Characterization of staphylococci

A total of 158 Staphylococcus strains from various sources were characterized by biochemical, physiological, and morphological tests. Numerical taxonomy was applied by using these features. Taxonomic analysis was done with programs run under the MVS-TSO system of the IBM 370 complex and PDP-10 system of the National Institutes of Health. DNA-DNA hybridization with nitrocellulose filters was done to compare selected atypical cultures with American Type Culture Collection reference strains. We found that the use of the nomenclature of Bergey's Manual (8th edition) to identify these strains by species was not adequate. DNA homology values supported the formation of Staphylococcus hyicus subsp. hyicus separate from Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus. The three tests that best separated these strains into four species were (i) tube coagulase (6-h or 24-h porcine plasma or 24-h Difco rabbit plasma), (ii) production of acetoin or acid aerobically from ribose, maltose, or trehalose, and (iii) growth in the presence of novobiocin. Four strains of S. hyicus subsp. hyicus (VII76, VII113, VII131, and VA519) gave typical enterotoxigenic responses in monkey-feeding tests but were negative for enterotoxins A through E, suggesting the presence of one or more new enterotoxins. Two coagulase-negative, heat-stable DNase-positive strains (D143 and ARM) could not be classified by either DNA-DNA hybridization or numerical taxonomy, and D143 was enterotoxigenic as measured by the monkey-feeding bioassay. DNA homology showed that strain FRI-698M was more closely related to S. epidermidis than to S. aureus, yet it produced enterotoxin D. These data suggest the occurrence of coagulase-negative enterotoxigenic strains that are not S. aureus; nonetheless, a positive tube coagulase test and heat-stable DNase test should together be useful for routine screening of most potentially enterotoxigenic staphylococci in foods.     

Rat Sertoli and spermatogenic cells express a similar gene, and its product is antigenically related to an outer dense fiber-associated protein.

We have previously reported that a heterodimeric protein secreted by rat Sertoli cells is antigenically related to a protein associated with outer dense fibers of the sperm tail. Therefore, we have explored the possibility that Sertoli and spermatogenic cells express a similar gene encoding a homologous protein. A Sertoli cell heterodimeric protein cDNA probe recognizes specific mRNA in pachytene and round spermatids fractionated by centrifugal elutriation; however, this specific mRNA was less prominent than in cultured Sertoli cells. In agreement with these observations, in situ hybridization experiments show that Sertoli cells are predominantly engaged in active heterodimeric protein mRNA synthesis, while meiotic prophase spermatocytes and spermatids also show significant but less abundant specific mRNA. Immunoblotting experiments demonstrate that, while Sertoli cells synthesize a heterodimeric protein consisting of two disulfide-linked components with molecular masses of 45 and 35 kD, both primary spermatocytes and round spermatids synthesize single 30 kD monomers not associated by disulfide linkage but recognized by antisera to Sertoli cell heterodimeric protein. Immunoblotting and immunogold electron microscopic studies show that antisera to Sertoli cell heterodimeric protein recognize a protein associated with outer dense fibers. This immunoreactivity was abolished by a 5-min pronase treatment, without affecting the integrity of outer dense fibers. Results of this study and previous studies demonstrate that both Sertoli and spermatogenic cells express a similar gene and that an antigenically related product encoded by this gene becomes associated with outer dense fibers during their assembly at spermiogenesis.     

Host tree resistance against the polyphagous wood-boring beetle Anoplophora glabripennis

Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae: Lamiini) is an invasive wood‐boring beetle with an unusually broad host range and a proven ability to increase its host range as it colonizes new areas and encounters new tree species. The beetle is native to eastern Asia and has become an invasive pest in North America and Europe, stimulating interest in delineating host and non‐host tree species more clearly. When offered a choice among four species of living trees in a greenhouse, adult A. glabripennis fed more on golden‐rain tree (Koelreuteria paniculata Laxmann) and river birch (Betula nigra L.) than on London planetree (Platanus × acerifolia (Aiton) Willdenow) or callery pear (Pyrus calleryana Decaisne). Oviposition rate was highest in golden‐rain tree, but larval mortality was also high and larval growth was slowest in this tree species. Oviposition rate was lowest in callery pear, and larvae failed to survive in this tree species, whether they eclosed from eggs laid in the trees or were manually inserted into the trees. Adult beetles feeding on callery pear had a reduced longevity and females feeding only on callery pear failed to develop any eggs. The resistance of golden‐rain tree against the larvae appears to operate primarily through the physical mechanism of abundant sap flow. The resistance of callery pear against both larvae and adults appears to operate through the chemical composition of the tree, which may include compounds that are toxic or which otherwise interfere with normal growth and development of the beetle. Unlike river birch or London planetree, both golden‐rain tree and callery pear are present in the native range of A. glabripennis and may therefore have developed resistance to the beetle by virtue of exposure to attack during their evolutionary history.     

T cells with FC receptors in myeloma; suppression of growth and secretion of MOPC-315 by T alpha cells

We have previously demonstrated that 1) BALB/c mice and patients with IgA myeloma developed a marked expansion of T cells with surface IgA-Fc receptors (T alpha cells), 2) the FcR alpha were induced by direct interaction of soluble myeloma IgA with T cells, and 3) the T alpha cells induced in IgA myeloma were Lyt-1-2+, IgA isotype-specific suppressor cells in normal immune responses. These findings established that the host with IgA myeloma responds to the large amounts of Ig produced by the tumor by activating an otherwise normal IgA isotype-specific suppressor circuit. In the present studies, we extend our previous observations and show that T alpha cells can suppress both the growth and secretion of MOPC-315 myeloma tumor cells. Thus, the isotype-specific immunoregulatory circuit activated in myeloma is capable of suppressing tumor cells as well as normal cells.     

A method for cultivating morphologically undifferentiated embryonic stem cells from porcine blastocysts

Variable conditions were tested to determine an in-vitro cultivation method for the formation of morphologically undifferentiated embryonic stem cells from the inner cell mass (ICM) derived outgrowth of porcine blastocysts. Although all 16 Day-9 embryos failed to form colonies, 14 such colonies were obtained from a total of 69 Day-10 embryos when they were co-cultivated with porcine uterine fibroblast (PUF) cells over a 6-day period. The best results were obtained in Dulbecco's modified Eagle medium (DMEM) with 10% fetal calf serum and 10% porcine serum supplemented with bovine insulin and beta-mercaptoethanol, in which six out of seven embryos formed adequate ICM-derived colonies. Since murine fibroblasts were not found to be suitable feeder cells in this procedure, an endocrine synergistic interaction, which promotes embryonic attachment and colony formation, between porcine blastocysts and PUF cells is hypothesized. Continued propagation of the ICM-derived cells was not dependent on these factors; a total of seven cell lines were obtained after three to five subsequent passages on murine feeder-layers that resembled morphologically undifferentiated embryonic cells.     

Endoglin Requirement for BMP9 Signaling in Endothelial Cells Reveals New Mechanism of Action for Selective Anti-Endoglin Antibodies

Endoglin (ENG), a co-receptor for several TGFβ-family cytokines, is expressed in dividing endothelial cells alongside ALK1, the ACVRL1 gene product. ENG and ACVRL1 are both required for angiogenesis and mutations in either gene are associated with Hereditary Hemorrhagic Telangectasia, a rare genetic vascular disorder. ENG and ALK1 function in the same genetic pathway but the relative contribution of TGFβ and BMP9 to SMAD1/5/8 activation and the requirement of ENG as a co-mediator of SMAD phosphorylation in endothelial cells remain debated. Here, we show that BMP9 and TGFβ1 induce distinct SMAD phosphorylation responses in primary human endothelial cells and that, unlike BMP9, TGFβ only induces SMAD1/5/8 phosphorylation in a subset of immortalized mouse endothelial cell lines, but not in primary human endothelial cells. We also demonstrate, using siRNA depletion of ENG and novel anti-ENG antibodies, that ENG is required for BMP9/pSMAD1 signaling in all human and mouse endothelial cells tested. Finally, anti-ENG antibodies that interfere with BMP9/pSMAD1 signaling, but not with TGFβ1/pSMAD3 signaling, also decrease in vitro HUVEC endothelial tube formation and inhibit BMP9 binding to recombinant ENG in vitro. Our data demonstrate that BMP9 signaling inhibition is a key and previously unreported mechanism of action of TRC105, an anti-angiogenic anti-Endoglin antibody currently evaluated in clinical trials.     

Inhibition of the Na,K-ATPase by fluoride. Parallels with its inhibition of the sarcoplasmic reticulum CaATPase.

Addition of lithium fluoride to a suspension of Na,K-ATPase undergoing turnover produced a slow (minutes) complete loss of ouabain-sensitive ATPase activity. Persistence of the effect in the presence of deferoxamine showed that fluoride inhibits independent of aluminum. The time course of onset of inhibition was adequately fit by a function corresponding to a monophasic transformation with a pseudo first-order rate constant (k(obs)). This constant varied hyperbolically with [Mg2+] (half-maximal effect at 9 mM Mg2+), whereas it increased with no sign of approaching saturation as the square of [F-], implying that inhibition requires binding of two fluorides/ATPase. The value of k(obs) was found to be increased by greater than 10-fold in the presence of potassium ([K+]1/2 = 0.6 mM) or ouabain. Sodium, ATP, and ADP, which favor the E1 form of the enzyme, had a protective effect. These results implicate the potassium-occluded MgE2(K2) complex as the main fluoride-susceptible form. Protection by Pi and orthovanadate suggests that fluoride exerts its effect at the phosphorylation site. Inhibition was reversible, although slowly, with t1/2 = 7 h at 37 degrees C. Sodium greatly accelerated reversal (t1/2 = 3 min with 150 mM Na+ present), and potassium antagonized this acceleration. The value of k(obs) for reactivation increased steeply with [Na+], with the sodium dependence being about the same at pH 8.0 as at pH 7.4. All of these effects have parallels to effects of fluoride on the sarcoplasmic reticulum CaATPase (Murphy, A. J., and Coll, R. J. (1992) J. Biol. Chem. 267, 5229-5235).     

A new model of the distal convoluted tubule

The Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) of the kidney is a key determinant of Na(+) balance. Disturbances in NCC function are characterized by disordered volume and blood pressure regulation. However, many details concerning the mechanisms of NCC regulation remain controversial or undefined. This is partially due to the lack of a mammalian cell model of the DCT that is amenable to functional assessment of NCC activity. Previously reported investigations of NCC regulation in mammalian cells have either not attempted measurements of NCC function or have required perturbation of the critical without a lysine kinase (WNK)/STE20/SPS-1-related proline/alanine-rich kinase regulatory pathway before functional assessment. Here, we present a new mammalian model of the DCT, the mouse DCT15 (mDCT15) cell line. These cells display native NCC function as measured by thiazide-sensitive, Cl(-)-dependent (22)Na(+) uptake and allow for the separate assessment of NCC surface expression and activity. Knockdown by short interfering RNA confirmed that this function was dependent on NCC protein. Similar to the mammalian DCT, these cells express many of the known regulators of NCC and display significant baseline activity and dimerization of NCC. As described in previous models, NCC activity is inhibited by appropriate concentrations of thiazides, and phorbol esters strongly suppress function. Importantly, they display release of WNK4 inhibition of NCC by small hairpin RNA knockdown. We feel that this new model represents a critical tool for the study of NCC physiology. The work that can be accomplished in such a system represents a significant step forward toward unraveling the complex regulation of NCC.