Purification and properties of ATPase from an alkalophilic Bacillus

ATPase was purified from an alkalophilic Bacillus. The enzyme has a molecular weight of 410,000 and consists of five types of subunits of molecular weights of 60,000 (α), 58,000 (β), 34,000 (γ), 14,000 (δ), and 11,000 (?). The subunit structure is suggested to be α₃β₃γδ?. The enzyme is activated by Mg²⁺ and Ca²⁺. The pH optima of the enzyme with 0.1 and 2.0 mm Mg²⁺ are 9 and 6, and those with 1 and 10 mm Ca²⁺ are 8–9 and 7, respectively. Ca²⁺-ATPase hydrolyzes only ATP, whereas Mg²⁺-ATPase hydrolyzes GTP and, to a lesser extent, ATP. The values of V and K_m of the enzyme with ATP in the presence of 10 mm Ca²⁺ or 0.6 mm Mg²⁺ at pH 7.2 are 17 or 0.5 units/mg protein and 1.2 or 0.3 mm, respectively. The enzyme with Mg²⁺ is appreciably activated by HCO^?₃. Relationship of the ATPase to the active transport system in the bacterium is suggested.     

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.     

Production and characterization of functional domains of human fibronectin expressed in Escherichia coli.

An efficient expression system was constructed in Escherichia coli that produced a 33-kDa fragment, C-274, of human fibronectin with a strong cell-adhesive activity. The entire sequence of the heparin-binding domain with 271 amino acids, H-271, was also expressed. Deletion analysis of the type III repeats showed that the heparin-binding site was at type III-13. The cell-adhesive activity of a fusion protein, CH-271, containing the cell- and the heparin-binding domains was twice that of C-274 when BHK but not B16-F10 melanoma cells were tested; H-271 alone was inactive. Recombinant proteins containing the CS1 sequence of the IIICS region were more active than C-274 and CH-271 with B16-F10. However, H-296, which contained both H-271 and CS1, was almost inactive with BHK. CH-296, which contained CS1 at the C-terminus of CH-271, was more active with B16-F10 than H-296 and C-CS1, which was produced by the deletion of H-271 from CH-296. Thus, the cell-binding domain was active with both kinds of cells. The heparin-binding domain promoted the adhesion of both kinds of cells only when linked to the cell-binding domain or CS1. CS1 was specific for the adhesion of B16-F10 but was not essential.     

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.     

Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin.

A physiologically diverse range of Gram-positive and Gram-negative bacteria was found to be susceptible to inhibition and inactivation by lactoferricin B, a peptide produced by gastric pepsin digestion of bovine lactoferrin. The list of susceptible organisms includes Escherichia coli, Salmonella enteritidis, Klebsiella pneumoniae, Proteus vulgaris, Yersinia enterocolitica, Pseudomonas aeruginosa, Campylobacter jejuni, Staphylococcus aureus, Streptococcus mutans, Corynebacterium diphtheriae, Listeria monocytogenes and Clostridium perfringens. Concentrations of lactoferricin B required to cause complete inhibition of growth varied within the range of 0.3 to 150 micrograms/ml, depending on the strain and the culture medium used. The peptide showed activity against E. coli O111 over the range of pH 5.5 to 7.5 and was most effective under slightly alkaline conditions. Its antibacterial effectiveness was reduced in the presence of Na+, K+, Mg2+ or Ca2+ ions, or in the presence of various buffer salts. Lactoferricin B was lethal, causing a rapid loss of colony-forming capability in most of the species tested. Pseudomonas fluorescens, Enterococcus faecalis and Bifidobacterium bifidum strains were highly resistant to this peptide.     

Construction and characterization of a fusion protein with epidermal growth factor and the cell-binding domain of fibronectin.

An efficient expression system was constructed for C-EGF, a fusion protein made of a fragment of the cell-binding domain of human fibronectin (FN) bound with epidermal growth factor (EGF). C-EGF was produced in Escherichia coli HB101 cells carrying the recombinant plasmid pCE102 as inclusion bodies, which were solubilized and refolded after purification. C-EGF had both cell-adhesive and EGF activities, so it might be more effective than EGF in therapeutic applications. This fusion system would be useful for the construction of a recombinant drug delivery system for cells that have fibronectin receptors (integrins).     

Quantitative analysis of the metabolism in the isolated pupal abdomens of the silkworm, Bombyx mori, infected with nuclear polyhedrosis virus

The rate of oxygen uptake and the amount of several main cell constituents were determined in NPV-infected and uninfected isolated pupal abdomens of the silkworm, Bombyx mori. The oxygen uptake in uninfected isolated abdomens increases for the first 2 days due to the effect of water injection, but thereafter it remains almost unchanged at a relatively low level. When isolated abdomens are infected with an NPV, the oxygen uptake increases markedly from 3 days postinfection onward, being about three times that of uninfected isolated abdomens at late stages of infection. The amounts of cell constituents analyzed in the present study change little in uninfected isolated abdomens throughout the experiment. Infection of NPV leads to a marked accumulation of both DNA and RNA, and a loss of glycogen. The amount of protein, lipid, and sugar are scarcely affected by NPV infection. These results indicate that the activity of metabolism in uninfected isolated abdomens is not only low but also stable, and that infection of NPV results in an activation of host cell metabolism. It is considered that such isolated pupal abdomens provide an excellent opportunity to analyze both the process of NPV replication and the alteration of host cell metabolism resulting from NPV infection.