Factors influencing the chlorine susceptibility of Mycobacterium avium,Mycobacterium intracellulare,and Mycobacterium scrofulaceum

The susceptibility of representative strains of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum (the MAIS group) to chlorine was studied to identify factors related to culture conditions and growth phase that influenced susceptibility. M. avium and M. intracellulare strains were more resistant to chlorine than were strains of M. scrofulaceum. Transparent and unpigmented colony variants were more resistant to chlorine than were their isogenic opaque and pigmented variants (respectively). Depending on growth stage and growth rate, MAIS strains differed in their chlorine susceptibilities. Cells from strains of all three species growing in early log phase at the highest growth rates were more susceptible than cells in log and stationary phase. Rapidly growing cells were more susceptible to chlorine than slowly growing cells. The chlorine susceptibility of M. avium cells grown at 30 degrees C was increased when cells were exposed to chlorine at 40 degrees C compared to susceptibility after exposure at 30 degrees C. Cells of M. avium grown in 6% oxygen were significantly more chlorine susceptible than cells grown in air. Chlorine-resistant MAIS strains were more hydrophobic and resistant to Tween 80, para-nitrobenzoate, hydroxylamine, and nitrite than were the chlorine-sensitive strains.     

Methods used to investigate a possible environmental source of Mycobacterium avium-intracellulare-scrofulaceum (MAIS) infection in farmed deer

Currently used methods for the culture of mycobacteria from contaminated material were found to be unsatisfactory in an investigation of a possible environmental source of Mycobacterium avium-intracellulare-scrofulaceum (MAIS) infection in a New Zealand deer farm. Different combinations of established procedures were investigated using soil spiked with a laboratory strain of M. avium. The most successful combination involved mixing the soil in nutrient broth (pH 8.0) containing Tween 80, incubating at 37 degrees C for 1 h to germinate sporing contaminants, treatment for 24 h with 1% cetylpyridinium chloride, followed by washing and culture on Lowenstein-Jensen slopes and incubation at 37 degrees C and 42 degrees C in approximately 5% CO2 atmosphere. This procedure allowed good recovery of M. avium while successfully inhibiting saprophytic mycobacteria and other soil organisms, and was chosen to process the deer farm samples. No mycobacteria resembling the deer strains were found in these samples.     

Effect of turbulent-flow pasteurization on survival of Mycobacterium avium subsp. paratuberculosis added to raw milk

A pilot-scale pasteurizer operating under validated turbulent flow (Reynolds number, 11,050) was used to study the heat sensitivity of Mycobacterium avium subsp. paratuberculosis added to raw milk. The ATCC 19698 type strain, ATCC 43015 (Linda, human isolate), and three bovine isolates were heated in raw whole milk for 15 s at 63, 66, 69, and 72 degrees C in duplicate trials. No strains survived at 72 degrees C for 15 s; and only one strain survived at 69 degrees C. Means of pooled D values (decimal reduction times) at 63 and 66 degrees C were 15.0 +/- 2.8 s (95% confidence interval) and 5.9 +/- 0.7 s (95% confidence interval), respectively. The mean extrapolated D72 degrees C was <2.03 s. This was equivalent to a >7 log10 kill at 72 degrees C for 15 s (95% confidence interval). The mean Z value (degrees required for the decimal reduction time to traverse one log cycle) was 8.6 degrees C. These five strains showed similar survival whether recovery was on Herrold's egg yolk medium containing mycobactin or by a radiometric culture method (BACTEC). Milk was inoculated with fresh fecal material from a high-level fecal shedder with clinical Johne's disease. After heating at 72 degrees C for 15 s, the minimum M. avium subsp. paratuberculosis kill was >4 log10. Properly maintained and operated equipment should ensure the absence of viable M. avium subsp. paratuberculosis in retail milk and other pasteurized dairy products. An additional safeguard is the widespread commercial practice of pasteurizing 1.5 to 2 degrees above 72 degrees C.     

Fluorescent acid-fast microscopy for measuring phagocytosis of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum by Tetrahymena pyriformis and their intracellular growth

Fluorescent acid-fast microscopy (FAM) was used to enumerate intracellular Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum in the ciliated phagocytic protozoan Tetrahymena pyriformis. There was a linear relationship between FAM and colony counts of M. avium cells both from cultures and within protozoa. The Ziehl-Neelsen acid-fast stain could not be used to enumerate intracellular mycobacteria because uninfected protozoa contained acid-fast, bacterium-like particles. Starved, 7-day-old cultures of T. pyriformis transferred into fresh medium readily phagocytized M. avium, M. intracellulare, and M. scrofulaceum. Phagocytosis was rapid and reached a maximum in 30 min. M. avium, M. intracellulare, and M. scrofulaceum grew within T. pyriformis, increasing by factors of 4- to 40-fold after 5 days at 30 degrees C. Intracellular M. avium numbers remained constant over a 25-day period of growth (by transfer) of T. pyriformis. Intracellular M. avium cells also survived protozoan encystment and germination. The growth and viability of T. pyriformis were not affected by mycobacterial infection. The results suggest that free-living phagocytic protozoa may be natural hosts and reservoirs for M. avium, M. intracellulare, and M. scrofulaceum.     

In vitro assessment of a direct transfer vitrification procedure for bovine embryos

We developed a simple vitrification technique for bovine embryos that could permit direct transfer. Embryos were produced in-vitro by standard procedures. The base medium for cryopreservation was a chemically defined medium similar to SOF + 25 mM Hepes and 0.25% fatty acid free bovine serum albumin (FAF-BSA) (HCDM2). In experiment 1, embryos were first exposed to 3.5M ethylene glycol (V1) for 1, 2 or 3 min at room temperature (20-24 degrees C), and then moved to 7 M ethylene glycol (V2) at 4 or 20-24 degrees C and loaded in 0.25-mL straws. After 45 s in 7 M ethylene glycol, straws were placed in liquid nitrogen. Embryos that were loaded at 20-24 degrees C had higher survival rates than those loaded at 4 degrees C (P<0.05). Exposure for 1 min was best for morulae, while 3 min was best for blastocysts. In experiment 2, blastocysts were handled at 24 degrees C and exposed to two concentrations of ethylene glycol in V1 (3.5 or 5 M) followed by V2 as in experiment 1, two warming temperatures (20 or 37 degrees C) and two post-warming holding times until culture (5 or 15 min). Exposure to 5 M ethylene glycol and warming at 37 degrees C was the optimal combination of procedures, and embryos survived well after 15 min in straws if warmed at 37 degrees C. In experiment 3, ethylene glycol concentration (3, 4 or 5 M) and exposure time (0.5 or 1 min) during two-step addition of cryoprotectant were studied for bovine morulae. In experiment 4, morulae were exposed to V2 for 30 or 45 s in HCDM2 or Vigro holding medium and then held in 22-24 degrees C air or 37 degrees C water post-warming. Experiment 5 was like experiment 4 except blastocysts were used. Overall survival rates of blastocysts in experiment 5 averaged 80% of non-vitrified controls after 48 h culture. The survival rates with in vitro-produced morulae in experiments 1, 3 and 4 were unacceptable. Vitrification solutions based on Vigro tended to result in higher survival than HCDM2 for blastocysts, but not morulae. In experiment 6, the survival rate in vitro of in vivo-produced morulae and blastocysts after two-step vitrification was nearly 100%. Our vitrification technique was very effective for in vitro produced blastocysts, but not for in vitro-produced morulae.     

Survival of Mycobacterium avium in drinking water biofilms as affected by water flow velocity, availability of phosphorus, and temperature

Mycobacterium avium is a potential pathogen occurring in drinking water systems. It is a slowly growing bacterium producing a thick cell wall containing mycolic acids, and it is known to resist chlorine better than many other microbes. Several studies have shown that pathogenic bacteria survive better in biofilms than in water. By using Propella biofilm reactors, we studied how factors generally influencing the growth of biofilms (flow rate, phosphorus concentration, and temperature) influence the survival of M. avium in drinking water biofilms. The growth of biofilms was followed by culture and DAPI (4',6'-diamidino-2-phenylindole) staining, and concentrations of M. avium were determined by culture and fluorescence in situ hybridization methods. The spiked M. avium survived in biofilms for the 4-week study period without a dramatic decline in concentration. The addition of phosphorus (10 microg/liter) increased the number of heterotrophic bacteria in biofilms but decreased the culturability of M. avium. The reason for this result is probably that phosphorus increased competition with other microbes. An increase in flow velocity had no effect on the survival of M. avium, although it increased the growth of biofilms. A higher temperature (20 degrees C versus 7 degrees C) increased both the number of heterotrophic bacteria and the survival of M. avium in biofilms. In conclusion, the results show that in terms of affecting the survival of slowly growing M. avium in biofilms, temperature is a more important factor than the availability of nutrients like phosphorus.     

The effect of chlorpromazine on the temperature and osmotic sensitivity of erythrocytes]

The effect of chlorpromazine on the development of cold shock in erythrocytes exposed to sodium chloride was shown to depend on the tonicity of the medium in which the cells were cooled from 37 degrees C down to 0 degrees C as well as on the amphipate concentration. After cooling of erythrocytes in a NaCl (0.75-1.5 M)-containing medium with chlorpromazine (7 x 10(-5) M, 2.1 x 10(-4) M and 3.5 x 10(-4) M) the hypertonic cold shock was inhibited, the protective effect of the amphipate being less pronounced at its increasing concentrations. After cooling of cells under conditions of moderate hypertonicity (0.3-0.6 M NaCl) no modifying effect of chlorpromazine on the sensitivity of erythrocytes to the temperature decrease from 37 degrees C down to 0 degrees C was manifested. However, under iso- and hypertonic conditions chlorpromazine used at 2.1 x 10(-4) M and 3.5 x 10(-4) M stimulated the cold shock development in erythrocytes. A sharp increase in the medium tonicity (up to 1.8-3.0 M and higher) the cells underwent isothermal hemolysis which was more expressed at 0 degrees C than at 37 degrees C. These data suggest that chlorpromazine significantly activates the hemolytic process at low temperatures.     

Re-activation of the peptidyltransferase centre of rabbit reticulocyte ribosomes after inactivation by exposure to low concentrations of magnesium ion.

1. The larger subrivosomal particles of rabbit reticulocytes retained full activity in the puromycin reaction and in poly(U)-directed polyphenylalanine synthesis after 4h at 0 degrees C when buffered 0.5M-NH4Cl/10-30mM-MgCl2 was the solvent. 2. Activity in the puromycin reaction was diminished to approx 10% after 15-30 min at 0 degrees C when the concentration of MgCl2 was lowered to 2mM. 3. Activity was not restored when the concentration of MgCl2 was raised from 2mM to 10-30 mM at 0 degrees C. However, activity was recovered as measured by both assay systems when the ribosome fraction was heated to 37 degrees C at the higher concentrations of MgCl2. 4. Recovery of activity was noted during the course of the polyphenylalanine synthesis in 50 mM-KCl/5mM-MgCl2/25mM-Tris/HCl, pH 7.6, at 37 degrees C. Re-activation was slow at 20 degrees C and below. 5. No more than about 5% of the protein moiety of the subparticle was lost in 0.5M-NH4Cl on decreasing MgCl2 concentration from 10mM to 2mM. No proteins were detected in the supernatant fractions by gel electrophoresis after ribosomes were separated by differential centrifugation. The supernatant fraction was not essential for the recovery of activity. However, at higher (e.g. 1M) concentrations of NH4Cl, proteins were split from the subparticle. 6. The loss and regain of activity found on lowering and restoring the concentration of MgCl2 at 0.5M-NH4Cl appears to arise from a conformational change that does not seem to be associated with a loss and regain of particular proteins. 7. A 2% decrease in E260 was noticed when the concentration of Mg2+ was restored, and the change in the spectrum indicated a net increase of approx. 100A-U base-pairs per subribosomal particle. 8. When the concentration of Mg2+ was restored, S20,W of the subparticle remained at 52+/- 1S until the sample was incubated at 37 degrees C when S20,W increased to 56 +/- 1S compared with the value of 58 +/- 1S for the subparticle as originally isolated.     

Expression profiles of low molecular mass heat shock proteins by Mycobacterium avium and Mycobacterium intracellulare

Closely related non-tuberculous mycobacterial species, Mycobacterium avium and Mycobacterium intracellulare, were compared for the profiles of their production of low molecular mass heat shock proteins at 45 degrees C, by performing polyacrylamide gel electrophoresis analysis of bacterial cell lysate proteins. All of the M. intracellulare but not M. avium strains potently increased the production of the 18-kDa heat shock protein, when cultured at 45 degrees C. Half of the M. intracellulare strains with high sensitivity to 45 degrees C produced not only the 18-kDa heat shock protein but also the 16-kDa heat shock protein at 45 degrees C. These findings indicate that M. avium and M. intracellulare differentially respond to 45 degrees C heat shock in terms of the production of low molecular mass heat shock proteins.     

Binding kinetics of ecotropic (Moloney) murine leukemia retrovirus with NIH 3T3 cells.

A quantitative analysis of the binding kinetics of intact Moloney murine leukemia retrovirus (MoMuLV) particles with NIH 3T3 cells was performed with an immunofluorescence flow cytometry assay. The virus-cell binding equilibrium dissociation constant (KD), expressed in terms of virus particle concentration, was measured to be 8.5 (+/- 6.4) x 10(-12) M at 4 degrees C and was three- to sixfold lower at temperatures above 15 degrees C. The KD of virus binding is about 1,000-fold lower than the KD of purified MoMuLV envelope. The association rate constant was determined to be 2.5 (+/- 0.9) x 10(9) M-1 min-1 at 4 degrees C and was 5- to 10-fold higher at temperatures above 15 degrees C. The apparent dissociation rate constant at 4 degrees C was 1.1 (+/- 0.4) x 10(-3) min-1 and was doubled for every 10 degrees C increase in temperature over the range tested (15 to 37 degrees C).     

Antigenicity of Candida tropicalis strain cells cultured at 27 and 37 degrees C

All cells of four Candida tropicalis strains IFO 0199 (Ct-0199), IFO 0587 (Ct-0587), IFO 1400 (Ct-1400), and IFO 1647 (Ct-1647), obtained by cultivation at 27 and 37 degrees C for 48 h in yeast extract-added Sabouraud liquid medium, showed the shapes of typical budding yeast and the same agglutination patterns against factor sera 1, 4, 5 and 6 in the commercially available kit 'Candida Check'. The cells of the C. tropicalis IFO 0589 strain display the same properties at 27 degrees C but formed hyphae at 37 degrees C. The cell wall mannan (Ct-0589-37-M) obtained from the strain cells cultured at 37 degrees C had lost most of its reactivity against factor sera 4, 5 and 6 in an enzyme-linked immunosorbent assay, in contrast to the mannan (Ct-0589-27-M) at 27 degrees C. The 1H-nuclear magnetic resonance patterns of the mannans obtained from the cells of the four C. tropicalis strains IFO 0199, IFO 0587, IFO 1400, and IFO 1647, obtained by cultivation at 37 degrees C, did not change compared to those at 27 degrees C. By contrast, the Ct-0589-37-M had significantly lost the beta-1,2-linked mannopyranose units, corresponding to the serum factors 5 and 6. These results show that the IFO 0589 strain is an unusual strain among the general C. tropicalis strains studied.     

Cross-bridge kinetics modeled from myoplasmic [Ca2+] and LV pressure at 17 degrees C and after 37 degrees C and 17 degrees C ischemia

We modeled changes in contractile element kinetics derived from the cyclic relationship between myoplasmic [Ca(2+)], measured by indo 1 fluorescence, and left ventricular pressure (LVP). We estimated model rate constants of the Ca(2+) affinity for troponin C (TnC) on actin (A) filament (TnCA) and actin and myosin (M) cross-bridge (A x M) cycling in intact guinea pig hearts during baseline 37 degrees C perfusion and evaluated changes at 1) 20 min 17 degrees C pressure, 2) 30-min reperfusion (RP) after 30-min 37 degrees C global ischemia during 37 degrees C RP, and 3) 30-min RP after 240-min 17 degrees C global ischemia during 37 degrees C RP. At 17 degrees C perfusion versus 37 degrees C perfusion, the model predicted: A x M binding was less sensitive; A x M dissociation was slower; Ca(2+) was less likely to bind to TnCA with A x M present; and Ca(2+) and TnCA binding was less sensitive in the absence of A x M. Model results were consistent with a cold-induced fall in heart rate from 260 beats/min (37 degrees C) to 33 beats/min (17 degrees C), increased diastolic LVP, and increased phasic Ca(2+). On RP after 37 degrees C ischemia vs. 37 degrees C perfusion, the model predicted the following: A x M binding was less sensitive; A x M dissociation was slower; and Ca(2+) was less likely to bind to TnCA in the absence of A. M. Model results were consistent with reduced myofilament responsiveness to [Ca(2+)] and diastolic contracture on 37 degrees C RP. In contrast, after cold ischemia versus 37 degrees C perfusion, A x M association and dissociation rates, and Ca(2+) and TnCA association rates, returned to preischemic values, whereas the dissociation rate of Ca(2+) from A x M was ninefold faster. This cardiac muscle kinetic model predicted a better-restored relationship between Ca(2+) and cross-bridge function on RP after an eightfold longer period of 17 degrees C than 37 degrees C ischemia.     

Prostaglandin E1 binding protein on the surface of primary cultured hepatocytes

Prostaglandin E1 (PGE1) was bound to primary cultured rat hepatocytes in a receptor-dependent manner in serum-free medium at 4 degrees C. When added at a concentration of 2 X 10(-9) M, maximal specific binding occurred within 60-90 min. Trypsin treatment of the cells reduced the binding capacity to about 50% of that of untreated cells. Scatchard-analysis of the binding data showed that the cells had an apparent dissociation constant of 1.2 X 10(-8) M and a binding capacity of 580 fmol (approximately 3.5 X 10(11) PGE1 receptors)/mg of protein. In experiments at 37 degrees C, maximal specific binding occurred within 5 min and was 6-7 times that at 4 degrees C, but the amount of bound PGE1 decreased rapidly after 5 min due to metabolism of PGE1 in the hepatocytes. Thin-layer chromatographic analysis showed that the material bound to the cell surface consisted of intact PGE1 and its metabolites at 37 degrees C, but PGE1 only at 4 degrees C.     

Monooxygenase activity of rat liver microsomes immobilized by entrapment in a crosslinked prepolymerized polyacrylamide hydrazide

Rat liver microsomes were immobilized by entrapment in a chemically crosslinked synthetic gel obtained by crosslinking prepolymerized polyacrylamide-hydrazide with glyoxal. Approximately 88% of the microsomal fraction was entrapped in the gel. The specific rate of O-demethylation of p-nitroanisole was used to assay the microsomal cytochrome P-450 activity of the immobilized microsomal preparations. The gel entrapped microsomes showed monooxygenase activity at 37 degrees C of Vmax = 2.3 nmol p-nitrophenol/min per nmol cytochrome P-450, similar to that of microsomes in suspension. The Km value for the p-nitroanisole-immobilized microsomal cytochrome P-450 system (1.2 X 10(-5) M) was rather close to that of microsomes in suspension (0.8 X 10(-5) M). Under the experimental conditions used the pH activity curve of the immobilized preparation was shifted towards more alkaline values by approx. 0.5 pH unit in comparison with microsomes in suspension. The rate of cytochrome c reduction by the immobilized microsomal system (11.7 nmol/min per mg protein) at 25 degrees C was considerably lower than that of the control (microsomes in suspension, 78 nmol/min per mg protein). Enzyme activity in both preparations showed the same temperature dependence at the temperature range of 10 to 37 degrees C. The immobilized microsomal monooxygenase system could be operated continuously for several hours at 37 degrees C provided that adequate amounts of an NADPH-generating system were added periodically. Under similar conditions a control microsomal suspension lost its enzymic activity within 90 min.     

Effective heat inactivation of Mycobacterium avium subsp. paratuberculosis in raw milk contaminated with naturally infected feces

The effectiveness of high-temperature, short holding time (HTST) pasteurization and homogenization with respect to inactivation of Mycobacterium avium subsp. paratuberculosis was evaluated quantitatively. This allowed a detailed determination of inactivation kinetics. High concentrations of feces from cows with clinical symptoms of Johne's disease were used to contaminate raw milk in order to realistically mimic possible incidents most closely. Final M. avium subsp. paratuberculosis concentrations varying from 10(2) to 3.5 x 10(5) cells per ml raw milk were used. Heat treatments including industrial HTST were simulated on a pilot scale with 22 different time-temperature combinations, including 60 to 90 degrees C at holding (mean residence) times of 6 to 15 s. Following 72 degrees C and a holding time of 6 s, 70 degrees C for 10 and 15 s, or under more stringent conditions, no viable M. avium subsp. paratuberculosis cells were recovered, resulting in >4.2- to >7.1-fold reductions, depending on the original inoculum concentrations. Inactivation kinetic modeling of 69 quantitative data points yielded an E(a) of 305,635 J/mol and an lnk(0) of 107.2, corresponding to a D value of 1.2 s at 72 degrees C and a Z value of 7.7 degrees C. Homogenization did not significantly affect the inactivation. The conclusion can be drawn that HTST pasteurization conditions equal to 15 s at > or =72 degrees C result in a more-than-sevenfold reduction of M. avium subsp. paratuberculosis.     

Ca2+- and Mg-ATP-dependent shape change of human erythrocyte ghosts and triton shells

Human erythrocyte membranes (ghosts) prepared from fresh blood changed in shape from spherical to crenated, when suspended in 10(-7)-10(-6) M Ca2+-EGTA buffers. Although the ghosts from long-stored ACD blood (10 weeks) were less sensitive to 10(-7)-10(-6) M Ca2+, the ghosts obtained from this blood after it had been preincubated with adenine and inosine for 3 h at 37 degrees C were highly sensitive to Ca2+. When these highly sensitive ghosts were incubated in 10 mM Tris-Cl buffer (pH 7.4) or 1 mM MgCl2 (pH 7.4) at 0 degrees C, they gradually lost Ca2+ sensitivity within 60 min, but they recovered Ca2+ sensitivity again after re-incubation with 2 mM Mg-ATP for 20 min at 37 degrees C followed by washing with 1 mM MgCl2 (pH 7.4). The shape of these highly Ca2+-sensitive ghosts immediately changed from crenate to disc on addition of 1 mM Mg-ATP even at 6 degrees C in the presence of 10(-7)-10(-6) M Ca2+. A similar shape change was also observed when ghosts treated with 0.5% Triton X-100 (Triton shells) were used. Triton shells from fresh blood ghosts or from long-stored blood ghosts which had been preincubated with 2 mM Mg-ATP for 20 min at 37 degrees C shrank immediately in the presence of 10(-6) M Ca2+ and then swelled on addition of 1 mM Mg-ATP. The specificity to ATP and the dependency on ATP concentration are in agreement with those of the ghost shape change at step 2 (Jinbu, Y. et al., Biochem biophys res commun 112 (1983) 384-390) [18]. These results suggest that cytoskeletal protein phosphorylation enhances sensitivity to Ca2+ and induces erythrocyte shape change in the presence of physiological concentrations of ATP and Ca2+.     

The hatching of common carp (Cyprinus carpio L.) embryos in response to exposure to different concentrations of cryoprotectant at low temperatures

The hatching performance of embryos of the common carp (Cyprinus carpio L.) was examined after 1, 7, 14, 21, or 28 days of storage at -8, -6, -4, -2, 0, 2, or 4 degrees C with different concentrations of methanol (0.5-7.0 M in 0.5 M steps) or varying concentrations of methanol in 0.1 M sucrose or trehalose. Preserved embryos failed to hatch after storage at -8 and -6 degrees C, regardless of the duration of storage or the concentrations tested. Likewise, there was no hatching out above 5.0 M concentration of methanol, even with the addition of sucrose or trehalose. After storage at 2 or 4 degrees C, the hatching rate was higher with mixtures of methanol (1.5 M) and trehalose (0.1 M) than with methanol plus sucrose or methanol alone. At 4 degrees C, the solution containing 1.5 M methanol supplemented with trehalose gave the highest hatching response of embryos stored for 14 days. Comparison of hatching after 24h of storage at the effective temperatures (-4, -2, 0, 2, and 4 degrees C) revealed that low concentrations of methanol were effective at high temperatures and high concentrations at sub-zero temperatures. The combination of 0.1 M trehalose with 1.5 M methanol gave the highest percentage hatching out both at 4 and 2 degrees C. At 0 degrees C, the highest percentage hatching occurred with 0.1 M trehalose plus 2.5 M methanol and at -2 and 4 degrees C, the best results were with 0.1 M trehalose plus 3.0 M methanol.     

Hepatocyte adhesion to carbohydrate-derivatized surfaces. I. Surface topography of the rat hepatic lectin

The rat hepatic lectins, galactose- and N-acetylgalactosamine-binding proteins found on the hepatocyte cell surface, mediate adhesion of isolated primary rat hepatocytes to artificial galactose-derivatized polyacrylamide gels. Biochemical and immunohistochemical techniques were used to examine the topographical redistribution of the rat hepatic lectins in response to galactose-mediated cell adhesion. Hepatocytes isolated from rat liver by collagenase perfusion had an average of 7 x 10(5) cell surface lectin molecules per cell, representing 30-50% of the total lectin molecules per cell, the remainder residing in intracellular pools. Hepatocytes incubated on galactose-derivatized surfaces, whether at 0-4 degrees C or 37 degrees C, rapidly lost greater than 80% of their accessible cell surface lectin binding sites into an adhesive patch of characteristic morphology. The kinetics of rat hepatic lectin disappearance were used to estimate a lateral diffusion coefficient greater than 9 x 10(-9) cm2/s at 37 degrees C, suggesting rapid and unimpeded lectin diffusion in the plane of the membrane. Indirect immunofluorescence labeling of adherent cells using antihepatic lectin antibody revealed a structured ring of receptors surrounding an area of exclusion (patch) of reproducible size and shape which represented approximately 8% of the hepatocyte cell surface. Notably, adherent cells, which had lost greater than 80% of their accessible surface binding sites, still endocytosed soluble galactose-terminated radioligand at greater than 50% of the rate of nonadherent control cells. No net movement of rat hepatic lectin from intracellular pools to the cell surface was found on cells recovered after adhesion to galactose-derivatized surfaces at 37 degrees C, suggesting that the physical size and/or lectin density of the patch was restricted by kinetic or topological constraints.     

Relationship between respiratory enzymes and survival of Escherichia coli under starvation stress in lake water

Survival, electron transport system (ETS) activity and the activity of NADH and succinate dehydrogenase of Escherichia coli ML30 were studied under starvation stress at different temperatures in a filtered-autoclaved lake water microcosm. ETS activity in E. coli declined rapidly at 30 degrees C but more slowly at 4 degrees and 15 degrees C over a 20 d starvation period. The decrease in ETS activity in E. coli only started after 6 d of incubation at 4 degrees C and 15 degrees C. Viability of E. coli, as determined by plate counts, declined faster at 37 degrees C than at the other temperatures and remained highest at 4 degrees C in filtered-autoclaved lake water. There was also a significant cell size reduction at 37 degrees C in filtered-autoclaved lake water but not at 4 degrees C. ETS activity after up to 16 d of starvation increased after the addition of nutrient broth to the filtered-autoclaved lake water at 15 degrees C and 30 degrees C suggesting that cells were still able to respond to nutrients, even after prolonged starvation. The response to the addition of nutrient broth, however, declined with the length of the starvation period. The activity of both succinate and NADH dehydrogenase declined over a 13 d starvation period. The loss of activity was fastest at 37 degrees C compared to lower incubation temperatures but even at 4 degrees C, a significant proportion of the activity was lost over the 13 d period.     

Interactions of cooling rate, warming rate, glycerol concentration, and dilution procedure on the viability of frozen-thawed human granulocytes

Difficulties in the successful freezing of human granulocytes could lie at two levels. One is that critical cryobiological variables have not yet been identified, the other is that the inconsistent results may be due to unusual biological aspects of the cell. This paper is concerned with the former. A prerequisite for the successful freezing of mammalian cells is the ability of the cell to tolerate cryoprotective levels of additive. The additive studied here was glycerol. Based on fluorescent staining with fluorescein diacetate, we found that 1 and 2 M concentrations are in fact chemically toxic at 22 degrees C. Superimposed on this toxicity is some osmotic sensitivity to the removal of the additive by other than slow dilution. The dilution procedure was selected on the basis of computer modeling of the osmotic response of the cells. The model requires a value for the permeability coefficient for glycerol. The value (4 X 10(-5) cm/min) was obtained by measuring the rate of increase of the volume of cells in hyperosmotic glycerol. The response of human granulocytes to freezing to -196 degrees C and thawing in 1 or 2 M glycerol was not unusual. The optimum cooling rate was 1-3 degrees C/min, and cooling at 10 degrees C/min or faster was especially deleterious if warming was slow (1 degree C/min) rather than rapid (188 degrees C/min). The FDA assay showed that some 75% of the cells survived freezing and thawing at optimum rates in 1 or 2 M glycerol; and some 50-60% remained viable after the glycerol had been removed, provided that the cells remained at 0 degrees C. However, granulocytes normally function at 37 degrees C. Because chemotaxis is considered a good assay of normal function, we developed a modified procedure capable of discriminating among random migration, enhanced random migration (chemokinesis), and directed cell migration (true chemotaxis). When frozen-thawed-diluted cells were incubated for 60 min at 37 degrees C, their survival, based both on the FDA assay and on the chemotaxis assay, was zero. In fact, a prior exposure of the cells to 2 M glycerol at 0 degrees C, even in the absence of freezing, resulted in a rapid loss in FDA viability when the cells were subsequently held at 37 degrees C for up to 60 min. Survivals based on FDA are usually reported to be considerably higher than survivals based on functional assays such as chemotaxis or phagocytosis.(ABSTRACT TRUNCATED AT 400 WORDS)