Temperature Effects on Legionella pneumophila Killing by and Multiplication in Phagocytes of Guinea Pigs

We examined the effects of temperature on the interaction between Legionella pneumophila and phagocytes of guinea pigs. The body temperatures of guinea pigs infected with a sublethal dose (1.2 × 10⁴ CFU) or a lethal dose (1.0 × 10⁵ CFU) of L. pneumophila elevated from 38.4±0.15 C to 40.2±0.42 C or 40.3 ± 0.62 C, respectively. The intracellular bacterial killing by and bacterial proliferation in the phagocytes were examined at 33, 37, 40, and 42 C, using in vitro culture systems of peritoneal macrophages or polymorphonuclear leukocytes (PMN) of guinea pigs. In all the macrophages incubated at different temperatures, significant intracellular bacterial killings were observed at 4 hr after in vitro phagocytosis. After 24 hr of incubation, there was about a 100-fold increase of CFU and the number reached a maximum after 48 hr of incubation in the macrophages incubated at 42 C as well as 37 and 40 C, suggesting that macrophages support the intracellular bacterial growth in hyperthermia. In the PMN, L. pneumophila CFU 4 hr or 12 hr after the infection were significantly lower at 42 C than those at 37 C (P<0.05), indicating that the bactericidal capacity of PMN was enhanced at 42 C compared to 37 C. However, in all the PMN incubated at different temperatures, there were about 10-fold increases of CFU 24 hr after the infection, suggesting that PMN as well as macrophages support intracellular bacterial growth in hyperthermia. The extracellular bacterial growth was examined at 33, 37, 40, and 42 C in buffered yeast extract (BYE) broth or RPMI 1640 medium containing 50% guinea pig serum as a permissive or non-permissive liquid medium for the bacterial growth, respectively. Inhibition of bacterial growth in BYE broth at 42 C, and a decrease of CFU in RPMI 1640 medium containing 50% guinea pig serum at 42 C were observed. In conclusion, hyperthermia may be beneficial by restricting extracellular bacterial survival, but it exerts no beneficial effect on the restriction of intracellular bacterial growth in phagocytes, though PMN showed enhanced initial killing at 42 C. These results suggest that fever, or hyperthermia itself, may not largely contribute as a nonspecific host defense early in the course of legionellosis.     

Effect of endocellular cryoprotectant upon polymorphonuclear neutrophil function during storage at low temperature.

The effect of cryoprotective agents dimethyl sulfoxide (DMSO), glycerol and ethylene glycol upon the function of polymorphonuclear neutrophils (PMNs) during storage between 0 ° and 4 °C was investigated.Increasing concentration of each cryoprotectant caused an increasing inhibition of chemotaxis with complete inhibition at 16.7%. At this concentration most PMNs were still able to exclude trypan blue dye. Chemotaxis was not inhibited if PMNs were exposed to 4.2 or 8.3% concentrations of cryoprotectants for 1 hr, and washed subsequently. However, the recovery of chemotaxis was not observed at 16.7% after 1 hr exposure to cryoprotectants. Moreover, a considerable number of PMNs could not exclude the dye. This would indicate that cells become fragile with cryoprotectants at a high concentration and the PMNs are easily damaged by washing. With 20 hr exposure PMNs, the inhibitory effect on chemotaxis was removed by washing when a 4.2% agent was used, but using an 8.3% agent, chemotaxis was not restored but PMNs exposed to DMSO displayed almost the same chemotaxis as a control. On the other hand, the ability of PMNs to ingest bacteria was not so markedly inhibited as the chemotaxis. With 1 hr exposure to cryoprotectants, the ingestive ability was hardly affected within 8.3%. As for 20 hr exposure, the same ingestive ability as that of a control was observed in all cases using a 4.2% concentration. However, using an 8.3% concentration, the DMSO-exposed PMNs retained a good ingestive ability.Judging from the above findings, DMSO would be suitable as a crypotrotective agent although the problem on toxicity remains to be resolved.     

Combined action of micafungin, a new echinocandin, and human phagocytes for antifungal activity against Aspergillus fumigatus

Micafungin, a new echinocandin, inhibits fungal cell wall beta-glucan synthesis. We postulated micafungin and host phagocytic cells could act together in damaging fungi. Using the metabolic XTT assay, micafungin alone (0.01 and 0.10 microg/ml) inhibited Aspergillus fumigatus germlings by 48% and 61%, respectively. Polymorphonuclear neutrophils (PMNs) inhibited germlings by 53%. Micafungin at 0.01 or 0.10 microg/ml and PMNs resulted in additive inhibition, 82% and 99%, respectively. Monocyte-derived macrophage (MDM) monolayers inhibited germling growth by 66%; micafungin (0.01 or 0.10 microg/ml) alone inhibited by 32% and 42%, respectively. MDMs and micafungin (0.01 or 0.10 microg/ml) caused an additive inhibition of growth, 85% and 95%, respectively. Hyphae were generated by incubation of conidia for 24 h with or without micafungin. PMNs alone, added to hyphae, inhibited growth by 19% in the subsequent 20 h. Hyphae generated in the presence of micafungin (0.10 microg/ml) and subsequently cultured with micafungin for 24 h inhibited growth by 64%. PMNs plus micafungin resulted in 82% inhibition. Monocytes alone inhibited hyphal growth by only 5%. Hyphae produced in the presence of micafungin (0.01 microg/ml) and incubated again with micafungin for 24 h inhibited growth by 47%; combination with monocytes resulted in 62% inhibition. These data indicate that micafungin inhibits growth of tissue forms of A. fumigatus, and phagocytes and micafungin together have an additive effect. These findings support the thesis that the greater efficacy of micafungin in vivo compared with in vitro could be due to combined effect of phagocytic cells and micafungin.     

Flow Cytometric Measurement of Bovine Milk Neutrophil Phagocytosis

A flow cytometric method for the evaluation of the phagocytic capacity of bovine milk polymorphonuclear neutrophils (PMN) is described. Milk PMN were isolated from stripping milk collected from udder quarters fitted with abraded intramammary devices (AIMD). A significant increase in the milk somatic cell count was observed in the stripping milk after the insertion of AIMD (308×103 and 1447×l03 cells/ml milk before respectively after the insertion of the AIMD, p < 0.001). PMN were also isolated from blood by a discontinous gradient of Percoli. Blood and milk PMN were incubated for 15 min with FITC-labeled bacteria in a ratio of 1 PMN:20 bacteria and a final serum concentration of 10 %. The number of extracellular bacteria and the percentage of phagocytic cells were measured by a flow cytometer. Percentage of phagocytized bacteria by milk PMN was significantly lower than that by blood PMN (p < 0.05). A smaller number of active phagocytes was present among cells isolated from milk than among cells isolated from blood. The phagocytic capacity of milk PMN reflects that of blood PMN in the same animal. A large variation in the phagocytic capacity of blood and milk PMN among animals was observed.     

Nitric oxide released from iNOS in polymorphonuclear leukocytes makes them deformable in an autocrine manner.

The objective of this study was to determine whether endogenous nitric oxide (NO) derived from reaction catalyzed by the inducible isoform of NO synthase (iNOS: NOS II) in polymorphonuclear leukocytes (PMNs) makes the PMNs deformable. Previous studies have shown that NO increases the deformability of PMNs and decreases the sequestration of PMNs in the lungs. However, there was little information regarding the effect of PMN-derived NO on the cells' deformability. In the present study PMNs were isolated from the blood of rats 24h after ip injection of saline (control) or lipopolysaccharide (LPS), and expression of iNOS in the PMNs of the LPS group was confirmed by immunocytochemistry. PMN deformability was evaluated by measuring the pressure generated during their passage through a microfilter at a constant flow rate. The nitrite/nitrate content of the solution in which the isolated PMNs were incubated was measured by the Griess method. In the control group, no iNOS was detectable in the PMNs, and the nitrite/nitrate level in the PMN incubation solution was low. Deformability was unchanged after incubation with specific iNOS inhibitor aminoguanidine, but decreased after incubation with N-formyl-methionyl-leucyl-phenyl-alanine. In the LPS group, PMN deformability was decreased compared to that of the control group. iNOS was detectable in the PMNs, and the deformability further decreased after incubation with aminoguanidine. These results suggest that endogenous NO generated during reactions catalyzed by iNOS in PMNs makes them deformable in an autocrine manner.     

Recovery,structure, and function of dog granulocytes after freeze-preservation with dimethylsulfoxide

The recovery, structure and function of dog granulocytes were determined before and after freeze-preservation. Leucocytes were isolated from defibrinated or anti-coagulated whole blood and subsequent erythrocyte sedimentation on a column of 2:1 dextran (6%)-isopaque (33.9%). Granulocytes isolated by these procedures were examined for changes in O₂ consumption associated with phagocytosis, in vitro directed migration (chemotaxis), bactericidal activity, and ultrastructure before and after freezing. Granulocytes were frozen in DMSO (7.5%) and autologous serum or HBSS-minus and 20% autologous serum at the rate of ?1 °C/min to ?80 °C and stored in liquid N₂ vapor.After freeze-preservation, O₂ consumption associated with phagocytosis was decreased by 54 and 64% for granulocytes isolated from defibrinated or from ACD-anticoagulated blood, respectively. Bactericidal activity is only slightly depressed in samples from either isolation method after freeze-preservation when compared to the prefreeze controls, but granulocytes isolated from defibrinated blood are significantly less effective in killing bacteria than those from ACD-anticoagulated blood. Chemotactic response after freeze-preservation was completely inhibited in granulocytes isolated from defibrinated blood. Exposure of granulocytes to ACD inhibited chemotaxis prior to freezing, but the granulocytes responded chemotactically after freeze-thaw and additional washing. The ultrastructure of granulocytes observed before and after freeze-thaw was similar for cells isolated by both methods. However, nuclear, cytoplasmic, and granular changes observed were slightly greater in granulocytes isolated from defibrinated blood. Dog granulocytes isolated by either method withstood freeze-preservation in DMSO to a degree not previously reported.It is concluded that dog granulocytes freeze-preserved by these methods are functional in vitro, but that phagocytic, directed migration, and bactericidal functions and ultrastructure are impaired to different degrees, according to the method of isolation and preparation for storage. These results indicate the need for continued investigation on the effects of storage variables on the preservation of granulocytes.     

Changes in porcine,ovine, bovine and equine blood progesterone concentrations between collection and centrifugation

The aim of this study was to determine if different methods of handling porcine, ovine, bovine and equine blood between collection and centrifugation influence measurable progesterone levels. A 2 × 2 × 5 factorial experiment was conducted for each species with heparin (with or without), temperature of incubation (4 and 22°C) and time of incubation (0, 6, 12, 24 and 48 h) as the main effects. Following centrifugation, plasma and serum samples were stored at ?20°C until progesterone concentrations were determined by radioimmunoassay. Method of handling porcine and equine blood between collection and centrifugation did not affect the levels of progesterone. However, heparinized blood held at 4°C resulted in the most consistent levels of progesterone over time. Progesterone levels were fairly consistent across time in the ovine blood by all methods of handling except heparinized blood incubated at 22°C. By 24 h after collection, plasma progesterone concentrations decreased by 50% for the ovine blood incubated at 22°C with heparin. Decreases were detected by all the methods of handling the bovine blood between collection and centrifugation. The rate of decline, however, was considerably faster for blood held at 22°C than blood held at 4°C. At 12–48 h after collection, the concentrations of progesterone averaged only 5% of the time 0 sample for blood incubated at 22°C. In contrast, at least 30% of the progesterone values in the time 0 sample were detected between 12 and 48 h of incubation for the blood held at 4°C.     

The antiteratogenic effects of hypo- and hyperthermia in trypan blue-treated chick embryos

Fertile White Leghorn chicken eggs (N = 174) were incubated under optimum conditions until the embryos had reached Hamburger-Hamilton stage 12 (about 48 hr). At that time, 20 μl of 1% trypan blue solution, dissolved in 0.85% NaCl (wt/vol) was injected through the yolk sac into the liquid yolk found just under the embryo. After injection, the eggs were separated into groups and returned to the incubator under control conditions (38°C), or at temperatures lower (35°C) or higher (41°C) than optimum.After an additional 24 hr of incubation, the embryos incubated at 35°C (N = 53) exhibited significantly fewer caudal hematomas (P < 0.02) than did embryos incubated at 38°C (N = 51). Similarly, embryos incubated at 41°C (N = 40) also exhibited significantly fewer caudal hematomas (P < 0.05) than did their corresponding (38°C) controls (N = 30). There was no significant difference between the 35°C group and their controls, or the 38°C group and their controls, in embryonic dry weight, dry weight of the area vasculosa, or crown-rump length. The only other significant difference detected between groups was a very slight but significant (P < 0.0005) decrease in Hamburger-Hamilton stage (0.4 stage unit) between embryos incubated at 35°C and the corresponding controls.Since incubation temperatures either above or below optimum result in a marked reduction in the teratogenic response to trypan blue treatment, we conclude that there exists a temperature optimum for the development of caudal hematomas.     

Viability of frozen rat granulocytes and granulocyte precursors

Mature rat polymorphonuclear leukocytes (PMNs) were frozen to ?196 °C, thawed, and tested for functional viability using a variety of criteria. The assays for functional viability included: qualitative and quantitative nitroblue tetrazolium tests for phagocytic activity, fluorometric tests for membrane integrity, chemotaxis, and bactericidal activity. Maximal survival was obtained when mature PMNs were frozen in the presence of 10% dimethyl sulfoxide (Me₂SO) and 5% hydroxyethyl-starch (HES) for cells cooled at ~10 °C per minute, followed by rapid warming. Maximal survival was obtained for granulocyte precursor cells (as measured by CFU-c) after freezing in the presence of 10% Me₂SO and cooling at ~10 °C per minute. The principal new findings for mature PMNs were: (i) there was a synergistic effect between intra- and extracellular protective additives; (ii) the optimal cooling rate increases from approximately 0.3 to 10 °C per minute when an extracellular protective agent, such as HES is included in the freezing media; (iii) the zwitterion buffer Hepes has a small but consistently beneficial effect on survival; (iv) granulocytes obtained from peripheral blood consistently show a higher functional survival after freezing (95%) than do PMNs obtained from a glycogen-induced peritoneal exudate (70%); (v) neither serum, plasma, nor other macromolecules are needed in the postt-haw dilution media to obtain high survival; and (vi) cells frozen using an optimized two-step protocol survived as well as those frozen using a continuous cooling protocol.     

Innate-immune responses of tilapia (Oreochromis mossambicus) exposure to acute cold stress

In this present study, Oreochromis mossambicus tilapia were transferred to cold water at 12°C for various time intervals (1, 4, 8, 24, and 48 hr) and its innate immune response was analyzed by studying cellular and humoral parameters. In vivo, alternative complement pathway activity in blood plasma was rapidly increased at 1 hr of cold water (12°C) exposure. Lysozyme activity and cortisol levels of plasma were increased at 4 and 1 hr, respectively. Surprisingly, only plasma cortisol levels remained unchanged through 24 hr of cold water transfer. Phagocytic ability, phagocytic capacity, and respiratory burst (RB) activity of head kidney (HK) leukocytes and splenocytes showed no any significant changes. In peripheral blood leukocytes, phagocytic capacity, and RB activity were increased at 24 hr of cold water exposure. The expressions of genes involved innate immunity in splenocytes and HK leukocytes of tilapia cold water exposure were analyzed, messenger RNA (mRNA) expressions of HSP70, HSP90, and immunoglobulin M failed to change upon exposure to cold stress. Major histocompatibility complex-I and II mRNAs were significantly increased in tilapia splenocytes at 1 hr of cold water transferred. Whereas myxovirus (Mx) expression was increased in splenocytes and HK leukocytes of tilapia after 1 hr of cold water exposed. Our result reveals that the exposure of tilapia to acute cold stress condition significantly enhances plasma acid phosphatase activity and both phagocytic capacity and RB activity. Furthermore, cold stress significantly stimulates Mx gene expression in splenocytes and HK leukocytes.     

Effect of time and temperature on bovine serum and plasma progesterone concentration

Whole blood, with and without anticoagulant, from 5 pregnant cows was incubated at 40°C for 0 (30 minutes after collection), 6 and 24 hours (hr) before the blood was centrifuged and the plasma or serum was frozen for later progesterone assay. Mean plasma progesterone concentration decreased from 6.6 ng/ml at 0 hr to 1.7 ng/ml at 6 hr (P < 0.01) and to 2.8 ng/ml at 24 hr (P < 0.01). Mean serum progesterone concentration decreased from 6.1 ng/ml at 0 hr to 3.9 ng/ml at 6 hr (P < 0.01) and to 4.4 ng/ml at 24 hr (P < 0.01). Whole blood samples with and without EDTA were also incubated at 4°C for 24 hr. Mean plasma progesterone concentration decreased from 6.6 ng/ml at 0 hr to 4.2 ng/ml at 24 hr (P < 0.01). Mean serum progesterone concentration decreased from 6.1 ng/ml at 0 hr to 4.7 ng/ml at 24 hr (P < 0.01). The incubation time and temperature of whole blood, from collection of blood to the separation of serum or plasma, significantly affects assayable concentration of progesterone.     

Inhibition of the accumulation of viral polypeptides in the densonucleosis virus-infected midgut of the silkworm,Bombyx mori,reared at a supraoptimal temperature

Effect of a supraoptimal temperature on the accumulation of viral polypeptides in the midgut was examined by immunoblot analysis in the larvae of the silkworm, Bombyx mori, infected with Bombyx densonucleosis virus type 2. In the larvae reared continuously at 25°C, viral polypeptides were first detected in the midgut at 2 days postinfection (pi) and in the feces at 4 days pi. When the larvae inoculated per os with the virus for 24 hr at 25°C were immediately shifted to 35°C, there were no detectable viral polypeptides in both the midgut and feces throughout the experiment. In the infected larvae shifted from 25° to 35°C at 48 hr pi, viral polypeptides preexisting in the midgut decreased to an undetectable level within 48 hr after the temperature shift, and no viral polypeptides were detected thereafter. Viral polypeptides in the feces of these larvae became detectable at 48 hr (4 days pi) after the temperature shift, as in the larvae at 25°C, and disappeared by 96 hr (6 days pi). These results indicate that a supraoptimal temperature inhibits accumulation of viral polypeptides in the midgut. It is likely that inhibited production of viral polypeptides rather than enhanced discharge of the infected midgut cells is responsible for the inhibited accumulation of viral polypeptides in the midgut at 35°C.     

Impaired alveolar macrophage function in smoke inhalation injury

The high incidence of both bacterial pneumonia and the adult respiratory distress syndrome (ARDS) associated with smoke inhalation injury (SII) may result, at least in part, from smoke-induced injury to the alveolar macrophage (AM). Specifically, we hypothesized that AM antimicrobial function, ability to phagocytose apoptotic PMNs, and capacity to prevent apoptosis in PMNs are impaired by smoke. To test these hypotheses, AMs were harvested by bronchoalveolar lavage from sheep before and after the animal was exposed to cotton smoke. The two populations of AMs were incubated with Pseudomonas aeruginosa (PSA) in vitro. Normal AMs (NAMs) phagocytosed a mean of 99 ± 11% of the PSA placed in their wells, whereas smoke-exposed AMs (SAMs) ingested only 60 ± 8%. NAMs killed 80 ± 8% of PSA ingested, whereas SAMs killed only 56 ± 16% (P < 0.05). When sheep PMNs, allowed to undergo apoptosis, were incubated with the two AM populations, 66 ± 3% of the NAMs and 40 ± 6% of the SAMs demonstrated phagocytosis of these apoptotic PMNs (P < 0.05). Fresh sheep PMNs were incubated in unconditioned media, NAM and SAM-conditioned media, and followed over 48 hr for the development of apoptosis and maintenance of viability. The NAM-conditioned media markedly prevented apoptosis and augmented PMN survival relative to the unconditioned and SAM-conditioned media (P < 0.05). The poor antimicrobial function known to be characteristic of apoptotic PMNs, together with the directly impaired antimicrobial function of AMs, may contribute to the infectious complications of Sll. If the PMNs recruited to the lung in Sll are not properly supported by the AMs following smoke injury, large numbers may undergo apoptosis. If not properly disposed of by these SAMs, the apoptotic PMNs could eventually lyse, releasing tissue toxins, resulting in escalation of lung injury and leading to ARDS. © 1995 Wiley-Liss, Inc.     

Interaction of dexamethasone and Salmonella enteritidis immune lymphokines on Salmonella enteritidis organ invasion and in vitro polymorphonuclear leukocyte function

Abstract We used an anti-inflammatory dose of dexamethasone (DEX) and Salmonella enteritidis (SE)-immune lymphokines (ILK) followed by oral SE challenge to chicks to determine the effects of these treatments on SE organ invasion and in vitro function of PMNs derived from peripheral blood. Endpoints included percent protection against SE organ invasion, numbers of peripheral blood PMNs, and in vitro PMN adherence, chemotaxis, and SE killing. SE organ invasion was significantly reduced in chicks treated with either ILK alone or DEX + ILK compared to controls. Chicks treated with either DEX alone or DEX + ILK responded with a significant increase in numbers of peripheral blood PMNs as compared to controls, while numbers of PMNs in the peripheral blood from chicks treated with ILK alone were not significantly increased. PMN adherence and percent SE killing by PMNs derived from chicks treated with either ILK alone or DEX + ILK were significantly increased compared to controls. Chemotaxis of PMNs derived from chicks treated with either ILK alone or DEX alone significantly increased 2-fold over control levels. Interestingly, chemotaxis of PMNs derived from chicks that received DEX + ILK was similar to controls. Generally, ILK abated the anti-inflammatory effects of DEX on PMNs in these assays, except for chemotaxis. We interpret these data to suggest that ILK may confer protection to chicks against the early phase of SE organ invasion by inducing an inflammatory response predominated by activated PMNs.     

Phagocytic and humoral immunity of the adult cotton boll weevil, Anthonomus grandis (Coleoptera: Curculionidae), to Serratia marcescens

This investigation determined phagocytic, lysozymal, and bactericidal defensive responses of adult laboratory-reared cotton boll weevils, Anthonomus grandis. Phagocytosis was first demonstrated in boll weevils at 3 hr following injection of live Serratia marcescens. Maximum phagocytosis was found in 16.4% of plasmatocytes at the end of 16 hr postinjection. Lysozyme activity was demonstrated in both inoculated and uninoculated boll weevils. Peak lysozyme activity of 6.9 μg/ml was found at 48 hr following inoculation of heat-killed Serratia marcescens. Bactericidal activity was demonstrated in inoculated boll weevils but not in uninoculated boll weevils. Peak bactericidal activity occurred at 24 hr following inoculation of heat-killed Serratia marcescens. Lysozymal and bactericidal activities were shown to be separate functions.     

Purification and partial characterization of novel penicillin V acylase from Acinetobacter sp. AP24 isolated from Loktak Lake,an Indo-Burma biodiversity hotspot

Members of the bacterial genus Acinetobacter have attracted great attention over the past few decades, on account of their various biotechnological applications and clinical implications. In this study, we are reporting the first experimental penicillin V acylase (PVA) activity from this genus. Penicillin acylases are pharmaceutically important enzymes widely used in the synthesis of semisynthetic beta-lactam antibiotics. The bacterium, identified as Acinetobacter sp. AP24, was isolated from the water of Loktak Lake (Manipur, India), an Indo-Burma biodiversity hotspot. PVA production was increased threefold in an optimized medium with 0.2% sodium glutamate and 1% glucose as nitrogen and carbon sources respectively, after 24 hr of fermentation at 28°C and pH 7.0 with shaking at 180 rpm. The enzyme was purified to homogeneity by cation-exchange chromatography using SP-sepharose resin. The PVA is a homotetramer with subunit molecular mass of 34 kD. The enzyme was highly specific toward penicillin V with optimal hydrolytic activity at 40°C and pH 7.5. The enzyme was stable from pH 5.0 to 9.0 at 25 °C for 2 hr. The enzyme retained 75% activity after 1 hr of incubation at 40°C at pH 7.5.     

Saccharification of cellolulose by the cellulolytic enzyme system of Thermonospora sp. I. Stability of cellulolytic activities with respect to time,temperature, and pH

The stabilities and optima with respect to temperature and pH of the β-glucosidase, Avicelase, and carboxymethylcellulase (CMCase) activity of Thermomonospora sp., in the culture filtrate, culture whole broth, and filtrate after sonication of culture solids, are reported. The β-glucosidase is cell associated and has an optimal activity at about pH 6.5 and 55°C. In the whole culture broth, it has a half-life of about 8 hr at 55°C and less than 1 hr at 60°C, while the half-life of the activity in the sonicated, cell-free filtrate is less than 1 hr at 55°C. The Avicelase and CMCase activities occur in the extracellular culture fluid and have optima at about pH 7.0 and 5.9, and 65 and 70°C, respectively. The CMCase activity is stable over 24 hr at 60°C, but declines by 50% in the same period at 65°C. The Avicelase activity declines by 15% over 24 hr at 55°C, and by 50% at 60°C. The highest pH studied (pH 7.3) was the most destabilizing for all three activities. The thermostable characteristics of the cellulases from Themomonospora appear to make them suitable for commercial saccharification processes operating at elevated temperatures.     

Seed dormancy-breaking and germination requirements ofDrosera anglica,an insectivorous species of the Northern Hemisphere

Seeds of Drosera anglica collected in Sweden were dormant at maturity in late summer, and dormancy break occurred during cold stratification. Stratified seeds required light for germination, but light had to be given after temperatures were high enough to be favorable for germination. Seeds stratified in darkness at 5/1 °C and incubated in light at 12/12 h daily temperature regimes of 15/6, 20/10 and 25/15 °C germinated slower and to a significantly lower percentage at each temperature regime than those stratified in light and incubated in light. Length of the stratification period required before seeds would germinate to high percentages depended on (1) whether seeds were in light or in darkness during stratification and during the subsequent incubation period, and (2) the temperature regime during incubation. Seeds collected in 1999 germinated to 4, 24 and 92 % in light at 15/6, 20/10 and 25/15 °C, respectively, after 2 weeks of stratification in light. Seeds stratified in light for 18 weeks and incubated in light at 15/6, 20/10 and 25/15 °C germinated to 87, 95 and 100 %, respectively, while those stratified in darkness for 18 weeks and incubated in light germinated to 6, 82 and 91 %, respectively. Seeds collected from the same site in 1998 and 1999, stratified in light at 5/1 °C and incubated in light at 15/6 °C germinated to 22 and 87 %, respectively, indicating year-to-year variation in degree of dormancy. As dormancy break occurred, the minimum temperature for germination decreased. Thus, seed dormancy is broken in nature by cold stratification during winter, and by spring, seeds are capable of germinating at low habitat temperatures, if they are exposed to light.     

Role of the Phagocyte in Host-Parasite Interactions VIII. Effect of Whole-Body X Irradiation on Nicotinamides, Lysosomal Enzymes, and Bactericidal Activities of Leukocytes During Phagocytosis

By studying the effects of whole-body X irradiation on phagocytosis, a correlation between the metabolic and bactericidal activities of leukocytes following X irradiation was demonstrated. The total nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) content of polymorphonuclear neutrolphils (PMN) isolated from irradiated guinea pigs increased significantly when compared to nonirradiated controls. The ratio of unreduced to reduced (NAD) generally increased in PMN isolated from irradiated animals. This occurred with both resting and phagocytizing cells. The ratio of unreduced to reduced NADP of resting PMN isolated from irradiated animals had a tendency to increase. However, in phagocytizing cells a significant decrease in the ratio was noted. The total acid and alkaline phosphatase and beta-glucuronidase increased up to about 10 days postirradiation. These lysosomal enzymes returned to approximately normal by the 17th day postirradiation. All three lysosomal enzymes (acid and alkaline phosphatases and beta-glucuronidase) were released from the granules at a significantly faster rate during phagocytosis after irradiation. The bactericidal activities of PMN isolated from irradiated animals gradually decreased, and in some cases increased growth of the organisms was observed. The uptake or association of bacteria with PMN isolated from irradiated animals varied with the postirradiation time. Generally, a correlation with bactericidal activities could be made. The data indicate that the bactericidal system in phagocytes consists of at least two agents, H(2)O(2) and myeloperoxidase.     

Chemoattractant-stimulated GTPase activity is decreased on membranes from polymorphonuclear leukocytes incubated in chemoattractant

Polymorphonuclear leukocytes, PMNs, incubated in a chemoattractant undergo a time-dependent decrease in responsiveness to the chemoattractant; i.e. they desensitize or adapt. We have examined the role of ligand-induced changes at early steps in signal transduction for adaptation of PMNs to chemoattractants. The chemoattractant stimulation of a pertussis toxin-sensitive GTPase activity on PMN membranes was used as an assay of signal transduction. We find a decreased basal GTPase activity and a decrease in the ability of N-formylnorleucylleucylphenylalanine (FN-LLP) to stimulate this activity on membranes prepared from PMNs incubated with the chemotactic peptide FNLLP. The basal GTPase activity is decreased by up to 70% and the peptide-stimulated GTPase activity by up to 95% on membranes from PMNs incubated for 20 min at 37 degrees C in 10(-7) M FNLLP. The decrease in peptide-stimulated GTPase activity cannot be accounted for by the decreased number of FNLLP receptors on the membranes. Rather, receptors that remain available for binding stimulate the GTPase activity with a decreased efficiency. The ligand-induced change in GTPase activity is not stimulus specific. GTPase activity stimulated by both C5a and LTB4 was decreased on membranes from PMNs incubated in FNLLP. The decrease in chemoattractant-stimulated GTPase activity is partially reversed if cells are subsequently incubated at 37 degrees C in the absence of peptide prior to membrane preparation. We detected no quantitative or qualitative change in either pertussis toxin substrates or immunoreactive G proteins when membranes from control and FNLLP-treated cells were compared.