The differential effect on two hybrid proteins of deletion mutations within the hydrophobic region of the Escherichia coli OmpA signal peptide

Oligonucleotide-directed site-specific mutagenesis was used to systematically shorten the hydrophobic region within the signal peptide of the Escherichia coli outer membrane protein OmpA. DNA encoding the wild type and mutant OmpA signal peptides were then fused in frame to DNA encoding the mature regions of Staphylococcus aureus nuclease A and TEM beta-lactamase. The ability of these signal peptides to direct processing of the resulting hybrid proteins was dependent on both their length and the protein to which they were fused. Deletion of two or more residues progressively slowed processing of pro-OmpA-nuclease. By contrast, pro-OmpA-beta-lactamase was less sensitive to the length of the hydrophobic region than to the nature of the deleted residue(s). Deletion of an Ala residue tended to reduce processing efficiency of pro-OmpA-beta-lactamase, while deletion of an Ile residue, together with the Ala residue, resulted in improvement. The loss of either 3 or 4 residues abolished processing of both hybrids. These data indicate that both the length as well as the identity of residues in the hydrophobic region are important. The relative importance of these two factors depends on the mature region of the protein being secreted.     

The Cold Box stem-loop proximal to the 5'-end of the Escherichia coli cspA gene stabilizes its mRNA at low temperature.

The 5'-end region of cspA mRNA contains a Cold Box sequence conserved among several cold-shock mRNAs. This region forms a stable stem-loop structure followed by an AU-rich sequence. Here we show that the Cold Box region is essential for the normal scale of cspA mRNA induction after cold shock because a deletion of the stem-loop significantly destabilizes the mRNA and reduces the cold shock-induced cspA mRNA amount by approximately 50%. The AU-rich track, however, slightly destabilizes the mRNA. The integrity of the stem is essential for the stabilizing function, whereas that of the loop sequence is less important. Overexpression of a mutant cspA mRNA devoid of both the AUG initiation codon and the coding sequence results in a severe growth inhibition at low temperature along with a derepression of the chromosomal cspA expression. Furthermore, the overexpressed RNA is stably associated with the 30 S and 70 S ribosomes. Our results demonstrate that the AUG initiation codon and the coding region containing the downstream box are not required for cspA mRNA to bind ribosomes and that the 5'-untranslated region by itself has a remarkable affinity to ribosomes at low temperature.     

Species-area curves and estimates of total species richness in an old-field chronosequence

Average species-area curves were generated for vascular plants in 20 old-fields that were sampled in 1983, 1989, and 1994. These curves were fit with a saturating function to estimate total species richness for each old-field. Additional estimates of total species richness were generated by fitting the same saturating function to subsets of the species area curves and with a first-order jackknife procedure. Estimates of total species richness were strongly correlated with observed species richness. There was limited evidence suggesting that greater sampling was necessary to identify the same proportion of species in older, more species-rich old-fields.     

The carpal tunnel syndrome as a probable primary deficiency of vitamin B6 rather than a deficiency of a dependency state

The mean basal specific activities and the mean % deficiencies of the activity of the glutamic oxaloacetic transaminase of the erythrocytes were identical (n.s.) for a group of eight patients with a severe carpal tunnel syndrome and for a group of eight university students. There was no significant difference in the increases in the specific activities for the patients and the students at 4 concentrations of pyridoxal 5′-phosphate. The apparent K_m for the patients and the students was 95 μM and 61 μM (n.s.) respectively. It is concluded from these data in conjunction with previous findings that the carpal tunnel syndrome is a deficiency disease of vitamin B₆, which is probably primary rather than one of a dependency state.     

Adaptations for feeding on rock surfaces and sandy sediment by the fiddler crabs (Brachyura: Ocypodidae) Uca tetragonon(Herbst, 1790) and Uca vocans(Linnaeus, 1758)

Two species of fiddler crab, Uca tetragonon(Herbst, 1790) and Uca vocans(Linnaeus, 1758), which belong to the subgenus Gelasimus, dwell on rocky shores and muddy–sandy tidal flats, respectively, in Phuket Is., Thailand. We investigated their feeding ecology in relation to the morphology of their feeding organs: minor food-handling chelipeds and maxillipeds. U. tetragononfed chiefly on rocks covered by filamentous green algae. U. vocansfed on the emerged sand and in shallow water along the shoreline and in pools. While feeding, both crabs made sand pellets beneath their mouthparts and discarded them, indicating that they divided the matter scooped up with their minor chelipeds into edible and inedible fractions by using the maxillipeds in the water passing through their buccal cavity. The morphology of maxillipeds hardly differed between the two species, which means that both species are flotation-feeders. The morphology of their minor chelipeds, however, differed: the tips of the dactyl and pollex were flat in U. tetragononand pointed in U. vocans.When the minor cheliped was closed, U. tetragononhad a hemispherical space in the distal one-fourth of the gape, which was closed by the framing keratin layers and a few setae of the dactyl and pollex. On the other hand, U. vocanshad an ellipsoidal space in the distal half of the gape. We consider these morphological characters to be adaptations to the different feeding substrates for retaining more food-laden sediment. We discuss the role of the setae on the minor chelipeds on the basis of the morphological differences between populations of U. tetragononin Phuket Is. and East Africa where the crab inhabits muddy–sandy tidal flats.     

Cross-sectional Area-corrected Compression Modulus of Food Gel

The linearity of the stress-strain relationship for food gel is limited to a very narrow range of the strain (usually less than 0.1 as a Cauchy measure). The reason is thought due to the change in cross-sectional area of the gel upon deformation. In this report, the cross-sectional area was approximately corrected of the compressed gel on the assumption that the gel expanded uniformly without changing its volume upon compression. In cases when the initial Young’s modulus was calculated from the thus-corrected area for some food gels, the linearity was increased for a wider range of strain.     

A dicarba analog of beta-atrial natriuretic peptide (beta-ANP) inhibits guanosine 3',5'-cyclic monophosphate production induced by alpha-ANP in cultured rat vascular smooth muscle cells

The synthesis and biological properties are described of [Asu7,23']-beta-ANP-(7-28) (Asu, L-alpha-aminosuberic acid), a dicarba analog of beta-atrial natriuretic peptide (beta-ANP, an antiparallel dimer of human alpha-ANP with the chains linked by 7-23' and 7'-23 disulfide bonds). This Asu-analog (referred to as analog III) displaced 125I-alpha-ANP specifically bound to cultured rat vascular smooth muscle cells (VSMC) with an apparent Ki of 2.1 x 10(-8) M, but did not stimulate formation of intracellular cGMP at 10(-8) -10(-5) M. Analog III inhibited the alpha-ANP-stimulated cGMP production in VSMC competitively with a pA2 value of 7.45 and behaved as an antagonist of alpha-ANP in rat aorta smooth muscle relaxation. In addition, beta-ANP was also shown to inhibit the alpha-ANP-induced cGMP production in a dose-dependent manner. The mechanism of action of beta-ANP is also discussed.     

Purification and characterization of immunoglobulin production stimulating factor derived from human B lymphoblastoid HO-323 cells

An immunoglobulin (Ig) production stimulating factor (IPSF) for hybridomas was found in spent medium of the human B lymphoblastoid cell line, HO-323. The IPSF was purified by serial use of DEAE chromatography, ultrafiltration, gel filtration and HPLC-DEAE chromatography. Purified IPSF was estimated to be a 410 k macro molecule by gel filtration, and contained three types of isomers which were separated from each other by native polyacrylamide gel electrophoresis. All of the isomers were, however, assumed to have the same protein components by SDS polyacrylamide gel electrophoresis.The IPSF was effective for human-human and mouse-mouse hybridomas producing IgM, but not for IgG producers in the experimental condition used here. Human-human hybridoma HF10B4, cultured in IPSF-containing medium, produced 20 times more IgM than in IPSF-free medium under serum-free conditions. The IPSF showed very little proliferation stimulating activity on HF10B4 cells.