Heat-labile Factor Research Articles

Nutritional evaluation of grain amaranth ( Amaranthus hypochondriacus) in broiler diets

The present study was conducted to evaluate the feeding and energy utilization values of raw and autoclaved grain amaranth ( Amaranthus hypochondriacus) in broiler diets. The grain amaranth sample used in the study contained (g kg −1): crude protein, 168; crude fat, 58; crude fibre, 60; ash, 26; calcium, 2.2; total phosphorus, 5.6; lysine, 10.1; methionine, 3.5. In trial 1, raw amaranth was incorporated into a maize-soyabean meal-meat meal diet at 0, 200, 400 and 600 g kg −1 levels and fed to broiler chicks from 7 to 16 days of age. Weight gains and feed intake were depressed ( P < 0.01) with increasing levels of raw amaranth in the diet. Feed/gain values were similar between birds fed on diets containing 0 and 200 g kg −1 amaranth, but increased ( P < 0.01) beyond 200 g kg −1 level of inclusion. Trial 2 was conducted to evaluate whether the growth-depressing effects of raw amaranth grain could be overcome by autoclaving (130°C for 1 h). Weight gains, feed intake and feed/gain of chicks fed on 200 and 400 g kg −1 autoclaved amaranth diets were similar ( P > 0.05) to those fed on the control diet. The apparent metabolizable energy (AME) of grain amaranth was improved by autoclaving. The AME (MJ kg −1 DM) values of raw and autoclaved amaranth were 11.85 ± 0.29 and 13.11 ± 0.26, respectively. The positive responses to autoclaving, in terms of feeding and energy utilization values, suggest the presence of heat-labile anti-nutritional factor(s) in raw amaranth and studies are warranted to identify these factors. The results indicate that processed A. hypochondriacus grain is a potentially useful energy supplement for poultry and can be incorporated in broiler diets at levels up to 400 g kg −1 without adverse effects on performance.

Nuclear localization signal of SV40 T antigen directs import of plasmid DNA into sea urchin male pronuclei in vitro.

Nuclear import of plasmid DNA mediated by a nuclear localization signal (NLS) derived from SV40 T antigen was investigated in a cell-free extract. In vitro assembled sea urchin male pronuclei were incubated in a 100,000g supernatant of a zebrafish fertilized egg lysate, together with fluorescently labeled plasmid DNA bound to NLS or nuclear import deficient reverse NLS (revNLS) peptides. After 3 hr, DNA-NLS, but not DNA-revNLS, complexes were bound around the nuclear periphery. We demonstrate that nuclear import of DNA-NLS complexes is a two-step process involving binding to, and translocation across, the nuclear envelope. Binding is ATP-independent, occurs at 0 degree C and is Ca(2+)-independent. By contrast, translocation requires ATP hydrolysis, Ca2+, is temperature dependent and is blocked by the lectin wheat germ agglutinin. Both binding and translocation are competitively inhibited by albumin-NLS conjugates, require heat-labile cytosolic factors, and are inhibited by N-ethylmaleimide treatment of the cytosol. Binding and translocation are differentially affected by cytosol dilutions, suggesting that at least two distinct soluble fractions are required for nuclear import. The requirements for NLS-mediated nuclear import of plasmid DNA are similar to those for nuclear import of protein-NLS conjugates in permeabilized cells.

The Secreted Products ofPorphyromonas gingivalisAlter Human Gingival Fibroblast Morphology by Selective Damage to Integrin-Substrate Interactions

Porphyromonas gingivalis is implicated in periodontal diseases characterised by connective tissue destruction. This study examines the damaging effects of products of P. gingivalis strain W83 on human gingival fibroblast (HGF) integrins and cytoskeletal components. Within 18 h of exposure of subconfluent monolayers of HGF to the culture supernatant of P. gingivalis , diluted from 1/2 to 1/32 (protein concentrations 5.15 to 0.32 mg/ml), morphological changes leading to significant detachment from the surface of the culture vessel (P<0.05) were observed. Loss of attachment was concentration-dependent, not accompanied by cell death and due primarily to heat-labile component(s) of the supernatant. These changes were preceded by rapid loss of α 5 and β 1 integrin subunit expression and fibronectin which all occurred within 60 min and became more marked on incubation for 18 h. Pretreatment of the culture supernatant with the protease inhibitors leupeptin (50 μM) or Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK, 2 mM) abolished hydrolysis of Nα-benzoyl-L-arginine p-nitroanilide (L-BApNA) but failed to prevent cell detachment or the loss of β 1 integrin expression, implicating heat-labile factor(s) other than arginine- or lysine-specific proteinases. Reduction in α v and β 3 integrin subunit expression was seen 18 h after exposure to culture supernatant (≥0.16 mg protein/ml) and loss of F-actin stress fibres at ≥1.28 mg protein/ml. However, the actin-binding protein vinculin remained unaffected under these conditions. These differential effects of P. gingivalis on the membrane-associated integrins and cytoskeletal components of human gingival fibroblasts may contribute to the pathogenic mechanisms involved in the loss of connective tissue which characterises destructive periodontitis. Keywords: human gingival fibroblast, integrin, Porphyromonas gingivalis .

Isolation of a heat-labile protein factor involved in the 3′-processing of H4 pre-mRNA

It is well documented that the snRNP particles play a role in the processing of histone pre-mRNAs. There is also evidence that in addition to the snRNP particles, at least two more proteins are involved in the in vitro 3′-processing of histone pre-mRNAs. The aim of the present work was the isolation and purification of one of these PF250 factors. Processing factor PF250 was isolated from HeLa nuclear extracts using ion exchange chromatography. Its ability to partially restore the processing activity of heat inactivated nuclear extract was demonstrated in vitro using labelled H4 pre-mRNA. Our results indicate that nuclear extracts exposed to 50°C for 15 min lacked any processing activity. Using a series of column chromatography purification steps we isolated a thermolabile protein factor that partially restored the activity of heat inactivated HeLa cell nuclear extracts. It can be concluded that besides U7 snRNP particles at least one more thermolabile protein factor is required for the maturation of H4 pre-mRNA in vitro. It has a molecular mass of 30 kDa and is inactivated by heating at 50°C. These characteristics make it a likely candidate to be a regulatory factor involved in pre-mRNA processing.

Evidence That the Soluble Factors Secreted by Activated Immune Cells Suppress Replication of Human Neurotropic JC Virus DNA in Glial Cells

Coordination of the immune response to viral infection and disease in the brain is believed to involve bidirectional interaction between the immune system and the central nervous system (CNS). Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the CNS that generally affects patients exhibiting an immunocompromised condition due to various illnesses. The human polyomavirus, JCV, which infects greater than 70% of the adult population is the etiological agent of this disease. Infection with JCV occurs during childhood and the virus remains in the latent state with no apparent clinical signals. However, under immunocompromised conditions, the virus enters the lytic cycle, and upon cytolytic destruction of glial cells, causes PML. To understand the molecular mechanism underlying immune regulation of JCV replication, we have developed a cell culture system and have investigated the effect of soluble factors from T-cell cultures on replication of JCV DNA in glial cells. Our data demonstrate that replication of JCV DNA in the presence of PMA-stimulated T-cell supernatant is substantially decreased in transfected glial cells. Heat-inactivation and size-fractionation studies revealed participation of a heat labile factor(s) which loses its maximum activity at 60° and ranges between 30 and 100 kDa in size. The unfractionated T-cell supernatant and the fraction enriched in 30- to 100-kDa proteins reduced the level of viral DNA replication during the early phase of the lytic cycle. These observations suggest that regulatory factors which are secreted by immune cells may modulate the level of JCV DNA replication in glial cells. The importance of these observations in reactivation of JCV in immunocompromised individuals and development of PML is discussed.

Immunomodulatory activities of whey fractions in efferent prefemoral lymph of sheep

Studies on the immunomodulatory activities of ruminant milk and colostral whey fractions were undertaken. By comparing with boiled colostral whey in a preliminary experiment, a putative heat-labile immunostimulatory factor for antibody responses was found to be present in ovine colostral whey. Studies were then undertaken in sheep in which the efferent prefemoral lymphatic ducts were cannulated bilaterally, and immune responses in the node were measured following subcutaneous injection in the flank fold of whey protein preparations of various purities. A significant sustained decline of efferent lymphocyte output was observed following injection with autologous crude milk whey or colostral whey preparations, but no changes were observed in interferon-gamma levels in lymph plasma. Two bovine milk whey fractions (lactoperoxidase and lactoferrin) of high purity were compared in bilaterally cannulated sheep. A transient decline over the first 6 h was seen in the efferent lymphocyte output and lymph flow rate after injection of both fractions. A significant difference was seen between the two fractions in interferon-gamma levels in lymph at 6 h after injection. However, no significant changes in the proportion of the various efferent lymphocyte phenotypes were seen following either treatment. Whereas both fractions showed a significant inhibitory effect in a dose-dependent manner on the proliferative response of T lymphocytes, but not B lymphocytes, to mitogenic stimulation in vitro, no similar changes were seen following in vivo stimulation with these two fractions.

Adherence to and damage of endothelial cells by Cryptococcus neoformans in vitro: role of the capsule.

Escape from the intravascular compartment is likely a critical step in the development of hematogenously disseminated cryptococcal infections, such as meningitis. The capsule of Cryptococcus neoformans is considered to be a virulence factor because of its antiphagocytic properties. To further investigate the role of the capsule in escape from the intravascular compartment, we used isogenic strain pairs, an acapsular mutant, and an encapsulated clinical isolate to determine the effects of the capsule of C. neoformans on adherence to, phagocytosis by, and damage of endothelial cells in vitro. Acapsular C. neoformans adhered significantly more to endothelial cells and caused greater endothelial cell injury than did encapsulated organisms. Coating of an acapsular strain with cryptococcal glucuronoxylomannan decreased both adherence to and damage of endothelial cells by 61.7% +/- 9.1% and 76.6% +/- 10.2%, respectively. Transmission electron microscopy demonstrated internalization of acapsular, but not encapsulated, organisms by endothelial cells. Internalization of an acapsular strain occurred through endothelial cell phagocytosis and was inhibited by cytochalasin D. Phagocytosis required a heat-labile serum factor, probably complement. These results suggest that acapsular or poorly encapsulated C. neoformans may be the form(s) that escapes from the vasculature during initiation of hematogenously disseminated disease.

Mechanisms of Enhancement of Natural Killer Activity by an Antibody to CD44: Increase in Conjugate Formation and Release of Tumor Necrosis Factor α

In this report, we found that an anti-CD44 mAb, S5, augmented conjugate formation between the effector cells (canine PBMC) and an NK-sensitive target cell line [canine thyroid adenoma carcinoma cell line (CTAC)], as determined by two different methods: conjugate enumeration using fluorescence microscopy and two-color flow cytometric analysis. However, no increase in conjugate formation was seen when an NK-resistant target was used. This enhancement of initial conjugate formation could not be blocked using an antibody to CD18 (60.3), indicating that S5 possibly acted through another adhesive molecule(s). But the NK killing activity can be partially blocked by 60.3, indicating that this molecule may be important for later adhesive events. We also found that S5 stimulated the release of a heat labile cytotoxic factor from canine PBMC. This factor was found to lyse only TNFα-sensitive targets (CTAC and L929 cell lines) and caused apoptosis in the target cells. Its bioactivity was neutralized by polyclonal antibody to TNFα. All of these observations were consistent with the fact that the factor was TNFα Our data suggested that the two mechanisms responsible for the enhancement in canine NK activity by an mAb to CD44 were an increase in conjugate formation and the release of TNFα.

Identification of C1q as the heat-labile serum cofactor required for immune complexes to stimulate endothelial expression of the adhesion molecules E-selectin and intercellular and vascular cell adhesion molecules 1.

To examine the role of complement components as regulators of the expression of endothelial adhesive molecules in response to immune complexes (ICs), we determined whether ICs stimulate both endothelial adhesiveness for leukocytes and expression of E-selectin and intercellular and vascular cell adhesion molecules 1 (ICAM-1 and VCAM-1). We found that ICs [bovine serum albumin (BSA)-anti-BSA] stimulated endothelial cell adhesiveness for added leukocytes in the presence of complement-sufficient normal human serum (NHS) but not in the presence of heat-inactivated serum (HIS) or in tissue culture medium alone. Depletion of complement component C3 or C8 from serum did not prevent enhanced endothelial adhesiveness stimulated by ICs. In contrast, depletion of complement component C1q markedly inhibited IC-stimulated endothelial adhesiveness for leukocytes. When the heat-labile complement component C1q was added to HIS, the capacity of ICs to stimulate endothelial adhesiveness for leukocytes was completely restored. Further evidence for the possible role of C1q in mediating the effect of ICs on endothelial cells was the discovery of the presence of the 100- to 126-kDa C1q-binding protein on the surface of endothelial cells (by cytofluorography) and of message for the 33-kDa C1q receptor in resting endothelial cells (by reverse transcription-PCR). Inhibition of protein synthesis by cycloheximide blocked endothelial adhesiveness for leukocytes stimulated by either interleukin 1 or ICs in the presence of NHS. After stimulation with ICs in the presence of NHS, endothelial cells expressed increased numbers of adhesion molecules (E-selectin, ICAM-1, and VCAM-1). Endothelial expression of adhesion molecules mediated, at least in part, endothelial adhesiveness for leukocytes, since leukocyte adhesion was blocked by monoclonal antibodies directed against E-selectin. These studies show that ICs stimulate endothelial cells to express adhesive proteins for leukocytes in the presence of a heat-labile serum factor. That factor appears to be C1q.

Coordinate regulation of nitric oxide and 1,25-dihydroxyvitamin D production in the avian myelomonocytic cell line HD-11.

Cells of the monocyte/macrophage lineage are capable of both nitric oxide (NO) and 1,25-dihydroxyvitamin D [1, 25-(OH)2D] production through expression of inducible nitric oxide synthase (iNOS) and a putative 25-hydroxyvitamin D (25-OHD)-1-hydroxylase, respectively. We have recently reported that 1,25-(OH)2D synthesis in the chick myelomonocytic cell line HD-11 is restricted by inhibition of iNOS. In the current set of experiments, measuring nitrite, a stable water-soluble secreted metabolite of NO as an index of iNOS activity and 1,25-(OH)2D3 in lipid extracts of cells incubated with 200 nM 25-OHD3 as an index of 1-hydroxylase activity, we demonstrate that NO and 1,25-(OH)2D production by HD-11 cells are temporally related, induced by the same kinds of activating agents, and coordinately regulated. NO and 1,25-(OH)2D3 production by HD-11 cells was stimulated severalfold by the macrophage stimulators interferon-gamma and lipopolysaccharide and by an autologous, nonlipid, heat-labile factor with an apparent molecular mass approximately 10,000 daltons. As expected NO synthesis was 1) dependent upon the presence of L-arginine in the extracellular medium, 2) subject to significant stimulation by Nw-hydroxy-L-arginine, an L-arginine-derived intermediate in NO biosynthesis, and by sodium nitroprusside, a non-L-arginine-dependent source of intracellular NO, and 3) inhibited by Nw-nitro-L-arginine methyl ester, a competitive inhibitor of iNOS. At high NO production rates, induced either by high-dose lipopolysaccharide or by sodium nitroprusside exposure, there was an apparent downturn in 1,25-(OH)2D3 synthesis, suggesting functional dependence of the 1-hydroxylase on NO but ultimate inhibition of 1,25-(OH)2D3 synthetic capacity at high levels of intracellular NO production. On the basis of these results we postulate that the macrophage 25-OHD-1-hydroxylation reaction may be dependent on iNOS-generated NO as a soluble source of electrons and regulated in an autocrine mode by a macrophage-derived NO stimulatory factor and NO itself.

Antibody Responses against Escherichia coli Heat-Labile Toxin and Colonization Factor Antigens I and II in Argentinian Children

Serum antibody responses against Escherichia coli heat-labile enterotoxin (LT) and colonization factor antigens (CFAs) I and II were studied in 84 children < 5 years old living in two communities. These villages differed in the quality of their drinking water. Children from both communities developed significantly increased antibody titers against LT and CFA/II but not against CFA/I during 3 months of follow-up. The magnitude of the anti-LT response was significantly higher in children from Zaiman than in those from Las Dolores. Antibody titers rose to maximum levels during the second year of age and reached relatively constant levels in children aged 2-5 years, probably due to repeated exposure to enterotoxigenic E. coli strains. Antibody levels of 30 children were followed for 2 years; increases in anti-LT and anti-CFA titers varied in the different age groups.

Complement Synthesis Influencing Factors Produced by Acute Myeloid Leukemia Blast Cells.

In a previous study, we found hypercomplementaemia in the sera of acute myeloid leukemia patients. In this study we show that the supernatants of mononuclear cells, derived from peripheral blood taken in the blastic phase, from patients with acute myeloid leukemia (CM-AML) increased the in vitro complement protein synthesis of HepG2 hepatocellular carcinoma cells. This effect of CM-AML was mediated by heat labile soluble factors and involved the synthesis of mRNA and protein. Inhibition experiments with anti-cytokine antibodies and immunoaffinity chromatography revealed that this effect of CM-AML is mostly mediated by IL-1 and IL-6.

Effects of Conditioned Medium from Different Cultured Cell Types on Aromatase Expression in Adipose Stromal Cells

To determine whether serum-free (SF) conditioned media (CM) from several human breast cancer cell lines and primary stromal cell cultures contain factor(s) that mimic the marked stimulatory effects of serum on aromatase activity and aromatase P450 (P450arom) gene expression in adipose stromal cells in culture (ASC) in the presence of dexamethasone (DEX). Adipose stromal cells, harvested from fresh adipose specimens, were grown to confluence, switched to SF media, and then incubated in the presence or absence of DEX with CM from T47-D breast cancer cells, pre-treated with or without 17 beta-estradiol (E2), and with CM from stromal cell cultures. Aromatase activity of the ASC was determined by the [3H] water release assay. Total RNA was isolated, and reverse transcription-polymerase chain reaction was performed to determine the expression of various 5'-termini. T47-D CM stimulated aromatase activity in a concentration-dependent manner, similar to that of serum, in ASC incubated with DEX. Estrogen potentiated this in a dose-dependent fashion. The ASC CM and endometrial stromal cell CM also markedly induced aromatase activity in ASC. Heat inactivation destroyed the stimulating ability of CM. The majority of P450arom 5'-termini expressed by ASC incubated with CM plus DEX contained the promoter I.4-specific sequence. Conditioned media from several breast cancer cell lines and primary stromal cell cultures can mimic the effects of serum in the presence of DEX to stimulate aromatase activity in ASC. These results suggest that undefined, heat-labile and proteinaceous factors are present in CM that stimulate P450arom expression in a fashion similar to that of serum.

Influence of osteoclasts and osteoclast-like cells on osteoblast alkaline phosphatase activity and collagen synthesis

Osteoblasts have been shown to modulate osteoclast activity, but the reverse process has not been investigated. In the current study conditioned medium (CM) was collected from osteoclasts and osteoclast-like cells and its effects on osteoblast alkaline phosphatase (ALPase) activity and collagen synthesis ([3H]proline hydroxylation) were determined. In primary chick osteoblasts, cultured chick embryo frontal bones, and UMR-106-01 cells, collagen synthesis and ALPase activity, but not [3H]thymidine incorporation, were inhibited by CM from chick marrow-derived giant cells, which possess some of the phenotypic characteristics of osteoclasts. However, collagen synthesis in chick embryo fibroblasts was not affected by giant cell CM. CM collected from cultures of chicken osteoclasts and human osteoclastoma cells and marrow-derived giant cells inhibited collagen synthesis in UMR-106-01 cells, but the effects of ALPase activity varied with the cell type. In contrast, mononuclear cell and fibroblast CM did not alter collagen synthesis. Initial characterization studies demonstrate that the inhibitor is a heat-labile factor with a molecular weight greater than 3500. In summary, authentic osteoclasts, tumor osteoclast-like cells, and chicken and human multinucleated giant cells produce a soluble factor that alters osteoblast collagen synthesis, suggesting that osteoclasts play a role in the modulation of osteoblast activity.

Coaggregation between Porphyromonas gingivalis and mutans streptococci.

Coaggregation occurred between Porphyromonas gingivalis and mutans streptococci. The coaggregation was completely inhibited by L-arginine, N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), and a trypsin inhibitor, and weakly inhibited by L-lysine, N-ethylmaleimide, lysozyme, and human whole saliva. The results of heat and proteinase K treatment suggested that a heat-labile proteinaceous substance of P. gingivalis and a heat-stable substance of mutans streptococci may play a role in the coaggregation. Mutans streptococci also aggregated in the presence of the heat-labile factor in the supernatant of P. gingivalis. The aggregation was also inhibited by L-arginine, TLCK, and a trypsin inhibitor.

Opsonic effect of globulins on phagocytosis of liposomes by bone marrow cells.

In recent years there has been a great surge in the development of particulate drug carriers for chemotherapy and diagnostic medicine. However, to realise the full potential of various drug carriers such as liposomes, it is necessary to understand how these particles are cleared from the circulation when injected intravenously, and what determines their recognition by the reticuloendothelial cells in the liver, spleen and bone marrow. In the circulation the drug carriers become coated with plasma components and this process is thought to determine their recognition by the reticuloendothelial cells. Liposomes coated in vifro with opsonins such as immunoglobulin, components of the complement system, fibronectin. C-reactive protein etc., when injected intravenously are cleared rapidly from the circulation (1). Recently we have demonstrated the presence of opsonins in serum which exhibit tissue specificity and described their properties which differentiate between 'liver-specific' and 'spleen-specific' opsonins responsible for the enhancement o f phagocytosis of liposomes by Kupffer cells and splenic macrophages respectively (2,3). However, so far there has been no clear indication to suggest the identity of the tissue-specific opsonin involved in phagocytosis of the injected liposomes. Bone marrow is one of the major organs of the reticuloendothelial system and plays a small but significant role in the clearance of foreign particulates including liposomes. In vifro serum is found to enhance the uptake of cholesterol-containing liposomes in the bone marrow cells. This opsonic activity of serum is destroyed when the serum is heated at 55°C for 30 min. This suggests that heat-labile complement may be involved in the phagocytosis of liposomes. However, when dialysed or EGTA-chelated serum is used, a complete loss of its opsonic activity occurs. Addition of the serum dialysate or divalent cations to the dialysed serum does not reinstall its lost opsonic activity (4). Thus these results shed doubt on the possibility of involvement of the complement and hence we searched for another heat-labile serum factor which may be responsible for enhancing phagocytosis of liposomes in these cells. Heat-labile pand y-globulin are known to enhance phagocytosis of certain strains of bacteria (1) and p-globulin is also known to enhance uptake of anionic liposomes into the perfused liver (5). Hence we examined the effect of various serum globulins on the uptake of neutral high cholesterol liposomes in the freshly prepared bone marrow Cells. The results in Table 1 show that y-globulin and immunoglobulin in the absence or presence of serum have little effect on the uptake of liposomes in the bone marrow cells, whereas p-globulin in the absence of serum stimulates the uptake of liposomes by 5-fold as compared to the control and it retains its opsonin activity in the presence of serum. Thus these results suggest that p-gloublin may be the heat-labile serum factor that enhances phagocytosis of liposomes in the bone marrow. Table 1

NAD-independent Actinobacillus pleuropneumoniae strains: Production of RTX toxins and interactions with porcine phagocytes

Actinobacillus pleuropneumoniae RTX toxin (Apx) production by A. pleuropneumoniae biotype 2 (NAD-independent) serotype 2 strains was studied. Western blot analysis of culture supernatants of all biotype 2 strains tested revealed the presence of a 103 kDa protein which reacted with a monoclonal antibody against ApxIIA. This protein was also recognized by sera of pigs infected with a biotype 2-serotype 2 strain. Furthermore, antibodies that could neutralize ApxIIA were present in these sera. Proteins corresponding to ApxIA or ApxIIIA were not detected. The effects of a biotype 1-serotype 2 and a biotype 2-serotype 2 strain and their metabolites on the oxidative activity of porcine pulmonary alveolar macrophages (PAM) and polymorphonuclear cells (PMN) were compared using a chemiliminescence (CL) technique. Viable bacteria of both biotypes stimulated the production of oxygen radicals by phagocytes. CL responses were higher for the biotype 1 than for the biotype 2 strain. After having reached a peak value, the oxidative activity decreased until a total inhibitions was achieved. Inactivated washed bacteria had no influence on the oxidative activity of phagocytes. In contrast, heat labile factors in culture supernatants of both biotypes stimulated and inhibited the oxidative activity of PAM in a dose-dependent manner. Dilutions of supernatant up to 1 32 of the biotype 2 strain and up to 1 512 of the biotype 1 strain were toxic for PAM, while dilutions from 1 64 to 1 128 of the biotype 2 strain and from 1 1024 to 1 4096 of the biotype 1 strain stimulated the oxidative activity. To evaluate the ability of PAM to eliminate low numbers of the biotype 1-serotype 2 and the biotype 2-serotype 2 strain, 100 CFU of each strain was incubated with 10 5 PAM. The bacterial growth and the viability of PAM were assessed as a function of time. The biotype 1 strain killed PAM more rapidly and multiplication of the bacteria was higher than for the biotype 2 strain.

Heat shock affects 5' splice site selection, cleavage and ligation of CAD pre-mRNA in hamster cells, but not its packaging in InRNP particles.

The effect of heat shock on the packaging and splicing of nuclear CAD pre-mRNA, a transcript expressed constitutively from a non heat-inducible promoter, was studied in vivo in Syrian hamster cells. While mild heat shock did not affect significantly the packaging of CAD RNA in 200S InRNP particles, it caused perturbation to splicing. First, the heat shock inhibited splicing of CAD pre-mRNA. Second, it affected 5' splice site selection by activating cleavage at a cryptic 5' splice site; yet ligation of the cryptic exon to the downstream proximal exon was not observed. Base complementarities of the cryptic site with U1, U5, or U6 snRNAs are comparable, or even better, than those with the neighboring normal site. Hence, the exclusion of the cryptic site under normal growth conditions cannot be attributed to weaker base pairing with these snRNAs. On the other hand, these results imply the involvement of a heat labile factor in the selection of the 5' cleavage site. The exclusion of the cryptic site at 37 degrees C and the aborted splicing at this site after heat shock may also be explained by a proposed nuclear checking mechanism that detects in-frame stop codons upstream of the 5' splice site, and aborts splicing at such sites to prevent the production of a defective message.

Factors affecting Yersinia enterocolitica (serotype O:8) viability in deliberately inoculated blood.

Interpretation of in vitro experiments using Yersinia enterocolitica in blood components requires information on factors affecting the organism's survival. Several factors were found to influence the survival of Y. enterocolitica (serotype O:8) in blood components. A 20-minute room-temperature incubation with plasma-containing components resulted in approximately 2 log10 inactivation. Inactivation could be prevented by preincubation treatment of the plasma at 55 degrees C for 1 hour, which suggests the involvement of heat-labile plasma factors. No antibacterial activity was observed in washed red cells during the 20-minute room-temperature incubation. However, Y. enterocolitica colony-forming units declined by up to 2 log10 in washed red cells during the first days of 4 degrees C storage. Use of a white cell-reduction filter on freshly inoculated samples removed approximately 1 log10 of the organism regardless of whether bacteria were suspended in saline or washed red cells. Thus, bacterial levels may be affected by plasma, cellular components, and white cell-reduction filters. However, caution should be exercised in interpreting in vitro spiking studies designed to investigate the potential benefits of white cell reduction to eliminate the growth of Y. enterocolitica because of potential differences between naturally infected and experimentally inoculated blood.

Cytokines in stools of children with inflammatory bowel disease or infective diarrhoea.

To determine the concentrations of interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF alpha) in stools from children. Stool samples from 14 healthy children, 32 children with inflammatory bowel disease, and 23 children with acute diarrhoea were emulsified in an equal volume of phosphate buffered saline and then centrifuged to produce a clear supernatant fluid. IL-6 and TNF alpha were measured by enzyme linked immunosorbent assay (ELISA). TNF alpha was detected in the stools of all 14 healthy children (12-130 pg/g stool), but IL-6 was detected only in three. Similar results were seen in children with inactive inflammatory bowel disease. Stool TNF alpha concentrations were raised in samples from children with active inflammatory bowel disease, but in most (11/18) of these samples IL-6 was undetectable. Stool samples contained a heat-labile factor which rapidly destroyed IL-6 immunoreactivity. Most children with diarrhoea had TNF alpha concentrations similar to those of healthy controls and most were also negative for IL-6. Three children with Shigella flexneri infection had extraordinarily high concentrations of both TNF alpha and IL-6 in their stools. There is constant low grade production of TNF alpha in the intestine of healthy people. Raised values are indicative of mucosal inflammation, but are not specific. Stool IL-6 is of little use in assessing mucosal inflammation because immunoreactivity is rapidly lost in stool samples.