A mutation in the folA promoter delays adaptation to minimal medium by Escherichia coli K-12

In its natural environment, Escherichia coli is subjected to frequent changes in nutrients and physical parameters such as temperature. Adapting to new conditions requires an orderly progression of events. We show that a folA mutant that overexpressed dihydrofolate reductase because of a C to T promoter mutation, adapted very slowly when transferred from rich medium at 37 degrees C to glucose minimal medium at 42 degrees C. It adapted more rapidly when serine and threonine were added. At 37 degrees C, it was more sensitive than its parent to serine, pyruvate and valine. We propose that the mutant does not increase synthesis of serine and threonine normally upon transfer to minimal medium. This results in a limited availability of serine, threonine and isoleucine during adaptation. Phenotypically, this is manifested as a growth delay at 42 degrees C and an increased sensitivity to isoleucine restriction at 37 degrees C.     

Studies on the mechanisms of macrophage activation. III. Investigations on the interaction of macrophage-activating factor with the macrophage membrane

A macrophage-activating factor (MAF), produced by concanavalin (Con A)-stimulated spleen lymphocytes, was tested by its capacity to induce mouse peritoneal exudate macrophages to destroy the intracellular parasite Leishmania enriettii. MAF could be removed from active media by adsorption on macrophages at 37°C and 4°C; the adsorption was not specific, as cells from various origins could similarly be used to deplete supernatants of their activity. Treatment of macrophages with trypsin resulted in loss of response to MAF, presumably due to inactivation of a membrane receptor for the lymphokine. In addition, trypsin treatment of macrophages, following a 6-h pulse, but not a 9-h-pulse, of MAF-rich medium, inhibited activation suggesting that triggering of the biochemical processes of activation required interaction between macrophages and the lymphokine of sufficient duration, and that once these processes were initiated, parasite destruction could proceed to completion in the absence of further exogenous lymphokine. MAF activity was enhanced by treatment of macrophages with al-antitrypsin suggesting a role for membrane-associated serine esterases in modulating the cell response to the lymphokine. High concentrations of Con A also impaired MAF-induced activation, presumably through binding of the lymphokine by the lectin.     

Binding and lysis of antibody-coated human erythrocytes by activated human monocytes

Human monocytes exposed to PHA-leukocyte-conditioned medium for 24 hr acquire markedly enhanced ADCC against antibody-coated human erythrocytes. Confluent monolayers of these activated monocytes were found to bind several fold the number of 51Cr-labeled antibody-coated erythrocytes as compared to monolayers formed from control monocytes (uncoated erythrocytes were not bound). The stoichiometry of this reaction indicated that activated monolayers bind approximately 3-5 erythrocyte targets per effector monocyte, whereas control monolayers bind less than 2. Bound target cells remain intact, affixed to the monocyte surface for up to 3 hr at 25 degrees C (they were not phagocytized); incubation at 37 degrees C resulted in both target cell adherence and 51Cr release suggesting that a proportion of target cells were lysed. A substantial fraction of target cells bound at 25 degrees C were lysed (activated greater than control monocytes) if the monolayers were warmed to 37 degrees C. These results indicate that target cell target cell binding can occur at both 25 and 37 degrees C, but lysis of bound targets requires incubation at 37 degrees C. Using this temperature distinction to examine binding and lysis independently, it was found that binding was abrogated by IgG Fc fragments, but could occur over a broad pH range (5-8), and in the presence of 2-deoxy-D-glucose, colchicine, and sodium azide. Lysis of bound targets, on the other hand, was blocked by inhibitors of glycolysis, microtubule/filament organization, cellular respiration, and serine esterase activity, as well as EDTA and low pH; lysis was unaffected by scavengers of extracellular H2O2 and superoxide radicals.     

Amino acid content of rat renal cortex and the response to in vitro incubation.

The concentration of aspartic acid, threonine, serine, glycine, and alanine is significantly higher in newborn rat renal cortex than in the adult tissue, while phenylalanine and histidine are higher in the adult. When adult cortical slices are placed in bicarbonate buffer at room temperature for 20 min there is a 30-60% decrease in the levels of all amino acids except for lysine, which is slightly higher, and methionine and serine, which do not change. Under the same conditions, newborn cortical slices reveal a similar decrease in only glycine, tyrosine, histidine, and the branched-chain amino acids. On subsequent in vitro incubation of the cortical slices at 37 degrees C for 120 min the concentrations in adult tissue remain at the lower values observed on removal from buffer at room temperature except that glutamic acid, glycine, and lysine levels decrease further and serine increases to the concentration found in fresh tissue. Newborn tissue when incubated at 37 degrees C for 120 min shows amino acid concentrations comparable to unincubated fresh tissue for all except aspartic acid, glutamic acid, serine, and phenylalanine, which reach levels higher than unincubated tissue. The ability of newborn tissue to maintain amino acid pools may play a role in the enhanced transport of some amino acids resulting from preincubation at 37 degrees C (Reynolds et al. Science 184: 68-69, 1974; Reynolds and Segal, Biochim. Biophys. Acta 406: 513-525, 1976).     

Degradation of Amyloid-related Serum Protein SAA by a Component Present in Rabbit and Human Serum

After incubation of protein SAA-containing rabbit serum at 37 degrees C overnight, the strength of the precipitation reaction against antiserum to SAA was decreased. This was at east partly due to enzymatic degradation of protein SAA. The enzymatic activity was not strongly associated with the SAA-high-density lipoprotein (HDL) complex, since it sedimented in the preparative ultracentrifuge at a density at which the SAA-HDL complex floats. The degrading component was obtained in concentrated form from rabbit and human sera by adsorption to Sepharose 4B. Degradation of human SAA by the human serum component was inhibited with disopropyl fluorophosphate, an inhibitor of serine proteases.     

Activation of kallikrein-kinin system in human plasma with purified serine protease from Dermatophagoides farinae

An aqueous extract from a mite culture, of Dermatophagoides farinae, activated prekallikrein to kallikrein in normal plasma. Crude protein preparation, obtained by ammonium sulfate precipitation (95% saturation) from the extract, exhibited high activity (0.81 units/mg protein) towards a synthetic substrate of coagulation factor XIIa, Boc-Gln-Gly-Arg-MCA, and had also activity to form kallikrein in human plasma deficient in coagulation factor XII. Treatment of the protein preparation with phenylmethylsulfonyl fluoride (PMSF), an inhibitor of serine enzyme, gave rise to inactivation of both activities. Thus, the serine protease specific for Boc-Gln-Gly-Arg-MCA in mite cultures of D. farinae was purified by ammonium sulfate precipitation and chromatographies on p-aminobenzamidine-sepharose CL-4B, DEAE-Toyopearl 650M, Sephadex G-75 superfine and Sephacryl S-200. The purified protease was homogeneous electrophoretically, and its molecular weight was estimated to be 30,000. The optimum pH and temperature were around 7.5 and 50 degrees C, respectively. The specific activity was 36 units/mg protein at pH 7.4 and 37 degrees C. The activity was completely inhibited by PMSF. The serine protease had the activity to activate the kallikrein-kinin system in normal human plasma.     

Modification of the structure and activity of lipid A in Yersinia pestis lipopolysaccharide by growth temperature

Yersinia pestis strain Yreka was grown at 27 or 37 degrees C, and the lipid A structures (lipid A-27 degrees C and lipid A-37 degrees C) of the respective lipopolysaccharides (LPS) were investigated by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. Lipid A-27 degrees C consisted of a mixture of tri-acyl, tetra-acyl, penta-acyl, and hexa-acyl lipid A's, of which tetra-acyl lipid A was most abundant. Lipid A-37 degrees C consisted predominantly of tri- and tetra-acylated molecules, with only small amounts of penta-acyl lipid A; no hexa-acyl lipid A was detected. Furthermore, the amount of 4-amino-arabinose was substantially higher in lipid A-27 degrees C than in lipid A-37 degrees C. By use of mouse and human macrophage cell lines, the biological activities of the LPS and lipid A preparations were measured via their abilities to induce production of tumor necrosis factor alpha (TNF-alpha). In both cell lines the LPS and the lipid A from bacteria grown at 27 degrees C were stronger inducers of TNF-alpha than those from bacteria grown at 37 degrees C. However, the difference in activity was more prominent in human macrophage cells. These results suggest that in order to reduce the activation of human macrophages, it may be more advantageous for Y. pestis to produce less-acylated lipid A at 37 degrees C.     

Novel observation of hot-cold-hot hemolysis exhibited by group B streptococci

Six human isolates of group B streptococci (GBS) were cultured on blood agar anaerobically at 37 degrees C for 18 h and then at 4 degrees C for 6 h and reincubated anaerobically at 37 degrees C for 6 h. Three of the strains showed a marked enlargement of the hemolysis zone compared with that obtained after hot-only (37 degrees C for 18 h) or hot-cold (37 degrees C for 18 h and then 4 degrees C for 6 h) treatment. Subsequent broth culture experiments revealed that enhanced hemolytic activity due to hot-cold-hot treatment was observed in all 6 GBS strains when cultured in the presence of starch.     

Demonstration of leukocyte inhibitory factor in human Schistosomiasis mansoni and its evaluation from patients in an endemic setting

Leukocyte migration inhibitory Factor (LIF) is produced by the peripheral blood mononuclear cells (PBMN) of most patients with Schistosoma mansoni infection upon their exposure to soluble egg antigens (SEA). PBMN of some patients also respond to adult worm (SWAP) antigens by LIF production. LIF is stable at 37 degrees C for 60 min but is sensitive to heating at 56 degrees C for even 30 min. The serine protease inhibitor PMSF destroyed LIF activity at concentrations of 10(-2) to 10(-3) M. Concanavalin A stimulated production of detectable levels of LIF by 8 hr, while SEA and SWAP did so by 15 and 39 hr, respectively. PBMN of healthy normal controls did not produce LIF upon exposure to SEA or SWAP. PBMN of a few field controls (stool negative subjects from an endemic area) produced detectable LIF activity when exposed to SEA or SWAP. PBMN from most infected (stool positive) patients from an endemic area produced LIF when exposed to SEA and only occasionally did so to SWAP. Previous studies have shown that most often only the PBMN of former, cured patients, and not chronically infected patients, produce the lymphokine activity termed mitogenic factor (MF). The current data indicate that because LIF is primarily produced by actively infected patients, its production may be controlled by different immunoregulatory mechanisms. Furthermore, although most SEA-related responses are highly immunoregulated in active, chronically infected patients, SEA appears to be a better stimulus for patient PBMN production of LIF than SWAP.     

Different antiviral activity and cell specificity of interferon preparations produced by mouse peritoneal cells at 37 degrees C and at 26 degrees C

Three sublines of mouse L cells and mouse embryo fibroblasts were used for determination of the antiviral activity of mouse interferons produced by nonadherent peritoneal exudate cells incubated either at 37 degrees C or at 26 degrees C. IFN produced at 37 degrees C or at 26 degrees C had the same antiviral activity in L Borgen, L929 cells. However, in MEC IFN-37 degrees had relatively higher activity than IFN-26 degrees. Of the interferon investigated only IFN-37 degrees exhibited antiviral activity in the established line of rat kidney cells. The IFN preparations showed no activity in the human and chicken cells. The studies on the sensitivity of viruses to both forms of IFN revealed that EMC and VSV viruses were equally sensitive to IFN-26 degrees C. However, the replication of EMC virus was more strongly inhibited by IFN-37 degrees than the multiplication of VSV virus.     

Characterization of the interaction between recombinant human interferon-gamma and its receptor on human polymorphonuclear leukocytes

The interaction of human recombinant interferon-gamma (rIFN-gamma) with human polymorphonuclear cells (PMN) was investigated. Bolton-Hunter radioiodinated rIFN-gamma bound to PMN in a specific and saturable manner. Eleven hundred binding sites were observed with a Ka of 0.56 x 10(10) M-1. Binding to PMN was rapid with a K1 of 9 x 10(5) M-1 sec-1 at 4 degrees C. At 37 degrees C binding was complete within 6 min. About 50% of bound ligand was internalized within 30 min at 37 degrees C. The receptor demonstrated moderate lability at 37 degrees C in culture. After 1 h at 37 degrees C, PMN lost 80% of their 125I-rIFN-gamma binding sites. This loss was reversed in part by the presence of interleukin-1 in the culture, but not tumor necrosis factor. These studies provide a framework for further investigation into the signalling process of rIFN-gamma on PMN.     

Adherence of Candida albicans to human buccal epithelial cells.

The adherence of Candida albicans to human buccal epithelial cells after 2 h at 37 degrees C was significantly greater in human saliva than in phosphate-buffered saline. in saliva, viable fungi adhered much better than did nonviable fungi, and this adherence was greater at 37 than at 25 degrees C. Viable yeasts, preincubated in saliva for 90 min at 37 degrees C before being washed and mixed with epithelial cells in phosphate-buffered saline, adhered better than nonviable yeasts or yeasts preincubated in phosphate-buffered saline. Enhanced adherence in saliva appeared to be associated with germination of the yeast cells. Conditions permitting germination (growth in tissue culture medium 199 at 37 degrees C but not at 25 degrees C) also supported enhanced adherence. After germination had occurred, the fungi could be killed with Formalin without interfering with their rapid and efficient adherence to epithelial cells. These data indicate that the enhanced adherence of C. albicans observed after incubation in saliva is related to changes in the fungi, rather than to a requirement for prolonged interaction between fungi and epithelial cells.     

Depressed mitogen responsiveness of lymphocytes at skin temperature.

The responsiveness of murine lymphocytes and human peripheral blood lymphocytes to phytohemagglutinin, concanavalin A, pokeweed mitogen, and endotoxin was tested in vitro at 32, 35, and 37 degrees C. The responses at 32 degrees C were delayed and often depressed. Mouse cells responded equally well at 35 and 37 degrees C. Human lymphocytes often responded more rapidly at 37 than at 35 degrees C. Since skin temperature, particularly that of the distal extremities, is usually 32 degrees C or less, a relative deficiency in cell-mediated immunity may exist in these sites. This may be part of the reason for the usual localization of certain infections, such as sporotrichosis, to these coller areas.     

Intracellular growth and cytotoxicity of Mycobacterium haemophilum in a human epithelial cell line (Hec-1-B).

We developed an in vitro model to study the temperature-regulated cytotoxicity and intracellular growth of Mycobacterium haemophilum in cultured human epithelial and endothelial cells. M. haemophilum associated with human epithelial and endothelial cells at similar rates when incubated at 33 and 37 degrees C, but only the epithelial cell line supported the multiplication of this organism. M. haemophilum grew equally well with epithelial cells at both temperatures. The aminoglycoside antibiotic amikacin was used to study the intracellular growth of M. haemophilum in the epithelial cells at 33 and 37 degrees C. Although an approximately equal number of bacteria were found within cells after 2 days of incubation at both temperatures, intracellular replication of M. haemophilum was 1,000-fold greater at 33 than at 37 degrees C. This intracellular multiplication was associated with destruction of the monolayers at 33 but not at 37 degrees C, and only culture filtrates from infected monolayers incubated at 33 degrees C were cytotoxic to fresh epithelial cell monolayers. This strain of M. haemophilum also produced contact-dependent hemolysis of sheep erythrocytes, demonstrating the possible presence of a cytolysin. These studies suggest that M. haemophilum has a preference for growth with cultured human epithelial cells. In addition, intracellular growth is best at 33 degrees C in epithelial cells, and this correlated with cytotoxicity at this temperature. This phenotype may be caused by induction of a soluble cytotoxic component, possibly a hemolytic cytolysin.     

Specificity and reversibility of chemotactic deactivation of human monocytes.

The chemotactic deactivation of human monocytes was studied to provide insight into the mechanism of chemotaxis. Deactivation was dependent on the dose of chemoattractant and time of incubation. A concentration in the cell suspension of 10(-8) M N-formylmethionylleucyl phenylalanine (FMLP) for 45 min at 37 degrees C led to 60% suppression of the subsequent specific chemotactic response. Higher concentrations of FMLP led to almost 100% specific suppression. Deactivation was specific under all conditions used. The response to a nonrelated chemoattractant, human serum-derived C5a, was unaffected by incubation in FMLP. Deactivation was also transient. If cells were deactivated at 37 degrees C with FMLP, they recovered within 6 h at 37 degrees C from this deactivation. Both phenomena, deactivation and recovery from deactivation, were temperature dependent. Monocytes could not be deactivated at 0 degrees C, and they did not recover from deactivation when kept at 0 degrees C. Thus, specific deactivation appears to require cellular metabolism, involving loss of receptors or blocking of a step between receptor occupancy and response.     

Effects of multiple freeze thaws and various temperatures on the reactivity of human immunodeficiency virus antibody using three detection assays

The effects of multiple freeze-thaw cycles and various temperatures (-20 degrees C, 4 degrees C, 25 degrees C, 37 degrees C) on the reactivity of human immunodeficiency virus (HIV) antibodies were evaluated using current ELISA, recombinant, and Western blot methodologies. Twenty consecutive freeze-thaw cycles and storage of specimens at -20 degrees C and 4 degrees C for 57 days resulted in no loss of HIV antibody reactivity nor false positive samples. Maintenance of clinical specimens at 25 degrees C and 37 degrees C for 57 days resulted in some loss of HIV antibody reactivity, but all positive and weakly reacting samples remained positive, and negative samples were unaffected.     

Cryoprotection of human oocytes: inappropriate exposure to DMSO reduces fertilization rates

Human oocytes were exposed to the cryoprotectant dimethyl-sulphoxide (DMSO) at either 4 or 37 degrees C. Subsequent fertilization of these oocytes showed that exposure to DMSO at 37 degrees C was associated with a greatly reduced fertilization rate when compared to untreated control oocytes, whereas no such reduction was seen in oocytes exposed to DMSO at 4 degrees C. The significance of these results for the potential cryopreservation of human oocytes is discussed.     

The effect of temperature on the interaction of Haemophilus ducreyi with human epithelial cells

To investigate if temperature affects the interaction of Haemophilus ducreyi with human epithelial cells, nine strains were used to evaluate the adhesion kinetics of the organism at 33 degrees C and 37 degrees C. The effect of the free toxin on the epithelial cells at those temperatures was also assessed. The cyto-adherence kinetics of H. ducreyi to the epithelial cells was significantly greater at 33 degrees C (10 times more) than at 37 degrees C in all seven clinical isolates tested. There was a significant difference in cell-associated H. ducreyi at 33 degrees C as compared with 37 degrees C. Control strains showed similar adhesion properties at both temperatures. However, the virulent strain CIP542 adhered in larger amounts than the avirulent strain A77. Electron microscopy revealed that there was more tissue necrosis at the lower than the higher temperature. The effect of the free toxin was the same at each temperature. However, strain A77 had significantly lower toxicity than strain CIP542 and the clinical isolates. These results suggest that H. ducreyi displays a temperature-dependent interaction with human epithelial cells, and this feature may play a role in the virulence of the organism in vivo. While the overall toxic effect of viable bacteria depends on the metabolic activity of the bacteria and is, therefore, higher at 33 degrees C than at 37 degrees C withthe same initial inoculum, the effect of the extracted toxin at molecular level with fixed concentrations is a temperature-independent event.     

The human lymphokine leukoregulin induces cell resistance to complement-mediated lysis.

Leukoregulin (LR) is a lymphokine secreted by human natural killer (NK) cells. Its effect on the susceptibility of K562 human erythroleukemic cells to lysis by antibody and complement was examined. As reported here, treatment of K562 cells with LR for 60 min at 37 degrees C confers on them resistance to complement damage. The LR-induced state of complement resistance is transient and the cells recover within 4-6 h unless a second dose of LR is added. The protective action of LR was observed using both conventional 51Cr-release and trypan blue inclusion assays. The protein synthesis inhibitors puromycin and cycloheximide and the protein kinase inhibitors tamoxifen, polymyxin B and W-7, could each block this action of LR. Fewer membrane attack complexes were found, following complement activation, on LR-treated than control cells. These results suggest that LR increases the capacity of K562 cells to down-regulate complement activation or repair the complement damage, possibly by inducing synthesis of defense proteins and/or activation of protective protein kinases.     

Pathogenesis of fever in delayed hypersensitivity: factors influencing release of pyrogen-inducing lymphokines.

In continuing studies on the pathogenesis of fever in states of delayed hypersensitivity, we have investigated the conditions for the release of an endogenous pyrogen (EP)-inducing lymphokine from draining-lymph-node lymphocytes of rabbits with delayed hypersensitivity to bovine gamma globulin. Using doses of 4 X 10(7) to 5 X 10(7) blood leukocytes (BL) as a source of EP, we found that ratios of about 5:1 of viable lymphocytes to BL were required to stimulate the BL to produce detectable amounts of EP in vitro. Both irradiated lymphocytes (1,700 R) as well as those from steroid-treated donors retained their ability to activate BL when incubated with antigen, properties consistent with activated "T" lymphocytes. In experiments to determine effects of temperature and duration of incubation on lymphokine release, the maximum EP-releasing activity was found to be present in supernatants of sensitized lymphocytes incubated with antigen for 18 h at 37 degrees C. These studies have confirmed that sensitized lymphocytes release a soluble, pyrogen-inducing lymphokine when incubated with antigen and further demonstrate that tissue macrophages (Kupffer cells) as well as BL can be activated to produce EP in vitro by this agent.