Expression of integrin subunits αv and β3 in acute lung inflammation

Integrin subunits v and 3 form a dimer, v3, which is expressed on normal neutrophils and endothelium. We investigated the expression of integrin subunits v and 3 in acute lung inflammation in Sprague-Dawley rats (n=5 each) following intratracheal challenge with Escherichia coli or Streptococcus pneumoniae, which induce neutrophil recruitment through different mechanisms. Control rats (n=5) were given endotoxin-free saline. Both bacterial challenges induced similar levels of recruitment of neutrophils in lungs. Western blots showed lower expression of integrin subunits v and 3 in lungs challenged with E. coli compared to those given S. pneumoniae. Immunohistochemistry and immunogold electron microscopy localized both integrin subunits in neutrophils and endothelium in the control and treated rat lungs. Quantitative immunohistochemistry showed that E. coli-challenged rat lungs contained a lower percentage of neutrophils expressing integrin subunits v and 3 compared to those challenged with S. pneumoniae (P<0.05). We conclude that E. coli infection decreased the percentage of neutrophils expressing integrin subunits v and 3 compared to S. pneumoniae infection. These data lay the foundation for further characterization of these integrin subunits in neutrophil migration specifically in S. pneumoniae infection that utilizes molecules other than 2 integrins for neutrophil recruitment.     

Evolutionary relationship between Enterobacteriaceae: comparison of the ATP synthases (F1F0) of Escherichia coli and Klebsiella pneumoniae

The ATP synthase complex of Klebsiella pneumoniae (KF₁F₀) has been purified and characterized. SDS-gel electrophoresis of the purified F₁F₀ complexes revealed an identical subunit pattern for E. coli (EF₁F₀) and K. pneumoniae. Antibodies raised against EF₁ complex and purified EF₀ subunits recognized the corresponding polypeptides of EF₁F₀ and KF₁F₀ in immunoblot analysis. Protease digestion of the individual subunits generated an identical cleavage pattern for subunits , , , , a, and c of both enzymes. Only for subunit different cleavage products were obtained. The isolated subunit c of both organisms showed only a slight deviation in the amino acid composition. These data suggest that extensive homologies exist in primary and secondary structure of both ATP synthase complexes reflecting a close phylogenetic relationship between the two enterobacteric tribes.Abbreviations ACMA 9-amino-6-chloro-2-methoxyacridine - DCCD N,N-dicyclohexylcarbodiimide - FITC fluorescein isothiocyanate - SDS sodium dodecyl sulfate - TTFB 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole     

Comparative amino acid sequence analysis of Thermotoga maritima β-glucosidase (BglA) deduced from the nucleotide sequence of the gene indicates distant relationship between β-glucosidases of the BGA family and other families of β-1,4-glycosyl hydrolases

The primary structure of the bglA gene region encoding a -glucosidase of Thermotoga maritima strain MSB8 was determined. The bglA gene has the potential to code for a polypeptide of 446 amino acids with a predicted molecular mass of 51545 Da. The T, maritima -glucosidase (BglA) was overexpressed in E. coli at a level comprising approximately 15–20% of soluble cellular protein. Based on its amino acid sequence, as deduced from the nucleotide sequence of the gene, BglA can be classified as a broad-specificity -glucosidase and as a member of the -glucosidase family BGA, in agreement with the results of enzymatic characterization of the recombinant protein. Comparative sequence analysis revealed distant amino acid sequence similarities between BGA family -glucosidases, a -xylosidase, -1,4-glycanases of the enzyme family F (mostly xylanases), and other families of -1,4-glycosyl hydrolases. This result indicates that BGA -glucosidases may comprise one enzyme family within a large enzyme order of retaining -glycosyl hydrolases, and that the members of these enzyme groups may be inter-related at the level of active site architecture and perhaps even on the level of overall three-dimensional fold.     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

Assessment of survival during starvation ofEscherichia coli andKlebsiella pneumoniae in artificial urine: analysis of the kinetics of colony formation

A kinetic model of colony formation was proposed by Hattori, based on a count of the colonies that appear on a plate in successive short intervals of time. In this model, three parameters (,t _r and N) are defined, which reflect the ability of a bacterium to yield colonies and allow us to described the dynamics of bacterial populations in soil and ofE. coli at different growth phases. In this paper we report a reparametrization of the kinetic model of colony formation, with the aim of facilitating more accurate calculation of andt _r. Moreover, we observed that during the starvation ofE. coli andK. pneumoniae in urine, can be used to assess survival, since this parameter clearly decreases during starvation. Retardation time values (t _r) were similar inE. coli andK. pneumoniae throughout the starvation experimental period.     

Oxygen isotope fractionation during respiration for different temperatures ofT. utilis andE. coli K12

Summary The temperature dependence of the oxygen isotope fractionation factor during respiration has been examined for two different microorganisms, namelyTorulopsis utilis andEscherichia coli K12 representing a yeast and a bacterium, respectively. The investigation covered a temperature range of 18° C, that is from 16° C to 34° C forT. utilis and from 19° C to 37° C forE. coli K12. Within this temperature range the fractionation factor ofT. utilis increases by 0.18; an insignificant change ( _{10° C} = 0.063;r = 0.067), whereas withE. coli K 12 an increase of 1.12; has been observed ( _{10° C} = 0.6;r = 0.55).     

Porosity and available water of temporarily waterlogged soils in a Quercus robur (L.) declining stand

Pedunculate oak (Quercus robur L.) is particularly sensitive to decline in clayey soils presenting a high-perched temporary water table. These soils induce two successive constraints in one-year cycle: water excess (and hypoxy) in winter and early spring, and water shortage in summer (water stress being more restrictive to oak). We determined the porosity and water properties of temporarily waterlogged clayey soils supporting forest stands of decliningQuercus robur trees in a 101yr-old oak stand in Belgium (50°06N, 4°16E). Roots unevenly colonized the soil down to 1.6m: fine roots (diameter<5mm) were mostly distributed on the surface horizons (0–0.3 m) and around 1.3m deep, despite dense clayey horizons appearing at 0.35m depth. Clay content below this depth reached 46–51. Illite and vermiculite were the dominant clay minerals. The clayey horizons exhibited marked shrink–swell properties: bulk density at 30kPa increased from 1.41 to 1.88gcm^–3 from the surface to 2m depth. There was also evidence of hypoxic conditions caused by water saturation of pore space in the rooting zone from October to mid-April. Extractable water (EW), calculated between moisture tensions of 5 and 1600kPa was 152.8mm. The level of perched temporary water table strongly depended on the seasonal rhythm of water uptake by trees and on the shrink–swell behaviour of soil.     

Variability of bacterial gene-directed enzyme production in human genetically deficient cells

Summary Human -galactosidase-deficient skin fibroblasts from a patient with generalized gangliosidosis (GM₁-gangliosidosis type I) were treated with phage plac DNA, coding for Escherichia coli -galactosidase (-D-galactoside galactohydrolase, EC 3.2.1.23). New -galactosidase activity detected in cell extracts of phage DNA-treated GM₁-gangliosidosis fibroblasts continued to vary considerably from one experiment to another. It behaved like the E. coli z-gene product upon immunochemical and physicochemical investigation. In some experiments the antigenic behavior of resultant -galactosidase activity in plac DNA-treated cells resembled that of mutant E. coli -galactosidase. Among the factors and variables that may be responsible for the variation in the results obtained here and elsewhere, low physical binding between prokaryotic mRNA sequences and fibroblast ribosomal RNA could play a part connected with effective translation. This hypothesis is discussed under the aspect of a comparison of the ribosomal binding site of lac z mRNA with the 3-terminus of the eukaryotic 18s ribosomal RNA, which shows limited possibilities for base-pairing interactions.More extensive possibilities for forming Watson-Crick base pairs between their initiation site and the eukaryotic ribosomal binding site exist for other prokaryotic messengers, such as those of Q-replicase, f 1-coat protein, or UDPG-4-epimerase.     

Polymorphism of the HLA DR1 haplotype in the Israeli population investigated at the serological,cellular, and genomic levels

In the present report, we used serological, cellular, and restriction fragment length polymorphism (RFLP) to investigate the DR1 haplotype in the Israeli population. We describe an Israeli homozygous typing cell (HTC), HLA-DwLVA, which defines a new lymphocyte-activating determinant associated with Bw65, DR1 and distinct from Dwl. The parents of this donor, non-Ashkenazi Algerian Jews, are first cousins and share HLA-Cw8, Bw65, BfS, DR1, DQw1, DPw4. No specificity could be assigned to HLA-DwLVA using the 91 Ninth Workshop HTCs. Two families and forty unrelated DR1 individuals were studied with DwLVA and a panel of DR1/Dw1 HTCs. HLA-DwLVA showed segregation as a single determinant within families. This new specificity was present in 24 out of 40 (60%) unrelated DR1 individuals, indicating that in the Israeli population DwLVA is the main lymphocyte-defined determinant associated with the serologically defined DRI specificity, in contrast to non-Jewish Caucasoids where DR1 is significantly associated with Dw1. The vast majority of DwLVA-positive carriers were also Bw65 carriers, indicating that Bw65, DR1, DwLVA may represent a typical allele combination in the Israeli population. The RFLP analysis established the correlation of certain RFLPs with Dw1 and DwLVA. In addition, we describe a cluster of RFLPs that may correspond to a new Dw subtype associated with DR1, for which no serological and cellular reagents have been described so far.     

Uptake of bacteriophage λ DNA in Escherichia coli by electroporation: An alternative to in vitro packaging

Summary By using a high field strength DC pulse of 15 kV/cm and a pulse duration of 5 ms for the transfection of E. coli by bacteriophage DNA, we obtained efficiencies of 1.1 × 10⁶ (pfu/g bacteriophage , DNA). This represents a 100-fold improvement over the traditional CaCl₂/heat shock method and is a viable alternative to the more costly in vitro packaging of recombinant bacteriophage DNA for the production of cDNA and genomic libraries.     

Host genes involved in the replication of single-stranded DNA phage ?K

Summary Using various replication mutants of E. coli, the host genes that participate in the replication of some K12-specific single-stranded DNA phages have been determined. Functional products of dnaE,-F,-G and -Z genes are required for the multiplication of K, whereas dnaA,-B,-C(D), H,-I and -P are dispensable for viral replication. In contrast with polB, recA, B, C, or xth functions, host rep activity is essential for K. At the restrictive temperature, the yield of K was markedly reduced in the ligts7 mutant and partially decreased in a polA ^ts strain. The phage K is thus less dependent on the host cells than X174 and A which require additionally the dnaB,-C(D) and -H functions. Replication of phage St-1 depends on dnaG and -Z gene products, but not on dnaP function. Although not much affected in polA ^ts host, growth of St-1 was significantly diminished in dnaF or ligts7 mutants.     

Dependence of transfection efficiency of calcium treated Escherichia coli cells on bacterial genotype and form of lambda DNA

Summary The transfecting activity of linear DNA is 100 times higher in calcium treated E. coli K 12 (ⁱ⁴³⁴) than in non-lysogenic strains: the levels of transfection are 1–2.10⁷ and 1–2.10⁵ infective centers per 1 g of DNA, respectively. The high efficiency of lysogenic cells transfection is not due to the spontaneously liberated helper phage. Evidently, it is called forth by transfecting DNA-prophage recombination or/and by inhibition of nuclease activity in lysogenic cells. Both ring forms DNA (supercoiled and open circles) show very low infectivity, if any, in calcinated cells.     

Large scale preparation of PA-oligosaccharides from glycoproteins using an improved extraction method

We have developed a new method for the large scale preparation of pyridylaminated (PA-) oligosaccharides from glycoproteins. Phenol/chloroform extration was adapted for the removal of protein and excess 2-aminopyridine, improving the efficiency of preparation. From a 2.5 g sample of human apo-transferrin, 25–30 mol of agalacto biantennary PA-oligosaccharide could be obtained. By increasing the concentration of PA-oligosaccharide substrate, we were able to detect a very low level ofN-acetylglucosaminlytransferase IV activity in CHO cell extracts.Abbreviations PA 2-aminopyridine - SDS sodium dodecyl sulfate - GlcNAc N-acetylglucosamine - GnT N-acetylglucosaminyltransferase - Gn,Gn-bi-PA GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine - Gn,Gn,Gn-tri-PA GlcNAc1-2(GlcNAc1-4)Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine - Gn,Gn,Gn-trí-PA GlcNAc1-2Man1-3({GlcNAc1-2(GlcNAc1-6)Man1-6})Man1-4GlcNac1-4GlcNAc-2-aminopyridine - Gn,(Gn),Gn-bi-PA GlcNAc1-2Man1-3(GlcNAc1-4)(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine     

Secretion of a 107 K dalton polypeptide into the medium from a haemolytic E. coli K12 strain

Summary Certain E. coli K12 strains are able to secrete a plasmid encoded 107 K protein into the culture medium. During exponential growth of the cells this protein represents approximately 1% of total cell protein.The presence of the 107 K polypeptide was demonstrated through the fortuitous use of strain MC4100. This gave a largely protein-free culture supernatant, presumably due to minimal lysis of whole cells. Pulse-labelling experiments showed that the secretion of the 107 K polypeptide reached a maximum during the stationary phase of growth, where it represented substantially more than 1% of total cell protein. The 107 K polypeptide is coded by the haemolytic plasmid pHly167, and appears to be related to a previously reported intracellular precursor form of the -haemolysin (Goebel and Hedgpeth 1982). However, additional extracellular factors appear to be required for -haemolysin activity since several nonhaemolytic mutants still secrete this protein.     

Further cytogenetic analyses of the Croatian triploid shallot “Ljutika” (Allium cepa var.viviparum, Alliaceae) and its comparison with the Indian triploid “Pran”

Feulgen and silver-stained karyotypes and meiosis of two triploid viviparous onion forms (Allium cepa var.viviparum), the Croatian Ljutika and the Indian Pran, were comparatively analyzed. The results of chromosome measurements show that Ljutika and Pran are karyologically not identical, although significant similarities were found in the morphology of their chromosomes. Five geographically distant clones of Ljutika showed good agreement in the number and gross morphology of the chromosomes and in the number and position of NORs and interphase nucleoli. Heterotrivalents were predominant in meiosis of Ljutika but a relatively high frequency of higher multivalents together with univalents and bivalents were also observed. The relationship between Ljutika and Pran and their possible origin are discussed.     

High-pressure induced recovery of β-galactosidases from immunoadsorbents: stability of antigens and antibodies. Comparison with usual elution procedures

A high pressure-induced process has been developed to recover antigens from immunoadsorbents, without denaturing either the antigen, or the antibodies bound onto supports. The addition of chemicals is not needed to recover the product. Two variants of a model antigen were considered: (i) the Escherichia coli-galactosidase and (ii) a chimerical E. coli-galactosidase. Two and three-dimensional immunoaffinity supports (membranes and beads) were used.     

Characterization of the effectors required for stable inheritance of Streptococcus pyogenes pSM19035-derived plasmids in Bacillus subtilis

The low-copy-number and broad-host-range pSM19035-derived plasmid pBT233 is stably inherited in Bacillus subtilis cells. Two distinct regions, segA and segB, enhance the segregational stability of the plasmid. Both regions function in a replicon-independent manner. The maximization of random plasmid segregation is accomplished by the recombination proficiency of the host or the presence of the pBT233 segA region. The segA region contains two open reading frames (or) [ and ]. Inactivation or deletion of or results in SegA^– plasmids. Better than random segregation requires an active segB region. The segB region contains two ors (or and or). Inactivation of either of the orfs does not lead to an increase in cell death, but or^– plasmids are randomly segregated. These results suggest that pBT233 stabilization relies on a complex system involving resolution of plasmid oligomers (segA) and on the function(s) encoded by the segB region.     

Effects of glycine supplement on protein production and release in recombinant Escherichia coli

Summary The effect of glycine supplement to growth media on protein expression and release in a recombinant strain RR1 of E. coli was investigated. Addition of glycine to the growth media in moderate amount (up to 1%) was observed to enhance significantly the release of periplasmic proteins from the cell to the broth. The extracellular activities of the model enzymes -amylase and -lactamase were increased by a factor of 16.3 and 3.8 respectively in the presence of glycine. These activities corresponded to about 50% of the total production for each protein. Furthermore, with glycine supplement the total enzyme activity of both -amylase, -lactamase as well as -galactosidase were increased by a factor of about 2.5. Cell growth characteristics and low extracellular activity of the cytoplasmic protein -galactosidase are indicative that glycine does not cause significant cell-lysis for a concentration below 0.7%.     

Genetic studies of the ribosomal proteins in Escherichia coli. XI. Mapping of the genes for L21, L27, S15 and S21 by using hybrid bacteria and over-production of these proteins in the merodiploid strains.

Summary Host capacity for growth of single-stranded DNA phages was investigated with several replication mutants of E. coli. In dnaL708, dnaM709 and dnaS707 mutants, multiplication of K was not restricted even at 42°C. In dnaM710 cells, however, growth of K was severely affected at 42°C but not at 33°C. Upon infection of K, parental replicative form was synthesized at the restrictive temperature, whereas subsequent step (replication of progeny replicative form) was blocked in the dnaZ strain. Growth of X174 and 3, as tested by transfection, was also thermosensitive in the dnaM710 mutant but not in the dnaL708, dnaM709 and dnaS707 strains. In contrast with , microvirid phages could grow in E. coli cells bearing the groPC259, groPC756 or seg-2 mutation.This paper is number 15 in the series entitled Sensivity of Escherichia coli to Viral Nucleic Acid