Contraction of filaments of Escherichia coli after disruption of cell membrane by detergent.

The osmotic pressure within a living bacterium creates stresses in the peptidoglycan that stretch the sacculus. We measured the amount of stretch by monitoring the shrinkage of growing cells of Escherichia coli after removal of the osmotic pressure by disruption of the phospholipid membranes with sodium dodecyl sulfate. Because the rods of the wild type are so short, length changes of filaments of longer than 7 microns were measured on phase-contrast micrographs. The filaments were prepared by growing ftsA and ftsI strains under permissive conditions in rich medium and then shifting them to 42 degrees C for 40 to 180 min. During this time, the mutant cells became elongated but did not divide. The growing filaments were mounted on a glass surface that had been treated with poly-L-lysine or RNase. The filaments were photographed before being treated with sodium dodecyl sulfate. The filaments were rephotographed at the time when the first change in phase contrast was noted. Some filaments were also measured at 10-min time intervals from 0 to 60 min. The reduction in phase contrast signaled the leakage of solutes and the loss of turgor pressure. The average length of the filaments decreased 17%. If the circumference were stretched to the same degree, then the surface area in vivo would be 45% greater than in the relaxed state. For comparison, a fully cross-linked monolayer of E. coli peptidoglycan in its most compact conformation could stretch up to 300% in achieving the most extended conformation possible without splitting covalent bonds.     

Bactericidal effect of hydrogen peroxide on Salmonella typhimurium in liquid whole egg

The effect of hydrogen peroxide on Salmonella typhimurium in whole egg was evaluated. The bactericidal effects observed on the test organism at 5 degrees and 20 degrees C were found to be similar. There was a 99% kill in the presence of 0.5% and 1.0% H2O2. Addition of the test organism and H2O2 after pre-heating the egg material at 40 degrees C for 15 min caused a rapid kill which was 10,000-fold greater than that produced by H2O2 alone.     

Variability of the turgor pressure of individual cells of the gram-negative heterotroph Ancylobacter aquaticus.

Cells of Ancylobacter aquaticus were observed under phase microscopy in a chamber to which a measured pressure could be applied. The initial collapse pressure (Ca), i.e., the lowest pressure needed to collapse the most pressure-sensitive gas vesicles, was measured for 69 cells. The cells were taken from cultures in low-density balanced exponential growth, and the experiments were performed quickly so that the bacteria were in a uniform physiological state at the time of measurement. The turgor pressure, Pt, is the difference between the pressure, C, that would cause collapse of vesicles when removed from the cell and Ca. In this paper we focus on the variability of Pt from cell to cell. Part of the observed variability of Ca was due to the variability of the collapse pressure of individual vesicles (standard deviation [SD] = 90 kPa), but because there were about 100 vesicles per cell and because a change in refracted light after the fifth vesicle (approximately) collapsed probably could be detected by the human eye, the pressure would only have an SD of 18.6 kPa due to this type of sampling error. The observed SD of Pt was 42 kPa, indicating that turgor pressure did vary considerably from cell to cell. However, the turgor pressure was independent of cell size. Statistical analysis showed that Pt would decrease 6.9 kPa over a cell cycle, but with too large an SD (19.9 kPa) to be significant. This implies that the observed change in Pt over the cell cycle is not statistically significant.     

Expression of antibody cDNA in murine myeloma cells: possible involvement of additional regulatory elements in transcription of immunoglobulin genes

Expression vectors for cDNA of the κ and λ1 chains of a monoclonal antibody directed against creatine kinase were introduced into murine myeloma cells. κ and γ1 cDNA were either under the control of the SV40 early promoter or of the cognate promoters and enhancers of the light- and heavy-chain genes. Secretion of immuno-reactive κ and γ1 chains into the culture medium was demonstrated with the SV40 promoter as well as with the cognate promoters. Expression of y 1 cDNA with the SV40 early promoter was about twice as high as with the heavy-chain promoter and enhancer. Expression of κ cDNA under the control of the S V40 early promoter was about 17 times higher than with the light-chain promoter and enhancer. These expression levels were compared to those of a genomic immunoglobulin (Ig) κ determinant, including introns. Such an entire κ gene led to expression of the light chain at levels double those with the κ cDNA construction using the SV40 promoter and about 35 times as high when using κ cDNA and the cognate promoter and enhancer. This result might indicate that, besides the cognate promoter and enhancer elements, other intragenic elements are involved in the regulation of Ig expression. However, the SV40 early promoter seems to be able to compensate for the absence of these postulated regulatory elements probably located in the introns.     

Responses of wild plants to nitrate availability

Summary Two annual species of Bromus, an invader (B. hordeaceus, ex B. mollis) and a non-invader (B. intermedius), were grown for 28 days in growth chambers, at 5 and 100 M NO ₃ ^- in flowing nutrient solution. No differences between the two species were observed at either NO ₃ ^- level, in terms of relative growth rate (RGR) or its components, dry matter partitioning, specific NO ₃ ^- absorption rate, nitrogen concentration, and other characteristics of NO ₃ ^- uptake and photosynthesis. The effects of decreasing NO ₃ ^- concentration in the solution were mainly to decrease the NO ₃ ^- concentration in the plants through decreased absorption rate, and to decrease the leaf area ratio through increased specific leaf mass and decreased leaf mass ratio. Organic nitrogen concentration varied little between the two treatments, which may be the reason why photosynthetic rates were not altered. Consequently, RGR was only slightly decreased in the 5-M treatment compared to the 100-M treatment. This is in contrast with other species, where growth is reduced at much higher NO ₃ ^- concentrations. These discrepancies may be related to differences in RGR, since a log-linear relationship was found between RGR and the NO ₃ ^- concentration at which growth is first reduced. In addition, a strong linear relationship was found between the RGR of these species and their maximum absorption rate for nitrate, suggesting that the growth of species with low maximum RGR may be partly regulated by nutrient uptake.     

The common src homology region 2 domain of cytoplasmic signaling proteins is a positive effector of v-fps tyrosine kinase function.

A conserved noncatalytic domain SH2 (for src homology region 2) is located immediately N terminal to the kinase domains of all cytoplasmic protein-tyrosine kinases. We found that the wild-type v-fps SH2 domain stimulated the enzymatic activity of the adjacent kinase domain 10-fold and functioned as a powerful positive effector of catalytic and transforming activities within the v-fps oncoprotein (P130gag-fps). Partial proteolysis of P130gag-fps and supporting genetic data indicated that the v-fps SH2 domain exerts its effect on catalytic activity through an intramolecular interaction with the kinase domain. Amino acid alterations in the SH2 domain that impaired kinase function interfered with association of the SH2 domain with the kinase domain. Deletion of a conserved octapeptide motif converted the v-fps SH2 domain from an activator to an inhibitor of tyrosine kinase activity. This latent inhibitory activity of v-fps SH2 has functional implications for phospholipase C-gamma and p21ras GTPase-activating protein, both of which have two distinct SH2 domains suggestive of complex regulation. In addition to regulating the specific activity of the kinase domain, the SH2 domain of P130gag-fps was also found to be required for the tyrosine phosphorylation of specific cellular proteins, notably polypeptides of 124 and 62 kilodaltons. The SH2 domain therefore appears to play a dual role in regulation of kinase activity and recognition of cellular substrates.     

Formation of zeatin allylic phosphate by the microsomal fractions of bulb disks of Iris × hollandica Tub. and tubers of Helianthus tuberosus L.

It is shown that in bulbous Iris zeatin originates from a nucleotide. This nucleotide is probably zeatin-allylic-phosphate, in which a phosphate group is attached to the isoprenoid side-chain of zeatin. The formation of zeatin-allylic-phosphate from t-zeatin and 8-[¹⁴C]-zeatin by the microsomal fractions of Iris bulb disks and Helianthus tubers was demonstrated. The responsible enzyme was partially purified. 5-AMP was found to be a phosphate group delivering substrate. Adenosine and adenine inhibited the enzyme reaction. The significance of the results is discussed in relation to cytokinin biosynthesis and the occurrence of bud blast in Iris.