerebellum express both Vim and GF. From P21 on, Vim expression is progressively rarer in all astrocytes except for Bergmann fibers, and GF-positive astrocytes become less numerous. These findings raise two issues: (a) the lineage and relationships of cells expressing Vim and GF; (b) Since GF-positive cells appear as axon ingrowth ceases, axons must grow in a terrain comprised of glial cells that have a different cytoskeletal composition (vimentin), reflecting a less differentiated state, than mature astrocytes or than the GF-rich astrocytes that proliferate after injury in adult CNS.     

Localization of glyoxylic acid-induced histofluorescence in surgically isolated medial basal hypothalamus of the rat

Summary Examination of glyoxylic acid-induced catecholamine histofluorescence in the hypothalamic median eminence of adult male rats revealed a linear pattern of fine varicosities coursing through the ependymal and fibrous zones, suggestive of juxtaposition to tanycytes. In order to determine the origin of these terminals, adult rats were subjected to complete isolation of the medial basal hypothalamus, using a small Halasz-Pupp knife. As rapidly as 24h after this deafferentation degenerative axon profiles were observed dorsal, as well as anterior and lateral, to the knife track. Occasionally at three days postoperatively, and routinely by seven days after surgery, fine-sized new fibres were seen passing through the knife wound. The linear profiles of varicosities observed in the normal median eminence remained traceable in the experimental preparations; the site of origin for these terminals therefore appears to be neurons of the arcuate (A12) and rostral periventricular (A14) regions. The results also indicate that fibres innervating the isolated area are capable of morphologically demonstrable new growth. The observations bear functional implications in assessing endocrine regulation following MBH isolation of the type used in this study.This study was supported by USPHS Postdoctoral Fellowship 5-F₂2-HD00630 (CT) and USPHS Grant NS-11642 (JRS). The authors wish to express their appreciation to Yvonne Cheung and Patricia Walker for technical assistance     

Inhibition of thymidine uptake in asynchronous HeLa cells by nitrobenzylthioinosine

Nitrobenzylthioinosine (NBMPR), an inhibitor of nucleoside transport by human erythrocytes, was found to be a potent inhibitor of thymidine uptake by asynchronous monolayer cultures of HeLa cells. Rates of thymidine uptake by the cultures at 20 °C were constant between 10 and 40 sec after thymidine addition and were assayed during this interval; TTP was the principal metabolite of thymidine and the thymidine phosphates accumulated at constant rates which extrapolated through time zero. The lack of an effect of NBMPR on thymidine kinase activity, or on the relative proportions of thymidine metabolites in cell extracts, indicated that NBMPR inhibited thymidine transport. When mediated entry (transport) was eliminated by 2 μM NBMPR, a significant diffusional component of thymidine entry was apparent. The mediated component of thymidine uptake exhibited Michaelis-Menten kinetics and apparent K_m and V_max values of 0.5 μM and 10–21 pmoles/min/10⁶ cells were obtained. When NBMPR-treated cells were transferred to NBMPR-free medium, inhibition of thymidine uptake persisted, suggesting that NBMPR was firmly bound to the transport inhibitory sites.     

Sexual preferences of male hamsters (Mesocricetus auratus) for conspecifics in different endocrine conditions

The behavioral responses of sexually experienced male hamsters toward a pair of anesthetized conspecifics were investigated. Males spent significantly more time licking, sniffing, and mounting neonatally and adult castrated males than intact males. Adult castrated males receiving oil injections were preferred over castrates receiving exogenous testosterone propionate (TP). Ovariectomized females were preferred over intact males, adult castrated males, or spayed females receiving exogenous TP. It was concluded that the absence of an androgen-dependent factor(s) renders an animal more sexually attractive.