Computer-assisted identification of anaerobic bacteria.

A computer program was developed to identify anaerobic bacteria by using simultaneous pattern recognition via a Bayesian probabilistic model. The system is intended for use as a rapid, precise, and reproducible aid in the identification of unknown isolates. The program operates on a data base of 28 genera comprising 238 species of anaerobic bacteria that can be separated by the program. Input to the program consists of biochemical and gas chromatographic test results in binary format. The system is flexible and yields outputs of: (i) most probable species, (ii) significant test results conflicting with established data, and (iii) differential tests of significance for missing test results.     

Conformational changes induced by integration host factor at origin gamma of R6K and copy number control.

We have investigated the role of integration host factor (IHF) in the replication of plasmid R6K by studying the maintainance of the plasmid in a strain of Escherichia coli that lacks both subunits of IHF and in an isogenic wild type strain and found that all three origins, alpha, beta, and gamma, were functional in the absence of IHF; however, loss of IHF reduced the copy number of those replicons initiating solely from ori gamma by 5-fold. Concomitant loss of direct repeats within the origin that bind the R6K replication initiator protein, Pi, resulted in a further reduction in copy number. Using gel mobility shift analysis, we showed that IHF bound specifically only to one site within the A/T rich region of the minimal origin adjacent to the Pi binding sites. The origin region possessed no intrinsic DNA curvature although IHF induced a strong bend upon binding. Combination footprinting with different orders of addition of Pi and IHF suggested that there was no cooperativity between the two proteins with regard to DNA binding. Hydroxyl-radical footprinting revealed hypersensitive asymmetric periodic cleavage sites within the origin region in the presence of IHF that extended over 200 base pairs and a localized perturbation of cleavage chemistry. The presence of periodic cleavages was dependent upon the presence of the wild type R6K origin sequence and was not observed when the IHF binding site was positioned adjacent to a heterologous sequence. We observed that the conformational changes induced by IHF upon binding to the R6K origin were negatively correlated with the observed decrease in copy number, and therefore, origin conformation altered by protein-DNA interaction may play an important role in the regulation of replication initiation.     

Human T cell activation by OKT3 is inhibited by a monoclonal antibody to CD44.

The CD44 molecule, also known as Hermes lymphocyte homing receptor, human Pgp-1, and extracellular matrix receptor III, has been shown to play a role in T cell adhesion and activation. Specifically, anti-CD44 mAb block binding of lymphocytes to high endothelial venules, inhibit T cell-E rosetting, and augment T cell proliferation induced by the CD2 or CD3-TCR pathways. We have characterized an anti-CD44 mAb (212.3) which immunoprecipitates a 90-kDa protein and is specific for CD44 as shown by peptide mapping and antibody competition studies. Interestingly, our studies with 212.3 demonstrate that this CD44-specific mAb completely inhibits T cell proliferation stimulated by the anti-CD3 mAb, OKT3. Inhibition is not a result of reduced cell viability, but is associated with 1) inhibition of IL-2 production, 2) inhibition of IL-2R expression, and 3) inhibition of OKT3-mediated increases in intracellular Ca2+ levels. In addition, 212.3 does not inhibit proliferation by the T cell mitogens PHA or PWM nor does it inhibit proliferation in a mixed lymphocyte reaction. Similar to other anti-CD44 mAb, 212.3 also augments T cell proliferation induced by mAb directed against the T11(2) and T11(3) epitopes of CD2. Thus, these studies describe a novel CD44-specific mAb (212.3) that inhibits T cell activation by OKT3 by blocking early signal transduction. Furthermore, these studies suggest that "receptor cross-talk" between the CD3-TCR complex and CD44 may regulate T cell activation.     

Identification of insulin-like growth factor-binding proteins in the circulation of four teleost fish species.

Insulin-like growth factor-binding proteins (IGF-BPs) were demonstrated in the circulation of four teleost fish species. In the coho salmon (Oncorhynchus kisutch), serum binding of 125I-labelled human IGF-I (125I-hIGF-I) was competitively inhibited by addition of excess recombinant bovine IGF-I (rbIGF-I) in a manner similar to that when rat serum was used. Western-ligand blot procedure using the same labelled hormone identified at least three major forms of IGF-BPs in the plasma of all four teleost species investigated: coho salmon, striped bass (Morone saxatilis), tilapia (Oreochromis mossambicus), and longjawed mudsucker (Gillichthys mirabilis). The first form is around 40-50 kDa, may be regulated by growth hormone (GH), and seems to be a good candidate for the fish version of mammalian IGF-BP3 (which is in the same size range and is GH-regulated). The second and third forms are 29 kDa and 31 kDa and are good candidates for the fish versions of mammalian IGF-BP1 and IGF-BP2, respectively, as they appear to be regulated by insulin and are in the same size range as their mammalian counterparts. Functionally different classes of circulating IGF-BPs may be conserved between fish and mammal.     

Indices of iron and copper status during experimentally induced,marginal zinc deficiency in humans

This study examined the effect of diet-induced, marginal zinc deficiency for 7 wks in 15 men (aged 25.3 +/- 3.3 yrs; mean +/- SD) on selected indices of iron and copper status. The regimen involved low-zinc diets based on egg albumin and soy protein with added phytate and calcium such that mean [phytate]/[Zn] and [phytate] X [Ca]/[Zn] molar ratios were 209 and 4116, respectively, for 1 wk, followed by 70 and 2000, respectively, for 6 wks. Subjects were then repleted with 30 mg Zn/d for 2 wks. Plasma copper, Cu,Zn-superoxide dismutase (Cu,Zn-SOD) activity in plasma and red blood cells (RBC), hemoglobin, hematocrit, and serum ferritin were determined weekly on fasting blood samples. Significant reductions (p less than 0.05) after 7 wks in RBC Cu,Zn-superoxide dismutase (49.5 +/- 7.2 vs 33.6 +/- 6.3 U/mg Hb) and serum ferritin (69.2 +/- 38.7 vs 53.8 +/- 33.7 micrograms/L) occurred; no comparable decline was noted for plasma Cu, hemoglobin, or hematocrit. Significant (p less than 0.05) but less consistent changes were also observed in plasma superoxide dismutase activity. None of the changes were associated with the decreases in plasma, urinary and hair zinc concentrations, and alkaline phosphatase activity in RBC membranes. Results indicate that the biochemical iron and copper status of the subjects was marginally impaired, probably from the dietary regimen that induced marginal zinc deficiency.     

Characterization of exopolymers of aquatic bacteria by pyrolysis-mass spectrometry.

Exopolymers from a diverse collection of marine and freshwater bacteria were characterized by pyrolysis-mass spectrometry (Py-MS). Py-MS provides spectra of pyrolysis fragments that are characteristic of the original material. Analysis of the spectra by multivariate statistical techniques (principal component and canonical variate analysis) separated these exopolymers into distinct groups. Py-MS clearly distinguished characteristic fragments, which may be derived from components responsible for functional differences between polymers. The importance of these distinctions and the relevance of pyrolysis information to exopolysaccharide function in aquatic bacteria is discussed.     

Ethylene production from alpha-oxo-gamma-methylthiobutyric acid is a sensitive measure of ligninolytic activity by Phanerochaete chrysosporium.

Production of hydroxyl radical in ligninolytic cultures was determined by measuring the alpha-oxo-gamma-methylthiobutyric acid-dependent production of ethylene gas. The results showed that the pattern of ethylene production was very similar to that of ligninolytic activity [[14C]lignin leads to 14CO2). Furthermore, nutritional parameters, which are known to affect ligninolytic activity, affected OH.-radical-dependent ethylene production in a similar fashion. The results indicate that assay for ethylene production from alpha-oxo-gamma-methylthiobutyric acid is a simple and sensitive measure of ligninolytic activity by Phanerochaete chrysosporium.     

Identification and preliminary characterization of Vibrio cholerae outer membrane proteins.

Outer membrane proteins of Vibrio cholerae were purified by sucrose density centrifugation and Triton X-100 extraction at 10 mM Mg2+. The proteins were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. V. cholerae outer membrane proteins presented a unique pattern when compared with the patterns of other gram-negative rods. There were 8 to 10 major bands (Mr 94,000 to 27,000), with most of the protein located in band 5 (Mr approximately 45,000), which thus appears to be the major structural protein of the outer membrane. Lipid and carbohydrate were associated with band 6.     

Ionic partial molar volumes of density gradient salts at finite concentrations.

A general method is presented for estimating concentration-dependent partial molar volumes for individual ions of heavy density gradient salts used in the isopycnic ultracentrifugation of charged macromolecules.