The development of functional neuromuscular junctionsin vitro: An ultrastructural and physiological study

Neuromuscular junctions were formedin vitro between rat spinal cord explants and myotubes. At various intervals after the spinal cord explants were added to the myotube culture (7 hr to 15 days of coculture), the presence of functional neuromuscular junctions was determined by recording miniature endplate potentials (mepps) from the myotubes contacted by a few neurites. Electron microscopical studies were conducted on identified myotubes in which mepps were recorded. Mepps were already found as early as 7 hr after coculture. The fine structure of these newly formed neuromuscular junctions was simple. No synaptic specializations were observed except the presence of a small number of synaptic vesicles in the nerve. The neuromuscular junctions differentiated during the coculture period. Synaptic vesicles formed a cluster at the prejunctional membrane with a localized density in the middle. Basal lamina started to form in 4-day-old cocultures and became continuous in cocultures of 10 days or longer. Clear postjunctional foldings were observed in 15-day-old cocultures. Higher mepp frequencies were correlated with more advanced ultrastructure.     

Chloride attachment negative-ion mass spectra of sugars by combined liquid chromatography and atmospheric pressure chemical ionization mass spectrometry

A method has been developed for the rapid determination of sugars, including molecular weight measurements, using high-performance liquid chromatography coupled with negative-ion, atmospheric-pressure chemical-ionization mass spectrometry. The chromatography was carried out on a 250 × 4 mm I.D. column packed with 7 μm NH₂-silica. The mobile phase consisted of a high percentage of methanol or acetonitrile with a small amount of chloroform. During the mass spectrometry, a strong base is formed from the polar solvent molecules by corona discharge, followed by ion—molecule reactions in the chemical ionization ion source (e.g. the methoxy anion is formed from methanol). This strong base reacts with the chloroform, generating chloride ions, which in turn react with the neutral sugar molecules as they elute from the chromatograph. The chloride ion and sugar interactions yield chloride-attachment ions, which are further stabilized by successive collisions. In this method, authentic monosaccharides and some oligosaccharides show dominant quasi-molecular ions, [M + Cl]⁻, with little fragmentation, and it is particularly useful for the molecular weight determination of sugars.     

Effect of the pH of culture medium on the alkalophilicity of a species of Bacillus

The amino acid incorporation and -aminoisobutyric acid (AIB) uptake of an alkalophilic Bacillus grown at pH 8.2 (the pH 8-bacteria) were much less pH dependent (less alkalophilic) than those of the organisms grown at pH 10.0 (the pH 10-bacteria), respectively. The rate of AIB uptake of the pH 10-bacteria was almost the same as that of the pH 8-bacteria, while the rate of amino acid incorporation of the pH 10-bacteria was higher than that of the pH 8-bacteria in alkaline environments. The colloidal titration with clupein showed that the amount of negative charge on the pH 10-bacteria was greater than that of the pH 8-bacteria in alkaline environments. Considerable difference in protein composition was observed between the membranes of the pH 8-and 10-bacteria, while no difference was observed in phospholipid composition.Abbreviations AIB Amino-isobutyric acid     

Experimental Approach Reveals the Role of alx1 in the Evolution of the Echinoderm Larval Skeleton

Over the course of evolution, the acquisition of novel structures has ultimately led to wide variation in morphology among extant multicellular organisms. Thus, the origins of genetic systems for new morphological structures are a subject of great interest in evolutionary biology. The larval skeleton is a novel structure acquired in some echinoderm lineages via the activation of the adult skeletogenic machinery. Previously, VEGF signaling was suggested to have played an important role in the acquisition of the larval skeleton. In the present study, we compared expression patterns of Alx genes among echinoderm classes to further explore the factors involved in the acquisition of a larval skeleton. We found that the alx1 gene, originally described as crucial for sea urchin skeletogenesis, may have also played an essential role in the evolution of the larval skeleton. Unlike those echinoderms that have a larval skeleton, we found that alx1 of starfish was barely expressed in early larvae that have no skeleton. When alx1 overexpression was induced via injection of alx1 mRNA into starfish eggs, the expression patterns of certain genes, including those possibly involved in skeletogenesis, were altered. This suggested that a portion of the skeletogenic program was induced solely by alx1. However, we observed no obvious external phenotype or skeleton. We concluded that alx1 was necessary but not sufficient for the acquisition of the larval skeleton, which, in fact, requires several genetic events. Based on these results, we discuss how the larval expression of alx1 contributed to the acquisition of the larval skeleton in the putative ancestral lineage of echinoderms.     

A Versatile Chromatographic Procedure for Purifying PS II Reaction Center Complex from Digitonin Extracts of Spinach Thylakoids

A versatile, two-step chromatographic method using DEAE-Toyopearl(Toyo Soda, Japan) is described for purifying photosystem IIreaction center complex from digitonin extracts of spinach thylakoidmembranes. The method is very simple and brings about an approximatefour-fold increase in the specific activity, on a chlorophyllbasis, of 2,4-dichlorophenol-indophenol photoreduction with1,5-diphenylcarbazide (to about 2,000 µ electron equivalentsper mg chlorophyll per h), with an approximate 40 percent recoveryin chlorophyll. The SDS-polyacrylamide gel electrophoresis performedin the presence of 4 M urea in the analyzing gel shows fourpolypeptide bands of the photosystem II reaction center of about47, 43, 30 and 9 kilodaltons. The absorption and fluorescence properties, as well as the pigmentand chemical compositions and the above mentioned polypeptideprofile of the purified complex are essentially identical withthose of the preparations isolated by the previously describedmethod (Satoh 1982). The digitonin solubilization of thylakoid membranes destroysthe water splitting machinery, so that the purified complexshows no oxygen evolving activity, even although 0.6–0.7atoms of manganese per 50 chlorophyll molecules still remain. (Received March 19, 1985; Accepted July 19, 1985)