Expression of the gene encoding protein A in Staphylococcus aureus and coagulase-negative staphylococci

Two shuttle vectors containing the gene for protein A (spa) from Staphylococcus aureus have been constructed to study expression of the gene in various strains of S. aureus and in the coagulase-negative species Staphylococcus epidermidis, Staphylococcus capitis, and Staphylococcus xylosus. One plasmid, pSPA15, contains the complete structural gene for protein A, which binds to the cell wall in various Staphylococcus species. The other plasmid, pSPA16, codes for a truncated protein A lacking the C-terminal part called region X. The latter is exclusively extracellular in all Staphylococcus species tested, which confirms the importance of region X for cell wall binding. The expression of the plasmid-coded protein A in various strains of S. aureus is strongly correlated to the expression of the chromosomal spa gene. The coagulase-negative species expressing plasmid-encoded protein A produce 12 to 30% of the amount coded by the chromosomal spa gene in S. aureus strains Cowan I and A676.     

Isolation of a yeast vector marked by the mutation of multiple resistance to antibiotics

The plasmid mutation AntR determining multiple resistance to antibiotics--tetracycline and cycloheximide in Saccharomyces cerevisiae was earlier obtained and genetically characterized. In this work we describe experiments on cytoduction and transformation, proving the localization of this mutation in the yeast 2 mu DNA. As a result of cotransformation of the sensitive cells carrying a double mutation in the gene LEU2 with the yeast vector marked by LEU2 and 2 mu DNA obtained from the yeast AntR mutant, the Leu+ AntR clones were selected. Though the primary co-transformans contain both plasmids in an unlinked state, we managed to get clones in which the markers AntR and LEU2 were linked. The putative recombinant molecules were cloned in Escherichia coli and then introduced into the yeast recipient cells, differing by the presence of the endogenous 2 mu DNA. Retransformation of cir0 cells results in the appearance of the clones in which LEU2 and AntR markers segregate together. Thus, the result of cotransformation and selection in vivo is that the mutation of multiple resistance was included into the yeast vector plasmid, presumably, in its 2 mu part.     

Effect of various gas atmospheres on the growth and survival of Campylobacter jejuni on beef

Pieces of fresh beef were inoculated with three strains of Campylobacter jejuni. The meat was then allocated to three treatments: (a) vacuum packaged, (b) packaged in an atmosphere of 20% CO2 + 80% N2, and (c) packaged into sterile Petri dishes in anaerobic cultivation boxes, which were filled with a gas mixture of 5% O2 + 10% CO2 + 85% N2. The packaging material in the first two treatments was PA 80/PE 100-PE 100/PA 80/PE 100. The survival of Campylobacter cells was followed at 37 degrees C, 20 degrees C and 4 degrees C for 48 h, 4 days and 25 days, respectively. At 37 degrees C the counts of two Campylobacter strains increased in each package treatment for 48 h. At 20 degrees C and at 4 degrees C the counts of the same two strains decreased by 1 to 2 log units and 0.5 to 1 log unit, respectively, during storage. The survival of the two strains was about the same in all package treatments. The third strain was the most sensitive of the strains studied. At 37 degrees C its numbers increased only in the optimal gas atmosphere; at 20 degrees C the strain was not detectable after 24 to 48 h storage and at 4 degrees C after 4 days storage. The aerobic plate counts were determined for all samples at the same time as Campylobacter counts. The high indigenous bacterial numbers of the meat samples did not appear to have a great effect on the survival or growth of campylobacters.     

Comparative anatomic studies of the vomeronasal complex and the rostral palate of various mammals

The structures of the rostral palate in regard to the vomeronasal complex of different species of mammals were studied. In all cases, we find a very interesting system of furrows which preserves a connection between the nasopalatine ducts and the preoral surroundings. For rodents, lagomorphs, Solenodon, Setifer, and Echinops, we find a special situation in this part of the palate. Here the incisors are not separated by a diastema nor the oral openings of the nasopalatine ducts are overgrown by a bipartite caudal branch of the rhinarium. The results of the anatomic studies of the vomeronasal complex and the rostral palate of the mammals investigated are discussed: First of all, some elements of the vomeronasal complex needed to be analysed in regard to structure and nomenclature, specifically the cartilago paraseptalis with its outer bar, the cartilago ductus nasopalatini and the cartilago palatina. Because of 2 criterions, the vomeronasal complex could be classified as either primitive or progressive. We find a primitive one in Didelphis, Tupaia, Solenodon, Oryctolagus, and all rodents. In contrast, the other insectivores studied and all primates show progressive structures at their vomeronasal complex. Finally, conclusions in regard to the function of the organs of Jacobson are derived from these studies. The significance of the "flehmen" mechanism for the functioning of the organs is questioned.     

Peroxisomes in sebaceous glands. IV. Aggregates of tubular peroxisomes in the mouse Meibomian gland

The occurrence of peroxisomes, their morphogenesis during the process of sebaceous transformation and their spatial relationship to the endoplasmic reticulum and lipid droplets were investigated by light and electron microscopy after visualization of the peroxidatic activity of catalase using an alkaline diaminobenzidine medium. The morphological alterations of peroxisomes display a characteristic sequence: During cellular differentiation, a remarkable proliferation of exclusively tubular, diaminobenzidine-reactive peroxisomes occurs. As maturation proceeds, an extensive elongation of tubular peroxisomes is seen. Concomitantly, they are densely packed in a regular, hexagonal arrangement and both the diameter and the catalase content gradually decreases. The most conspicuous feature of mature glandular cells are numerous highly organized aggregates of tubular, almost unstained peroxisomes with a diameter of 50 nm, arranged in a hexagonal pattern. They resemble adjacent tubular profiles of smooth endoplasmic reticulum. However, membrane continuities between these two compartments were never observed. During lethal disintegration peroxisomes subsequently decrease in number, probably by rapid sequestration within autophagolysosomes. The role of tubular peroxisomes in the biosynthesis of wax esters in the mouse Meibomian gland is discussed.     

Isolation of acid-resistant urinary trypsin inhibitors by high performance liquid chromatography and their characterization by N-terminal amino-acid sequence determination

Two crude fractions of acid-resistant trypsin inhibitors (apparent molecular masses 44 and 20 kDa, respectively) were prepared from human urine by gel permeation chromatography. From both preparations the pure inhibitors were isolated by high performance liquid chromatography (HPLC). Their N-terminal amino-acid sequences were determined and compared with those of HI-30 and HI-14 as isolated by reversible binding to either immobilized trypsin or immobilized chymotrypsin. The N-terminal amino-acid sequence of the high-molecular mass inhibitor UI-I isolated by HPLC was identical with those of HI-30 and UI-C-I isolated via immobilized trypsin or chymotrypsin, respectively. The low-molecular mass inhibitors UI-II and UI-C-II differ from HI-14 by the N-terminal extension Glu-Val-Thr-Lys-when obtained by HPLC or by the extension Thr-Lys-when obtained via immobilized chymotrypsin, respectively. The comparison of these N-termini with the amino-acid sequence of HI-30 (Ala1-...-Val16-Thr-Glu-Val-Thr-Lys-HI-14) defines the low molecular urinary trypsin inhibitors as proteolytic degradation products of the high-molecular urinary inhibitor. Proteolysis may occur at different bonds. The existing discrepancies in molecular architecture and in molecular masses of the urinary trypsin inhibitors are discussed.     

Balanced hereditary polymorphism and the mortality from cardiovascular diseases in the populations of 17 countries of Europe. I. A correlation analysis

The correlation analysis of ratios between six polymorphic genetic systems (ABO, MNSs, Rh, Hp, Gm, HLA) and mortality from ishemic heart disease, brain vascular lesions, and hypertensive disease in 17 European populations has been made. A statistically significant correlation has been established between the populational frequency of most of the 50 phenotypes and genes under study, and mortality. The qualitative structure of correlations and their quantitative expression depend on the cause of death, age and sex. The possible mechanisms of relationship between the genetic populational differences and mortality from cardiovascular diseases are discussed.     

Alcaligenes eutrophus hydrogenase genes (Hox)

Mutants of Alcaligenes eutrophus H16 lacking catalytically active soluble hydrogenase (Hos-) grew very slowly lithoautotrophically with hydrogen. Mutants devoid of particulate hydrogenase activity (Hop-) were not affected in growth with hydrogen. The use of Hos- and Hop- mutants as donors of hydrogen-oxidizing ability in crosses with plasmid-free recipients impaired in both hydrogenases (Hox-) resulted in transconjugants which had inherited the plasmid and the phenotype of the donor. This indicates that the structural genes which code for the hydrogenases reside on plasmid pHG1. The Hox function of one class of Hox- mutants could not be restored by conjugation. These mutants exhibited a pleiotropic phenotype since they were unable to grow with hydrogen and also failed to grow heterotrophically with nitrate (Hox- Nit-). Nitrate was scarcely utilized as electron acceptor or as nitrogen source. Hox- Nit- mutants did not act as recipients but could act as donors of the Hox character. Transconjugants derived from those crosses were Hox+ Nit+, indicating that the mutation which leads to the Hox- Nit- phenotype maps on the chromosome. Apparently, the product of a chromosomal gene is involved in the expression of plasmid-encoded Hox genes. We observed that the elimination of plasmid pHG1 coincided with the occurrence of multiple resistances to various antibiotics. Since Hox+ transconjugate retained the antibiotic-resistant phenotype, we conclude that this property is not directly plasmid associated.     

Characterization of the interchain and intrachain interactions between the binding sites of the free regulatory moiety of protein kinase I

The interaction between the four binding sites (two A sites and two B sites) of the regulatory subunit dimer of protein kinase I (RI2) was studied. The rate of association of c[3H]AMP to site B was slower when site A had already been occupied. Occupation of site A also retarded the rate of dissociation of c[3H]AMP from site B. This site A-B interaction was intrachain since it was observed also for a monomeric fragment of RI2. Thus, each monomer of RI2 must have one A site and one B site. Quantitative analysis of the rate constants for cAMP binding to variously liganded RI2 suggested little or no thermodynamic coupling between site A and B. This conclusion was supported by equilibrium binding data. Occupation of one A site retarded the dissociation of c[3H]AMP from the A site of the other subunit (interchain interaction). The rate kinetic constants as well as equilibrium binding data indicated a positively cooperative site A-A interaction. The interaction between cAMP and either site was enthalphy-driven (25 degrees C), the process being accompanied by a loss of entropy. The thermodynamic parameters did not support the occurrence of an abrupt conformational change at a certain level of ligandation of RI2. Half-maximal saturation of either site occurred at 1-2 nM cAMP (37 degrees C, pH 7.0, 0.15 M KCl). The concentration of RI2 did not detectably influence any binding parameters. Aging of RI2 produced a form with minimally, if at all, altered Mr, but which showed a more rapid release of c[3H]AMP bound to site B.     

Contrasting roles of tau and microtubule-associated protein 2 in the vinblastine-induced aggregation of brain tubulin

Two different proteins, tau and microtubule-associated protein 2 (MAP 2), are able to stimulate tubulin polymerization into microtubules in vitro, but it is not certain if both proteins act by the same mechanism. We have examined the effects of tau and MAP 2 on the vinblastine-induced polymerization of tubulin into spiral filaments. In the presence of tau, vinblastine induced extensive aggregation of tubulin as shown by a large increase in turbidity. The increase in turbidity was accompanied by the formation of large numbers of spirals composed of a filament 40-60 A in diameter. The rate and extent of this aggregation into spirals were dependent on the concentrations of tubulin, tau, and vinblastine. Unlike normal microtubule assembly, this type of aggregation was not inhibited by colchicine or podophyllotoxin. In contrast, MAP 2, even at high concentrations, was less effective than tau at promoting the vinblastine-induced increase in turbidity of tubulin. In fact, MAP 2 strongly inhibited the effect of tau. These results indicate that tau and MAP 2 interact differently with the tubulin molecule in the presence of vinblastine and suggest that the two proteins may play different roles in regulating or promoting microtubule assembly. Vinblastine may thus be a useful probe in analyzing the modes of interactions of tau and MAP 2 with tubulin.