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Characterization, localization, and sequence of F transfer region products: the pilus assembly gene product TraW and a new product, TrbI.

The traW gene of the Escherichia coli K-12 sex factor, F, encodes one of the numerous proteins required for conjugative transfer of this plasmid. We have found that the nucleotide sequence of traW encodes a 210-amino-acid, 23,610-Da polypeptide with a characteristic amino-terminal signal peptide sequence; in DNA from the F lac traW546 amber mutant, the traW open reading frame is interrupted at codon 141. Studies of traW expression in maxicells in the presence and absence of ethanol demonstrate that the traW product does undergo signal sequence processing. Cell fractionation experiments additionally demonstrated that mature TraW is a periplasmic protein. Electron microscopy also showed that F lac traW546 hosts do not express F pili, confirming that TraW is required for F-pilus assembly. Our nucleotide sequence also revealed the existence of an additional gene, trbI, located between traC and traW. The trbI gene encodes a 128-amino-acid polypeptide which could be identified as a 14-kDa protein product. Fractionation experiments demonstrated that TrbI is an intrinsic inner-membrane protein. Hosts carrying the pOX38-trbI::kan insertion mutant plasmids that we constructed remained quite transfer proficient but exhibited increased resistance to F-pilus-specific phages. Mutant plasmids pOX38-trbI472 and pOX38-trbI473 expressed very long F pili, suggestive of a pilus retraction deficiency. Expression of an excess of TrbI in hosts carrying a wild-type pOX38 plasmid also caused F-pilus-specific phage resistance. The possibility that TrbI influences the kinetics of pilus outgrowth and/or retraction is discussed.

Amino Acid Sequence

Role of Ly-6A/E and T cell receptor-zeta for IL-2 production. Phosphatidylinositol-anchored Ly-6A/E antagonizes T cell receptor-mediated IL-2 production by a zeta-independent pathway.

Ly-6A/E molecules were originally implicated in regulation of T cell activation because anti-Ly-6A/E mAb induce IL-2 production. More recently we have shown that anti-Ly-6A/E also inhibits IL-2 production induced by anti-CD3. In the present study we used mutant and transfected cell lines that varied in expression of Ly-6A/E or TCR-zeta to test whether the positive and negative modulations of IL-2 production by anti-Ly-6A/E occur by distinct mechanisms. Anti-Ly-6A/E inhibited anti-CD3-induced IL-2 production for Ly-6E.1-transfected EL4J cells, but did not affect IL-2 production of the parental Ly-6A/E-negative EL4J cells. These results indicate that TCR-mediated IL-2 production can occur in the absence of Ly-6A/E expression and establish that anti-Ly-6A/E-induced inhibition of IL-2 production was the result of antibody binding to Ly-6A/E. As expected, MA5.8 (zeta-negative) or CT108 (zeta-truncated) variants of the 2B4.11 T cell hybridoma did not produce IL-2 when stimulated with anti-Thy-1 or anti-Ly-6A/E mAb. In contrast, anti-Ly-6A/E inhibited anti-CD3-induced IL-2 production by MA5.8 and CT108. Furthermore, anti-Ly-6A/E-induced IL-2 production was restored for zeta-transfected MA5.8. Thus, although induction of IL-2 by anti-Ly-6A/E depends on zeta expression, inhibition of IL-2 by anti-Ly-6A/E occurs by a zeta-independent mechanism. Interestingly, anti-Ly-6A/E, but not anti-Thy-1, inhibited anti-CD3-induced IL-2 production by MA5.8 and Ly-6E.1-transfected EL4J. Therefore, inhibition of IL-2 production by anti-Ly-6A/E was not a general property of a mAb binding to a phosphatidylinositol-linked molecule, as has been suggested for induction of IL-2 production. Taken together these data suggest that the molecular mechanisms of induction and inhibition of IL-2 production by anti-Ly-6A/E are separable and expression of TCR-zeta is one variable that distinguishes these two pathways.

Animals

Interleukin (IL)-4 production by human T cells: differential regulation of IL-4 vs. IL-2 production.

We have examined the regulation of interleukin (IL)-4 production by human peripheral blood T cells. Production of IL-4 was shown to be regulated differently from IL-2 and interferon(IFN)-gamma production. Stimulation of peripheral blood lymphocytes with anti-CD3, anti-CD2, anti-CD28, Phorbol 12-myristate 13-acetate (PMA) or IL-2 as a single stimulant did not induce IL-4 production. However, combinations of anti-CD2 with either anti-CD28 or IL-2 resulted in IL-4 production, peaking at days 3-4. Stimulation with anti-CD3 instead of anti-CD2 gave similar results, but was less potent. After days 3-4, IL-4 levels decreased, most likely due to consumption of IL-4. PMA profoundly affected cytokine production, it enhanced IL-2 production by at least tenfold, whereas, in the same cell population, IL-4 production was almost completely inhibited. This was observed at the protein as well as at the mRNA level. In contrast, agents that increase intracellular cAMP levels inhibited IL-2 production but left IL-4 production unaffected. IFN-gamma production behaved similar to IL-2 production but the effects were less outspoken.

Antigens, Differentiation, T-Lymphocyte

Master standard data quantity food production code. Macro elements for synthesizing production labor time.

Preparation procedures of standardized quantity formulas were analyzed for similarities and differences in production activities, and three entrée classifications were developed, based on these activities. Two formulas from each classification were selected, preparation procedures were divided into elements of production, and the MSD Quantity Food Production Code was applied. Macro elements not included in the existing Code were simulated, coded, assigned associated Time Measurement Units, and added to the MSD Quantity Food Production Code. Repeated occurrence of similar elements within production methods indicated that macro elements could be synthesized for use within one or more entrée classifications. Basic elements were grouped, simulated, and macro elements were derived. Macro elements were applied in the simulated production of 100 portions of each entrée formula. Total production time for each formula and average production time for each entrée classification were calculated. Application of macro elements indicated that this method of predetermining production time was feasible and could be adapted by quantity foodservice managers as a decision technique used to evaluate menu mix, production personnel schedules, and allocation of equipment usage. These macro elements could serve as a basis for further development and refinement of other macro elements which could be applied to a variety of menu item formulas.

Animals

Regulation of reaginic antibody production in mice. I. Suppression by antigen of IgE antibody production in vitro.

Regulation of IgE production by antigen in a primed murine splenic lymphocyte culture system was described. Maximum IgE antibody production was found to occur when cells were cultured in the absence of exogenously added antigen. A cells and T lymphocytes did not affect the production of anti-DNP IgE antibody. By using a hapten-carrier antigen system (DNP-EA) for priming mice in vivo, it was found that the production of anti-DNP IgE by spleen cells in vitro was inhibited by hapten when coupled to homologous (EA) or heterologous (BGG) carrier, and was not enhanced or inhibited by homologous carrier. Anti-DNP IgE antibody production by cultures depleted of macrophages or T lymphocytes was found to be as sensitive to the suppressive effects of hapten as was the IgE production by whole spleen cell cultures. Both IgM and IgG secondary anti-DNP PFC responses in vitro were enhanced by the presence of the homologous hapten-carrier or carrier alone. DNP-BGG had no effect on the anti-DNP IgM or IgG PFC responses of the cultures. These data suggest that endogenous production of antibody (IgM or IgG) was not responsible for the observed suppression of the IgE response in vitrol The experimental results presented indicate that the regulation of the IgE production by antigen in the primed mouse splenic lymphocyte cultures was a consequence of the direct interaction of hapten with IgE B cells.

Animals

IL-1 production as a regulator of G-CSF and IL-6 production in CSF-producing cell lines.

We previously demonstrated that colony stimulating factor (CSF)-producing cell lines co-produce interleukin-1 (IL-1) and IL-6 in addition to CSFs. In the present study, we examined the role of IL-1 production in three human tumour cell lines producing granulocyte (G)-CSF, IL-1 and IL-6. Addition of anti-human IL-1 alpha antiserum to the culture caused a 90-62% reduction of G-CSF and a 85-44% reduction of IL-6 production, respectively, as evaluated by enzyme immunoassay in all three cell lines. The decrease of G-CSF and IL-6 production by the anti-IL-1 alpha antiserum was also confirmed at the level of mRNA expression. The anti-IL-1 alpha antiserum did not affect the growth of these cell lines. Excess recombinant IL-1 alpha exogenously added to the culture enhanced G-CSF and IL-6 production in all three cell lines. However, IL-1 alpha had little effect on the growth of these three cell lines. Neither anti-IL-6 nor anti-G-CSF antibodies affected the production of the other cytokines. These results indicate that IL-1 alpha regulates G-CSF and IL-6 production in these tumour cell lines, and suggest that the IL-1 production plays an important role in CSF-producing tumours.

Cell Division

Erythropoiesis in multiple myeloma: defective red cell production due to inappropriate erythropoietin production.

We investigated the pathophysiology of erythropoiesis in 62 patients with multiple myeloma and examined whether it would establish a rational basis for the treatment of their anaemia with recombinant human erythropoietin. Erythropoietin (Epo) production was evaluated by serum levels and erythropoiesis was quantitated by serum transferrin receptor (TfR) levels, both assessed relative to the degree of anaemia. Instead of the expected stimulation of erythropoiesis in response to anaemia, haematocrit correlated positively with marrow erythropoietic activity, indicating that the mechanism of anaemia was primarily defective red cell production. Erythropoiesis decreased and anaemia worsened significantly with advancing clinical stage. 25% of the patients had inadequate Epo production and this proportion increased to 50% in stage 3. Inappropriate Epo production was seen in 60% of patients with renal impairment but was also observed in a number of patients with normal renal function. Erythropoiesis correlated strongly with the adequacy of Epo production, particularly in advanced disease. We conclude that most myeloma patients have defective red cell production even in the absence of massive marrow infiltration and that inappropriate Epo production contributes to their anaemia.

Adult

Production of prostaglandins by the pseudopregnant rat uterus, in vitro, and the effect of tamoxifen with the identification of 6-keto-prostaglandin F1alpha as a major product.

1 Prostaglandin production by rat uterus homogenates has been studied, in vitro, on days 2 to 13 of pseudopregnancy. 2 The highest production of prostaglandins occurred on day 5. 3 The amounts of prostaglandins F and D formed were higher than the amounts of prostaglandin E on every day studied. 4 The ratios of prostglandins F and D to prostaglandin E produced steadily decreased up to day 6. It then increased with the highest values occurring between days 10 and 13, 5 Progesterone levels in peripheral plasma increased rapidly from days 2 to 5, remained high up to day 9, then steadily decreased between days 10 and 13. 6 The anti-oestrogenic drug, tamoxifen administered on day 2, significantly inhibited the increase of prostaglandin production which occurred on day 5. Prostaglandin E production was inhibited more than the production of prostaglandins F and D. 7 Analysis of the uterine extracts by gas chromatography and mass spectrometry showed prostaglandin F2alpha, F1alpha (in trace amounts), E2 and D2 to be present. 8 The major product detected was 6-keto-prostaglandin F1alpha. Its identification forms an addendum to the paper. 9 Also present as a major product was 6(9)-oxy-11,15-dihydroxyprosta-7,13-dienoic acid.

Animals

Enhancing effect of mouse peritoneal exudate cells and their products on antibody productivity of hybridoma cells: application of in vivo factors to in vitro culture.

Mouse peritoneal exudate cells induced by casein enhanced in vitro antibody production rate per cell of a hybridoma in co-culture. Culture supernatant of the exudate cells also enhanced three-fold the antibody productivity when added to cultures of a hybridoma at 10% (v/v). Hence the enhancement of antibody productivity by the exudate cells seemed to be caused by soluble enhancing factors secreted by the exudate cells. The exudate cells maximally secreted the enhancing factors when harvested from mice on day 4 of the induction period following the injection of casein. A semi-continuous culture of the hybridoma demonstrated the applicability of the culture supernatant to enhance antibody production by producing a two-fold increase over the control for seven days when supplemented with the supernatant at 5%. Significant amounts of interleukin-6 were detected in culture supernatant of the exudate cells. Interleukin-6 obtained from other sources enhanced the antibody productivity two-fold when added to the hybridoma culture at the concentration of 5 unit/ml. Interleukin-6, therefore, is expected to be one of the principal antibody enhancing factors secreted by the exudate cells. Other interleukins examined, that is, interleukin-1 to -5 did not enhance the antibody productivity.

Animals

Influence of medium composition on the cephalosporin C production with a highly productive strain Cephalosporium acremonium.

Cephalosporin production by a highly productive Cephalosporium acremonium strain was carried out and optimized by fed-batch operation in a 40 l stirred tank reactor using a complex medium containing 30-120 g l-1 peanut flour. The concentrations of cephalosporin C (CPC) and its precursors: penicillin N (PEN N), deacetoxy cephalosporin C (DAOC), and deacetyl cephalosporin C (DAC) were monitored with an on-line HPLC. The concentrations of amino acids valine (VAL), cysteine (CYS), alpha-amino adipic acid (alpha-AAA), the dipeptide alpha-amino-adipyl-cysteine (AC), and the tripeptide alpha-amino-adipyl-cysteinyl-valine (ACV), were determined off-line by HPLC. The RNA content and dry weight of the sediment as well as the oxygen transfer rate (OTR) and the CO2 production rate (CPR) were used to calculate the cell mass concentration (X). The influences of peanut flour (PF) and the on-line monitored and controlled medium components: glucose (GLU), phosphate, methionine (MET) as well as the dissolved oxygen (DOC) on the cell growth, the product formation, and the pathway of cephalosporin C biosynthesis were investigated and evaluated. When the glucose fed-batch cycle was optimized and oxygen transfer limitation was avoided (DOC greater than 20% of the saturation value), high process performance (103.5 g l-1 X, 11.84 g l-1 CPC, a maximum CPC productivity of 118 mg l-1 h-1, and the whole concentration of the beta-lactam antibiotics CPC, DAC, DAOC, PEN N 17.34 g l-1) was achieved by using 100 g l-1 PF in the medium with the optimum concentration of phosphate (260-270 mg l-1) and a low glucose concentration (less than 0.5 g l-1). The cultivations with different medium concentrations demonstrated that the product formation was directly proportional to the cell mass concentration. On the average, the cell mass-based yield coefficient of CPC: YCPC/X amounted to 0.115 g CPC per g cell mass.

Acremonium

Epstein-Barr virus BCRF1 gene product (viral interleukin 10) inhibits superoxide anion production by human monocytes.

Due to its similar biological activities to interleukin 10 (IL-10), Epstein-Barr virus (EBV) BCRF1 gene product (viral IL-10: vIL-10) has recently been recognized as an analogue of authentic IL-10. Preincubation of human monocytes with vIL-10, like human IL-10, induced smaller amounts of interferon-gamma (IFN-gamma) mRNA in activated human peripheral blood mononuclear cells (PBMNCs) than nonpreincubation, indicating that vIL-10 acts principally on monocytes. Since the activation of monocytes and their generation of oxidative products are regulated by various cytokines, we examined the effects of vIL-10 on superoxide anion (O2-) production by human PBMNCs and monocytes. Not only PBMNCs but also monocytes preincubated with vIL-10 showed a smaller production of O2-. Inhibition was achieved in a dose-dependent fashion and increased gradually after incubation with vIL-10. Additions of IFN-gamma, macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF), which prime monocyte activation and induce O2- production, were also affected by the reciprocal effect of vIL-10. Thus, vIL-10 production by EBV-infected cells may be involved in the development of EBV-related disorders.

Base Sequence

Mineral content of dairy products. I. Milk and milk products.

Sixteen kinds of dairy products were analyzed for five major minerals and four trace minerals. Commercial samples of fluid milk, cream, concentrated milks, cultured products, butter, and frozen desserts were also analyzed for fat, solids, protein, and minerals. Calcium, magnesium, sodium, and potassium were determined by atomic absorption, and phosphorus was determined colorimetrically. Flameless atomic absorption was used to determine iron, zinc, copper, and manganese. The data generally agreed with most recently published values. However, for the trace minerals, the data differed widely in some instances. Manufacturing practices and added ingredients produced considerable variations in mineral content of market samples. These variations, however, could be limited by selection of products, so that they would not preclude the use of dairy products in diets in which mineral composition must be controlled. The coefficients of variation, which indicate the variability that can be expected for each product, generally were high for sherbet and ice milk and low for low-fat milk and skim milk.

Animals

Production of interferon in the murine mixed lymphocyte culture. II. Interferon production is a T cell-dependent function, independent of proliferation.

Interferon production occurs after two days of culture in murine mixed lymphocyte cultures (MLC). This was demonstrated in various combinations of mouse spleen cells differing at the major histocompatibility (H-2) locus. Interferon production could be demonstrated in one-way MLC when F1/parent combinations were used and in reactions in which one partner was treated by puromycin. After treatment of both cell populations with mitomycin C, interferon production occurred in the absence of lymphoproliferation. Interferon production in response to alloantigen did not occur in spleen cell cultures of nude mice and in cultures treated by anti-theta antiserum plus complement indicating that interferon production is a T cell-dependent function.

Animals

[The behavior of polyphosphates during production and storage of long keeping milk products (author's transl)].

Polyphosphates added as stabilizers in the production process if liquid sterilized milk products are completely hydrolyzed to mono- and diphosphates. The diphosphate is further hydrolyzed during storage of the products. The extent of hydrolysis of diphosphate depends upon the processing conditions and may vary from production to production. During spray-drying of milk, the polyphosphates added are only partially hydrolyzed; further degradation of the polyphosphates takes place during storage.

Animals