Radioactive labeling of proteins in cultured postimplantation mouse embryos II. Dose and time dependency

Summary The conditions for optimum incorporation of radioactive amino acids into proteins of cultured postimplantation mouse embryos were investigated under the aspect of using these proteins for two-dimensional electrophoretic separations and fluorography. The aim was to obtain highly radioactively labeled proteins under conditions as physiological as possible. Mouse embryos of Days 8, 10, and 11 of gestation were cultured in Tyrode’s solution. Incubation time and concentration of [³H (or¹⁴C)]amino acids in the culture medium were varied over a broad range. Embryos were prepared with placenta and yolk sac or without any embryonic envelopes. After culturing, the physiologic-morphologic state of the embryos was registered on the basis of several criteria. The radioactivity taken up by the total protein of each embryo was determined and calculated in disintegrations per minute per milligram protein per embryo. To approach our aim, embryos of different developmental stages had to be cultured under different conditions. A good compromise for Day-8, Day-10, and Day-11 embryos was: embryos prepared with yolk sac (opened) and placenta, 150 μCi radioactive amino acids added per milliliter medium, incubation for 4 to 5 h. For maximum labeling of proteins it is advisable to culture Day-10 embryos without embryonic envelopes under particular conditions. This work was supported by grants from the Deutsche Forschungsgemeinschaft awarded to the project K1 237/3-2 (Systematic analysis of cell proteins).     

Application of Asymmetric Alternating Voltage Pulse Series for Investigation of the Action Potential in Chara

An alternating (asymmetric) bipolar staircase voltage clamppulse series was used to investigate the action potential inChara corallina. Using this protocol, we found that the actionpotential was present in the hyperpolarized region of the current-voltagerelationship of the whole cell membrane. Effects of strong hyperpolarizingvoltage steps, during the excitation process, can thus be studied. (Received August 22, 1989; Accepted October 14, 1989)     

Enzymes Involved in Anaerobic Polyethylene Glycol Degradation by Pelobacter venetianus and Bacteroides Strain PG1

In extracts of polyethylene glycol (PEG)-grown cells of the strictly anaerobically fermenting bacterium Pelobacter venetianus, two different enzyme activities were detected, a diol dehydratase and a PEG-degrading enzyme which was characterized as a PEG acetaldehyde lyase. Both enzymes were oxygen sensitive and depended on a reductant, such as titanium citrate or sulfhydryl compounds, for optimal activity. The diol dehydratase was inhibited by various corrinoids (adenosylcobalamin, cyanocobalamin, hydroxocobalamin, and methylcobalamin) by up to 37% at a concentration of 100 μM. Changes in ionic strength and the K⁺ ion concentration had only limited effects on this enzyme activity; glycerol inhibited the enzyme by 95%. The PEG-degrading enzyme activity was stimulated by the same corrinoids by up to 80%, exhibited optimal activity in 0.75 M potassium phosphate buffer or in the presence of 4 M KCI, and was only slightly affected by glycerol. Both enzymes were located in the cytoplasmic space. Also, another PEG-degrading bacterium, Bacteroides strain PG1, contained a PEG acetaldehyde lyase activity analogous to the corresponding enzyme of P. venetianus but no diol dehydratase. Our results confirm that corrinoid-influenced PEG degradation analogous to a diol dehydratase reaction is a common strategy among several different strictly anaerobic PEG-degrading bacteria.     

Continuous microbial production ofl-leucine with cell retention

Summary Continuous production ofl-leucine was carried out withCorynebacterium glutamicum, strain ATCC 13032 starting from-ketoisocaproic acid as the precursor, glucose as the carbon source and ammonium sulphate as the nitrogen source, with biotin in a mineral medium. By means of cross-flow microfiltration or centrifugal separation for cell retention in continuous fermentation an increase in cell density was achieved and the product solution was obtained cell-free. The cells were concentrated to over 70 g cell dry mass/1. In experiments of up to 42 days, conversion rates of 85%–99% andl-leucine yields of 85%–93% were achieved. With a substrate residence time of 3.6 h, 114 mmol/1l-leucine was produced with a space-time yield of 97 g/1 per day. A scale-up of the fermentation volume from 4 to 1001 provided comparable results.     

An env gene derived from a primary human immunodeficiency virus type 1 isolate confers high in vivo replicative capacity to a chimeric simian/human immunodeficiency virus in rhesus monkeys.

To explore the roles played by specific human immunodeficiency virus type 1 (HIV-1) genes in determining the in vivo replicative capacity of AIDS viruses, we have examined the replication kinetics and virus-specific immune responses in rhesus monkeys following infection with two chimeric simian/human immunodeficiency viruses (SHIVs). These viruses were composed of simian immunodeficiency virus SIVmac239 expressing HIV-1 env and the associated auxiliary HIV-1 genes tat, vpu, and rep. Virus replication was assessed during primary infection of rhesus monkeys by measuring plasma SIVmac p27 levels and by quantifying virus replication in lymph nodes using in situ hybridization. SHIV-HXBc2, which expresses the HIV-1 env of a T-cell-tropic, laboratory-adapted strain of HIV-1 (HXBc2), replicated well in rhesus monkey peripheral blood leukocytes (PBL) in vitro but replicated only to low levels when inoculated in rhesus monkeys. In contrast, SHIV-89.6 was constructed with the HIV-1 env gene of a T-cell- and macrophage-tropic clone of a patient isolate of HIV-1 (89.6). This virus replicated to a lower level in monkey PBL in vitro but replicated to a higher degree in monkeys during primary infection. Moreover, monkeys infected with SHIV-89.6 developed an inversion in the PBL CD4/CD8 ratio coincident with the clearance of primary viremia. The differences in the in vivo consequences of infection by these two SHIVs could not be explained by differences in the immune responses elicited by these viruses, since infected animals had comparable type-specific neutralizing antibody titers, proliferative responses to recombinant HIV-1 gp120, and virus-specific cytolytic effector T-cell responses. With the demonstration that a chimeric SHIV can replicate to high levels during primary infection in rhesus monkeys, this model can now be used to define genetic determinants of HIV-1 pathogenicity.     

HSP25 in isolated perfused rat hearts: Localization and response to hyperthermia

Recent investigations concentrate on the correlation between the myocardial expression of the inducible 70-kDa heat shock protein (HSP70i) by different stress conditions and its possible protective effects. Only few studies have focused on the involvement of small heat shock proteins in this process. We analyzed the location of the small heat shock protein HSP25 in isolated cardiomyocytes as well as its location and induction in isolated perfused hearts of rats. By immunofluorescence microscopy HSP25 was found to colocalize with actin in the I-band of myofibrils in cardiomyocytes of isolated perfused hearts as well as in isolated neonatal and adult cardiomyocytes. Hyperthermic perfusion of isolated hearts for 45 min resulted in modulation of different parameters of heart function and in induction of HSP25 and HSP70i. Temperatures higher than 43°C (44–46°C) were lethal with respect to the contractile function of the hearts. Compared to control hearts perfused at 37°C, significant increases during hyperthermic perfusion at 42°C and 43°C were obtained for heart rate, contraction velocity and relaxation velocity. In response to hyperthermia at 43°C and after subsequent normothermic perfusion for 135 min at 37°C, left ventricular pressure, contraction velocity and relaxation velocity remained significantly elevated. However, heart rate returned to control values immediately after the period of heat treatment. HSP25 is constitutively expressed even in normothermic perfused hearts as shown by Western blotting. Hyperthermia increased the content of HSP25 only in the left ventricular tissue. In contrast, HSP70i was strongly induced in all analyzed parts of the myocardium (left ventricle, right ventricle, septum). Our findings suggest a differential regulation of HSP25 and HSP70i expression in response to hyperthermia in isolated perfused hearts. The constitutively expressed HSP25 seems to be located adjacent to the myofibrils which implies a specific role of this protein even under unstressed conditions for the contractile function of the myocardium.     

Skeletal formation in the modern but ultraconservative chaetetid spongeSpirastrella (Acanthochaetetes) wellsi (demospongiae,porifera)

Summary The modern hadromerid coralline spongeSpirastrella (Acanthochaetetes) wellsi exhibits a unique secondary high-Mg calcite (>19 mol % MgCO₃) basal skeleton. The basal skeleton is constructed of bundles of elongated crystals more or less tangentially orientated. The initial formation of these crystals is controlled by soluble highly acidic aspartic and glutamic-rich (40%) macromolecules. The skeletal mineralization occurs in four different loci: in the top of the calicles, at the tabulae, on collagenous anchor fibres, and within closed spaces between the tabulae. The clicle walls are formed on the uppermost top of the basal skeleton as a continuous process. Based on long term stainings with Ca²⁺-chelating fluorochroms (calcein, chlorotetracyclines) the growth rate of this sponge is extremely low with ca. 50–100μm/a. The skeletal formation takes places outside the sponge, within a narrow zone (300–500 nm) between the basopinacoderm and the mature basal skeleton. The sponge produces thread-like folded templates (‘spaghetti fibres’) of 0,5–2 μm size, the shape controlling insoluble organic matrix. These templates become mineralized in a first step as MgCO₃, then are stretched. A soluble organic matrix is also secreted, and remains are included inside the mineralized skeleton. This organic matrix consists of in a complex mixture containing small very acidic proteins (5, 13, 31 KD; 40% Asp and Glu and therefore most probably Ca²⁺-binding) and high molecular weight glycoproteins among several other organic compounds. The mature crystals are high-Mg calcites. During calcification large cells with large reserve granules (LCG) are always present in a tight connection with the basopinacoderm. These cells form also the collagenous anchor fibres. Primary tabulae are formed by a non-collagenous organic sheet. Calcification happens only when LCG cells are enriched on the organic sheet. Randomly oriented high-Mg calcite crystals are growing on the collagenous anchor fibres. The same type of the mineralization is observed within the spaces of the tabulae. This particular case of mineralization is controlled by decaying sponge tissue (ammonification). The δ¹³C values are in equilibrium with the ambient sea water and vary between +3.2 and +2.8 ‰. The mode of mineralization of the basal skeleton can be described as biologically induced resp. matrix mediated.     

Interrelationship of renal vascular development and nephrogenesis

Within the cortex region of the neonatal rabbit kidney the developing microvasculature was investigated by means of two endothelium-detecting antibodies (EnPo 1 and EC1). Rows of antibody-labelled cells were found within tissue regions that had previously been described as avascular. We conclude that these vessel-like structures detected by EnPo 1 and EC1 are capillary precursors without lumina. Furthermore, beneath the fibrous capsule within the morphologically homogeneous mesenchyme two cell populations can be discriminated by use of differential antigen expression. The EnPo 1 antigen, which is abundant on endothelial cells and podocytes at different developmental stages, was detected on a subpopulation of mesenchymal cells. These cells were exclusively detected surrounding the tip of the collecting duct ampulla. Due to the unique specificity of EC1 and EnPo 1 the process of microvascular development can be readily followed on serial optical sections gained by laser scan microscopy. (1) Adjacent to EnPo 1-positive mesenchymal cell islets vessel-like structures are found that are in contact with the differentiated vasculature. (2) The renal vesicle is enclosed by a network of vessel-like structures establishing contact with differentiated vessels. (3) No guidance of invading capillary sprouts toward the developing glomerulus and nephron is required, since vascular elements already accompany the earliest detectable nephron stage.