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.     

Immunolocalization of hyalin in sea urchin eggs and embryos using an antihyalin-specific monoclonal antibody.

Monoclonal antibodies were raised against purified cortical secretory vesicles (CVs) from the eggs of Strongylocentrotus purpuratus. One of the monoclonal antibodies (MAb 69-10, an IgA) was shown by immunofluorescence labeling of intact and detergent-lysed CVs to be directed against a CV content antigen. Immunoblot analysis of CVs revealed that MAb 69-10 bound to a major CV polypeptide with an Mr similar to that of hyalin (i.e., 300,000). MAb 69-10 was subsequently shown to bind to purified hyalin prepared from S. purpuratus and to cross react with hyalin prepared from Lytechinus pictus. Immunogold labeling on thin sections of unfertilized S. purpuratus eggs showed that hyalin was localized to the electron-lucent portion of CVs. This result is in agreement with the labeling pattern obtained by Hylander and Summers (Dev Biol 93:368-380, 1982) using polyclonal antihyalin antibodies. In fertilized eggs and later-stage embryos, hyalin was observed to be located on the external surface of the embryo. MAb 69-10 should be useful in studies of the structure of hyalin and its function in morphogenesis.     

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.     

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.     

Neuronal responses to purinoceptor agonists in the leech central nervous system

Exracellular nucleotides like ATP and its derivatives are possible chemical messengers in vertebrate nervous systems. In invertebrate nervous systems, however, little is known about their role in neurotransmission. We have studied the reponse of identified neurones of the leech Hirudo medicinalis to the purinoceptor agonist ATP, ADP, AMP, and adenosine using conventional intracellular microelectrodes and whole-cell patch-clamp recording. Bath application of the agoinsts depolarized the different neurons, but not neuropil glial cells. The most effective responses (up to 10 mV) were observed with ATP (100 μM) or ADP (100 μM) in the noxious and touch cells. In most neurons the nonhydrolyzable ATP derivative ATP-γ-S (5 μM) induced larger depolarizations that 100 μM ATP, indicating that most of the potency of ATP is lost presumably due to its degradation by ectonucleotidases. In medial noxios cells, ATP (100 μM) induced an inward current of 1.7 ± 1.1 nA at a holding potential of ?60 mV. The ATP-induced current-voltage relationship showed an inward rectification and a reversal potential close to 0 m V. In a Na⁺-free extracellular solution, the ATP-induced inward current decreased and in a Na⁺- and Ca²⁺-free saline only a small residual current persisted. The possible P₂ purinoceptor antagonist suramin did not antagonize the ATP-induced current, but itself evoked an inward current and a conductance increase. We conclude that ATP activates nonselective cation channels in medial noxious cells of the leech with the order of potency of purinoceptor agonists ATP ≥ ADP > AMP. The results suggest that these cells express purinoceptors of the P₂ type. 1994 John Wiley & Sons, Inc.