Identification by heteroduplex analysis of an invertible element (Min) common among IncM group plasmids

The analysis of heteroduplexes between EcoRI digested DNA derived from IncM plasmids in the transfer-off and in the transfer-on state revealed an internal loop of about 1 kbp on a distinct large EcoRI fragment. The presence of an invertible element (Min) is suggested which enables the formation of conjugative pili at 30 degrees C, but switches off the pilus formation at 37 degrees C incubation temperature.     

Evaluation of dithiothreitol (DTT) for inactivation of IgM antibodies.

A newly introduced sulphydryl compound, dithiothreitol (DTT), is evaluated for its optimal conditions of inactivation of IgM antibodies. The maximal effects of DTT reagent are observed when its final concentrations are between 0.0025 M and 0.005 M, pH between 7.0 and 8.0, and incubation at 37 degrees C. Concentrations over 0.01 M, pH values greater than 8.0, and an incubation temperature over 40 degrees C resulted in a gel formation of the specimen. Examination of both 'cold' and 'warm' type antibodies demonstrated that the results obtained by the DTT reagent are in close agreement with those obtained by 2-mercaptoethanol reagents and DEAE Sephadex treatment. Since the procedure is simple and rapid and lacks offensive odour, DTT is recommended for routine use in blood banking for the inactivation of IgM antibodies.     

Optimal conditions for Fc receptor-ligand interaction.

The temperature and time dependence for Fc receptor-ligand interaction have been investigated in a murine lymphoblastoid cell line. The binding of antibody-coated erythrocytes or radioiodinated heat-aggregated IgG was evaluated at varying time points during incubation at 4, 21 or 37 degrees C. Maximum rosette formation was observed by 10 min incubation at 21 degrees C if cell suspensions were first centrifuged at 200 g for 2 min. Longer incubation periods were required in the absence of centrifugation. The kinetics of aggregate IgG binding were similar to rosette formation without centrifugation. The results demonstrate that receptor-ligand interaction in this system is both temperature and time-dependent.     

Effects of age, ambient temperature, and heat-stable Escherichia coli enterotoxin on intestinal transit in infant mice.

Some interrelationships among age, ambient temperature, intestinal transit, and enterotoxigenic Escherichia coli infection were studied in an infant mouse model. The transit of dye in the small intestine was accelerated during the response to heat-stable E. coli enterotoxin. Transit in the small intestine of normal mice accelerated with increased age (from less than 17 h to 8 days old) and accelerated with increased ambient temperature (from 25 to 37 degrees C). Transit was more rapid in the jejunum than in the ileum throughout the range of experimental conditions studied. E. coli strains that do not produce any of the pili known facilitate intestinal colonization were cleared from the small intestine more rapidly at 37 degrees C than at 25 degrees C. This clearance was thought to be due to accelerated transit at the higher temperature. In contrast, a strain of E. coli that produces K99 (pili previously shown to facilitate intestinal colonization in other species) was not cleared from the small intestine and colonized more intensively at 37 degrees C than at 25 degrees C. Intensified colonization by this strain was thought to be due to increased production of K99 at the higher temperature. It was suggested that sluggish intestinal transit may also be characteristic of the neonates of other species and be one of the factors predisposing them to intestinal colonization by enteropathogens. It was speculated that this predisposition may be enhanced if the neonates are chilled. However, the effect of ambient temperature on intestinal transit in homeothermic neonates such as pigs, calves, and humans may be different from that in mice because neonatal mice are poikilothermic.     

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.     

Temperature and pH effect on lindane removal by Streptomyces sp. M7 in soil extract

This work was conducted to study the removal of gamma-hexachlorocyclohexane (lindane) in a soil extract liquid medium (SE) by Streptomyces sp. M7 and to determine the influence of pH and temperature on bacterial growth and pesticide removal in this medium. When Streptomyces sp. M7 was cultured in SE supplemented with lindane 100 microg l(-1 )at different initial pH, the maximum growth was observed at pH 7 and the microorganism was not able to grow at pH 5 and 9; the highest pesticide removal (70.4%) by Streptomyces sp. M7 was noted at an initial pH of 7 at 4 weeks of incubation. The maximum removal (70% approximately) was observed when the microorganism was incubated in SE at 30 degrees C; although the optimal temperature for Streptomyces sp. M7 growth, with and without lindane, was 25 degrees C, and for the pesticide removal was 30 degrees C. The results of this study suggest that this actinomycete strain appears as an effective alternative in the remediation of lindane polluted sites.     

Comparison of modifications of the rosette test for detecting T lymphocytes.

Experiments were carried out with blood lymphocytes from 30 healthy subjects and 70 patients with chronic lymphocytic leukemia (CLL). Results of the rosette test with sheep red blood cells (SRBC) were compared by the methods of: I) Wybran (incubation at room temperature, reading after 3 hr); II) Jondal (incubation at temp. 310.15K (37 degrees C) and 277.15K (4 degrees C), reading after 24 hr; III) Weiner et al. (neuraminidase-treated SRBC, incubation at 273.15K (0 degrees C), reading after 15 min; IV) as in method III, but reading after 24 hr. The method of Wybran gave lowest results, statistically significantly differing from the results obtained by the other methods. The other modifications gave results that did not differ significantly. Neuraminidase treatment of SRBC accelerates formation of stable rosettes and this method is recommended in cases where a brief period of incubation is required. In other cases, the method of Jondal may be used.     

Growth of Tahyna virus at low temperatures.

Replication of two Tahyna virus strains in the Aedes albopictus mosquito cell line was studied in the temperature range from 6 to 28 degrees C. The virus grew in this temperature range; its replication rate was related to the temperature of incubation. At lower temperatures the virus titres increased more slowly and did not reach as high maximum values as at higher temperatures. Short increase in incubation temperature resulted in a short increase in the titre of virus previously incubated at 10 degrees C, but not of virus previously incubated at 15 degrees C. At 10 and 15 degrees C, the released virus was demonstrated for more than 300 days past infection (p.i.).     

The effect of M13 phage infection upon the F pili of E. coli

The infection of male strains of E. coli with low multiplicities of M13 phage (10 plaque-forming units/cell) causes no detectable loss of cell-associated F pili as measured by electron microscopy or the ability to attach R17 phage. Larger multiplicities of M13 infection, however, lead to a rapid loss of F pili at physiological temperatures but not at 4 °C, the loss occurring between 2 and 10 min post infection. The foregoing phenomenon is apparently unrelated to the synthesis of M13-specific or host proteins, since inhibition of protein synthesis in such cultures does not prevent the observed loss of F pili. Additional studies revealed that the disappearance of cell-associated F pili in M13-infected cultures is accompanied by the appearance of F pili fragments in the culture medium, suggesting that infection with large amounts of M13 phage causes a breakage or release of F pili from the cell. These results are discussed in terms of a conduction model for M13 phage infection.     

Detection and quantification of type C viral proteins in tissues and sera with an enzyme immunoassay.

The in vitro adhesion of three uropathogenic strains of Escherichia coli to epithelial cells from the periurethral area (area surrounding the urethral orifice) of women with and without a history of recurrent urinary tract infections was investigated. All strains showed a specific mannose-resistant hemagglutination restricted to human erythrocytes. Since only a few hundred periurethral cells were used in each test, gentle methods were required. Optimal results were obtained with bacteria grown for 16 h at 37 degrees C in nutrient broth without shaking. The binding of bacteria seemed to be irreversible under the conditions studied, since repeated washings of the epithelial cells after incubation did not decrease the number of adhering bacteria. Chloramphenicol was used to control the number of added bacteria in the incubation system. A difference in the adhesive capacity of periurethral cells of infection-prone and healthy individuals was most evident at concentrations of 2.5 x 10(9) bacteria/ml. Electron microscope studies indicated that pili mediated the adhesion. Adhesion was correlated with the mannose-resistant hemagglutination of human erythrocytes, indicating that the pili were not type 1 pili. Day-to-day variations in the adhesiveness of the bacteria were reduced by selecting well-adhering bacteria with the aid of in vitro passage on periurethral cells or human erythrocytes, and by exclusion of bacteria with low hemagglutination ability.     

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.     

Temperature enhancement of syncytium formation by HIV and Sendai virus.

Syncytium formation, the characteristic cytopathic effect (CPE) of the human immunodeficiency virus (HIV) and cell fusion by Sendai virus, is accelerated by increasing the ambient temperature to values at which normal metabolic activity is inhibited. Uninfected C8166, CEM, and H9 cells were absorbed at 4 degrees C onto monolayers of H9 cells chronically infected with HIV and incubated subsequently at either 37 degrees C or 45 degrees C. Similarly chick and human erythrocytes and Hela cells were agglutinated with Sendai virus at 4 degrees C before incubation at temperatures of up to 50 degrees C. With both viruses the rate of cell fusion was directly related to temperature. Since membrane fluidity is dependent on the phase-transition temperature points of the membrane lipids it is proposed that sufficient membrane fluidity is essential for cell fusion to occur. The implication of these observations on the cytopathology of HIV is discussed.     

Attachment pili from enterotoxigenic Escherichia coli pathogenic for humans.

Pili from enterotoxigenic Escherichia coli pathogenic for humans have been isolated by adsorption to the surface of erythrocytes followed by thermal elution. The pili are composed of two protein subunits with molecular weights of 13,100 and 12,500 as determined by sodium dodecyl sulfate-gel electrophoresis. These pili also bind to human buccal cells under temperature conditions (37 degrees C) which prevent the binding of these pili to the erythrocytes. Analogous temperature effects on binding have previously been observed with whole bacterial cells. This binding can be inhibited by antiserum prepared against the isolated pili.     

Further studies of a humoral chemotactic abnormality in glomerulonephritis

Chemotactic activity in the sera of patients with glomerulonephritis was compared under three simultaneously performed conditions: (1) incubation with buffer at 37 degrees C (CF-UNACT); (2) incubation with immune complexes at 37 degrees C (CF-ACT); (3) immediate heating at 56 degrees C (CF-56 degrees C). In all cases the generation of chemotactic factors was terminated by standard 'heat-inactivation' at 56 degrees C. Patients' CF-UNACT was similar to that of controls; patients' CF-ACT was significantly less than controls', but patients' CF-ACT and CF-56 degrees C was significantly greater than controls'. Patients CF-ACT and CF-56 degrees C were largely C5-dependent and were quantitatively similar. These divergent abnormalities could not be explained by spontaneous in vivo or in vitro (i.e., blood clotting) generation of complement chemotactic factors, the absence of Hageman factor-dependent chemotactic activity, or the presence of humoral inhibitors in patients' sera. It appears that inital 56 degrees C heating liberates C5-dependent chemotactic activity, a procedure that is usually believed to block its formation. Terminal 56 degrees C heating after 37 degrees C incubation did not generate such activity in CF-UNACT. The duration or sequence of heating at 56 degrees C, or both, are critical determinants for final expression of chemotactic activity in patients' sera, when viewed in relation to 37 degrees C incubation with immune complexes or buffer.     

One more probable structural transition in potato virus X virions and a revised model of the virus coat protein structure

We found that a 2-h incubation of potato virus X (PVX) virions in 10 mM Tris-HCl buffer pH 7.5 at -20 degrees C results in a strong but reversible drop in virion stability. Under these conditions, the PVX virions are completely disrupted by low (starting from 50 mM) concentrations of LiCl and CaCl(2) but not of NaCl. Incubation of PVX samples with 0.05-2 M LiCl at +4 degrees C did not result in virion disassembly and the virions were not disrupted upon incubation at -20 degrees C in 10 mM Tris-HCl buffer pH 7.5 without LiCl. We suggest that a 2-h incubation of the PVX virions at -20 degrees C in 10 mM Tris-HCl pH 7.5 results in a structural transition in the virus particles. A revised model of the three-dimensional organization of coat protein subunits in the PVX virions is proposed. This two-domain model explains better the high plasticity of the PVX CP structure.     

Covalent binding of aminopropanehydroxydiphosphonate to glutaraldehyde residues in pericardial bioprosthetic tissue: stability and calcification inhibition studies

Calcification has limited the clinical utility of bioprosthetic heart valves fabricated from either glutaraldehyde-pretreated bovine pericardium or porcine aortic valves. Aminopropanehydroxydiphosphonate (APDP), covalently bound to residual aldehyde groups in glutaraldehyde-treated pericardial bioprosthetic tissue, has been shown to inhibit cardiovascular calcification in the rat subdermal model. Using 3H-labeled glutaraldehyde (GLUT) at a concentration of 0.02 M and 0.14 M 14C-labeled APDP, we assessed the effects of GLUT incubation temperature (4 degrees or 25 degrees C) and pH of the GLUT incubation solution (pH 4.0, 7.4, or 10.0) on the GLUT incorporation step and subsequent APDP binding (24 hr 25 degrees C) into bioprosthetic valve (BPV) tissue (bovine pericardium). Increased incorporation of GLUT and APDP occurred at lower GLUT incubation temperature (GLUT, 346.05 +/- 1.9 nM/mg, 4 degrees C vs 259.76 +/- 1.39 nM/mg, 25 degrees C; APDP, 57.56 +/- 4.43 nM/mg, 4 degrees C vs 36.36 +/- 0.46 nM/mg, mean +/- standard error, at 25 degrees C). There also was a greater incorporation of GLUT but not APDP at the higher glutaraldehyde pretreatment pH (GLUT, pH 10.0, 213.73 +/- 73 nM/mg vs pH 4, 132.08 +/- 43 nM/mg; APDP, pH 10.0, 51.41 +/- 12 nM/mg vs pH 4.0, 49.97 +/- 6 nM/mg). In vivo studies revealed that all groups with treated BPV implanted for 21 days in male 3-week-old CD rats demonstrated a loss of both GLUT (12-50%) and APDP (48-64%) compared to preimplant content. BPV implant calcification was significantly inhibited in all groups treated with APDP compared to control Ca2+ (5.54 +/- 2.1-9.64 + 1.2 micrograms/mg, APDP pretreated, vs 93.64 +/- 11.65 micrograms/mg, control; P less than or equal to 0.001) despite the progressive loss of both GLUT and APDP with time. It is concluded that preincubation of BPV tissue in GLUT at lower temperature (4 degrees C) and higher pH (10.0) enhanced BPV GLUT uptake and subsequent APDP covalent binding. In addition, in the rat subdermal model, BPV tissue calcification was markedly inhibited by APDP, despite a significant loss of bound drug.     

Growth of SV40, adeno 7 and SV40-adeno 7 viruses in monkey and human cells at 29° and 37°C

Adeno 7 virus replicated well in human diploid (LEP) cells but only to a low degree in green monkey kidney (GMK) cells at 37 degrees C; it did not replicate in either system at 29 degrees C. At 37 degrees C SV 40 virus replicated well in GMK cells but only moderately in LEP cells; at 29 degrees C it did not replicate in either system. SV 40-adeno 7 hybrid grew in both GMK cells and LEP cells at 37 degrees C. At 29 degrees C this virus replicated in GMK cells but not in LEP cells. While the formation of V-antigen generally corresponded to the infectious virus production in the respective system, considerable differences were encountered in the T-antigens production. Adeno 7 T-antigen was detected earlier and in a higher percentage of GMK cells than in the fully permissive LEP cells and its formation was only slightly influenced by the incubation temperature. SV 40 T-antigen was more efficiently formed in GMK cells than in LEP cells. At 29 degrees C SV 40 T-antigen was only found in GMK cells and was detected later than at 37 degrees C. The difference in the formation of SV 40 T-antigen in GMK cells infected with SV 40 and SV 40-adeno 7 hybrid virus was further analyzed. The results obtained suggest that an early step of the virus-cell interaction, but neither virus attachment nor penetration, was involved.     

Activity and stability of recombinant human superoxide dismutase in buffer solutions and hypothermic perfusates

The stability of recombinant human superoxide dismutase (r-hSOD) in buffer solutions was studied in solutions at various pH and temperatures. Additionally, we studied the effects of incubation with proteases, serum and two types of hypothermic perfusates. R-hSOD was stable in the pH range of 6-11 and at temperatures up to 80 degrees C for 30 min. R-hSOD activity was not affected by incubation with trypsin, aminopeptidase M or serum for 2 h. R-hSOD activity determined at various temperatures (4-37 degrees C) did not vary remarkably. R-hSOD in hypothermic perfusates was stable at 4-37 degrees C for 24 h.     

Cellular pathway(s) of antigen processing in fish APC: effect of varying in vitro temperatures on antigen catabolism.

Studies were conducted to determine the effects of varying in vitro temperatures during cellular processing of T-dependent antigens by catfish antigen-presenting cells (APC). Strategy involved pulsing of long-term monocyte lines (used as APC) with antigen at different temperatures for various periods of time and then fixing and coculturing with autologous peripheral blood leukocytes (PBL) as responders at a permissive temperature (i.e., 27 degrees C). Results showed that APC incubated with antigen at low, nonpermissive, but physiologically relevant, temperatures (11 and 17 degrees C) elicited secondary proliferative responses by autologous PBL. However, responses elicited with APC pulsed at 11 and 17 degrees C required longer exposure to the antigen prior to fixation (i.e., greater than or equal to 8 h compared to 5 h, the optimal incubation time at 27 degrees C). Further, there was sufficient cell-associated antigen during a short-term pulsing period (1-2 h) at 11 and 17 degrees C to provide efficient presentation after subsequent incubation of the APC at 27 degrees C for an additional 10 h before fixation. In contrast, APC pulsed with antigen at an extremely low, physiologically irrelevant, temperature (4 degrees C) for extended periods of time did not elicit the proliferation of autologous responders unless antigen pulsing was carried out for 1-2 h and the APC subsequently shifted to 27 degrees C for an additional 10 hr. Antigen uptake assays revealed significant and similar levels of internalized antigen at 11, 17, and 27 degrees C, but not at 4 degrees C. However, intracellular degradation and formation of trichloroacetic acid-soluble antigen catabolites at 11 and 17 degrees C appeared to occur at a slower rate compared to APC at 27 degrees C. Significantly, primary anti-hapten plaque-forming cell responses were also observed with PBL cocultured with APC pulsed with hapten-carrier conjugates at 11, 17, and 27 degrees C. Consequently, the previously observed suppression of primary T-cell responses in fish at low, physiologically relevant, temperatures is not due to impaired antigen processing or presentation.