Assessment of the cell viability of cultured Perkinsus marinus (Perkinsea), a parasitic protozoan of the Eastern oyster, Crassostrea virginica, using SYBRgreen-propidium iodide double staining and flow cytometry

A flow cytometry (FCM) assay using SYBRgreen and propidium iodide double staining was tested to assess viability and morphological parameters of Perkinsus marinus under different cold- and heat-shock treatments and at different growth phases. P. marinus meront cells, cultivated at 28 degrees C, were incubated in triplicate for 30 min at -80 degrees C, -20 degrees C, 5 degrees C, and 20 degrees C for cold-shock treatments and at 32 degrees C, 36 degrees C, 40 degrees C, 44 degrees C, 48 degrees C, 52 degrees C, and 60 degrees C for heat-shock treatments. A slight and significant decrease in percentage of viable cells (PVC), from 93.6% to 92.7%, was observed at -20 degrees C and the lowest PVC was obtained at -80 degrees C (54.0%). After 30 min of heat shocks at 40 degrees C and 44 degrees C, PVC decreased slightly but significantly compared to cells maintained at 28 degrees C. When cells were heat shocked at 48 degrees C, 52 degrees C, and 60 degrees C heavy mortality occurred and PVC decreased to 33.8%, 8.0%, and 3.4%, respectively. No change in cell complexity and size was noted until cells were heat shocked at >or=44 degrees C. High cell mortality was detected at stationary phase of P. marinus cell culture. Cell viability dropped below 40% in 28-day-old cultures and ranged 11-25% in 38 to 47-day-old cultures. Results suggest that FCM could be a useful tool for determining viability of cultured P. marinus cells.     

Polystyrene embedding: a new method for light and electron microscopy.

Polystyrene embedments of histological specimens can be obtained with a solution of 1:14 polystyrene-toluene, 5% benzyl alcohol and 1% dibutyl phthalate, allowing the solvent to evaporate in polyethylene containers for 2-3 days at 58 C. The resulting blocks are easily cut into truncated pyramids, each containing a piece of tissue, which are then glued to a Plexiglas support. Drying is completed at 80 C for 20 hr. The pyramids can then be sectioned to produce thick sections with a steel knife or to produce semi- or ultrathin sections with a glass knife. A 10% paraldehyde solution is used to mount the light microscopy sections on a slide heated on a hot plate to 80 C; these can be treated with the same techniques used with paraffin sections. The results are of high quality. Semithin sections of tissues fixed for electron microscopy can be stained directly after mounting, or by a wider range of stains once the polystyrene has been removed by organic solvents. In electron microscopy, the ultrathin sections obtained with the usual techniques are highly electron beam-resistant and given acceptable results.     

Effects of time and temperature during attachment of sections to microscope slides on immunohistochemical detection of antigens.

To study the effects of time and temperature on attachment of tissue sections to microscope slides, we examined the intensity of immunohistochemical staining of selected antigens in nine different neoplastic and normal tissues after attaching sections at different times and temperatures. Typically, both the temperature and time are minimized when tissue sections attached to slides; however, suboptimal times and temperatures during attachment may result in either loss of tissue due to poor attachment or the necessity for inconvenient staining regimens. Using standard immunohistochemical techniques, 5 microm tissue sections were attached at 58 degrees C for 1, 4 and 24 hr. In a separate study, 5 microm tissue sections were attached for 16 hr at 58, 68 and 80 degrees C. The intensity of staining decreased slightly when the tissue sections were heated at 80 degrees C for 16 hr, but there was little or no decrease when tissues were heated at 68 degrees C or lower for 16 hr, or at 58 degrees C for up to 24 hr.     

Application of scanning electron microscopy to x-ray analysis of frozen- hydrated sections. III. Elemental content of cells in the rat renal papillary tip

The electrolyte and water content of cellular and interstitial compartments in the renal papilla of the rat was determined by x-ray microanalysis of frozen-hydrated tissue sections. Papillae from rats on ad libitum water were rapidly frozen in a slush of Freon 12, and sectioned in a cryomicrotome at -30 to -40 degrees C. Frozen 0.5- micrometer sections were mounted on carbon-coated nylon film over a Be grid, transferred cold to the scanning microscope, and maintained at - 175 degrees C during analysis. The scanning transmission mode was used for imaging. Structural preservation was of good quality and allowed identification of tissue compartments. Tissue mass (solutes + water) was determined by continuum radiation from regions of interest. After drying in the SEM, elemental composition of morphologically defined compartments (solutes) was determined by analysis of specific x-rays, and total dry mass by continuum. Na, K, Cl, and H2O contents in collecting-duct cells (CDC), papillary epithelial cells (PEC), and interstitial cells (IC) and space were measured. Cells had lower water content (mean 58.7%) than interstitium (77.5%). Intracellular K concentrations (millimoles per kilogram wet weight) were unremarkable (79-156 mm/kg wet weight); P was markedly higher in cells than in interstitium. S was the same in all compartments. Intracellular Na levels were extremely high (CDC, 344 +/- 127 SD mm/kg wet weight; PEC, 287 +/- 105; IC, 898 +/- 194). Mean interstitial Na was 590 +/- 119 mm/kg wet weight. CI values paralleled those for Na. If this Na is unbound, then these data suggest that renal papillary interstitial cells adapt to their hyperosmotic environment by a Na-uptake process.     

Binding and release of radiolabeled eukaryotic initiation factors 2 and 3 during 80 S initiation complex formation.

The AUG-dependent formation of an 80 S ribosomal initiation complex was studied using purified rabbit reticulocyte initiation factors radiolabeled by reductive methylation. The radiolabeled initiation factors were as biologically active as untreated factors. Reaction mixtures containing a variety of components (AUG, GTP, Met-tRNAf, initiation factors, and 40 S and 60 S ribosomal subunits) were incubated at 30 degrees C and then analyzed on linear sucrose gradients for the formation of ribosomal complexes. The results show that both eukaryotic initiation factor (eIF)-3 and the ternary complex (eIF-2.GTP.Met-tRNAf) bind independently to the 40 S subunit and each of these components enhances the binding of the other. All of the polypeptides of eIF-2 and eIF-3 participate in this binding. Formation of an 80 S ribosomal complex requires eIF-5 and 60 S subunits in a reaction that is stimulated by eIF-4C. Both eIF-2 and eIF-3 are released from the 40 S preinitiation complex during formation of the 80 S initiation complex. Release of eIF-2 and eIF-3 does not occur and 80 S ribosomal complexes are not formed if GTP is replaced by a nonhydrolyzable analog such as guanosine 5'-O3-(1,2-mu-imido)triphosphate. Despite a variety of attempts, it has not yet been possible to demonstrate binding of eIF-4C, eIF-4D, or eIF-5 to either 40 S or 80 S ribosomal complexes.     

Effects of long-term storage at different temperatures on conidia of Botrytis cinerea Pers.: Fr.

Survival of Botrytis cinerea conidia was studied after storage without pretreatments at different temperatures (-80 degrees C, -20 degrees C, 4 degrees C and 21 degrees C). Germination tests performed during 3 years showed that viability at 21 degrees C was completely lost after 1 month. Conidia stored for 30 months at -80 degrees C, -20 degrees C and 4 degrees C were able to germinate, respectively, at 79%, 8% and 0.2%. Changes in adenylate level, energy charge and respiration (O(2) consumption) made on each set of conidia were correlated to the germination rate. The 30-month-old stored conidia showed differences in pathogenicity tests on apples. While the pathogenic aggressiveness of conidia stored at -80 degrees C was almost the same as for fresh conidia, it decreased with increasing temperature of storage. An ultrastructural study made on conidia stored for 30 months at -80 degrees C has shown the emergence of a new wall layer in a retraction zone of the cytoplasm by comparison to fresh conidia. However, the integrity of the cytoplasmic content was maintained. The effects of low temperature storage, maintenance of cell integrity and pathogenicity of conidia of B. cinerea are discussed.     

Determination of aldoses and ketoses by GC-MS using differential derivatisation

A method has been established by which to determine aldoses and ketoses in plant material simultaneously. Monosaccharides were extracted by sonication with 80% ethanol and sugar oximes formed by treatment of the resultant extract with hydroxylamine and pyridine at 90 degrees C. After reaction, one aliquot of the product was derivatised with acetic anhydride at 90 degrees C, whilst a second aliquot was silylated with HMDS and TMCS at 80 degrees C. Both reaction mixtures were analysed by GC-MS in the SIM mode. Quantivation was linear within the range 1-4 microg/mL and the detection limit for monosaccharides was 5-25 ng/mL. The absolute recoveries were between 73.0 and 90.2% and the RSDs were 3.1-10.0%. This method was applied to analyse the free monosaccharides in Lyceum barbarum L.; eight monosaccharides were present in amounts between 0.26 and 368.65 microg/mg.     

Analysis of single nucleotide polymorphisms in the promoter region of interleukin-10 by denaturing high-performance liquid chromatography.

Interleukin-10 (IL10), an anti-inflammatory cytokine, has been implicated in a variety of immune- and inflammatory-related diseases. We investigated the following SNPs: -1082, -819, -592 in the promoter region of IL10 in a normal (control) population and selected diseases: breast cancer (BrCa), systemic lupus erythematosus (SLE), and B-cell chronic lymphocytic leukemia (B-CLL) by denaturing high-performance liquid chromatography (DHPLC) and found distinct genotype and haplotype patterns. DHPLC was performed using the Transgenomic WAVE instrument, a mutational discovery tool that allows for high throughout analysis of SNPs. The principle of DHPLC is based on separation of homo- and heteroduplex formation of individual polymerase chain reaction products at specific melting temperatures and set gradients. The melting temperature selected for each SNP was based on size and sequence of the polymerase chain reaction product (for -1082, 57 degrees C; for -819, 58 degrees C; and for -592, 59.2 degrees C). Before fragment mutational analysis, all samples were denatured at 95 degrees C and slowly reannealed to allow for reassociation of different strands. Heteroduplex samples were easily distinguished from homoduplex samples. In order to identify wild type from homozygous mutant, two homoduplex polymerase chain reaction samples had to be mixed together, denatured at 95 degrees C and reannealed. The homozygous mutant, when combined with wild type, displayed a double peak on chromatogram. Once distinct chromatograms were established for each of the SNPs and the nucleotide changes confirmed by sequencing, genotype and haplotype frequencies were tabulated for the groups studied.     

Receptor-mediated endocytosis of a ricin-colloidal gold conjugate in vero cells. Intracellular routing to vacuolar and tubulo-vesicular portions of the endosomal system

We have prepared a conjugate (Ri-Au) of the toxic plant protein ricin and colloidal gold (particle size 5 nm) and used it for internalization studies in monolayer cultures of Vero cells. The Ri-Au conjugate was very stable, with only little release of ricin ([125I]Ri) from the gold particles within a pH range of 4.5-8.0. Within 2 h at 37 degrees C, only very little intracellular degradation of the ricin preparation ([125I]Ri-Au) occurred. The cells bound the same proportion of native ricin ([125I]Ri) and Ri-Au from the medium, and the kinetics of toxicity (decrease in cellular incorporation of [3H]leucine) of [125I]Ri and [125I]Ri-Au were also comparable. At 4 degrees C, the cell-surface binding of Ri-Au was continuous and distinct, as revealed by electron microscopy. This binding was specific, since almost no Ri-Au surface binding occurred at 4 degrees C in the presence of 0.1 M lactose or 1 mg/ml native (unlabelled) ricin. Within the first 30 min of warming prelabelled cells to 37 degrees C, the amount of surface-associated Ri-Au decreased considerably (from 150 to 60 gold particles per micron cell surface in 40 nm sections). Coated pits and vesicles were involved in the internalization of Ri-Au, and within 5-30 min at 37 degrees C Ri-Au had been delivered to vacuolar and tubulo-vesicular portions of the endosomal system, and later also to lysosomes. Analysis of very thin (ca 20 nm) serial sections revealed that most of the tubulo-vesicular elements were separate structures not connected to the membrane of the vacuolar portion. Data here presented indicate that our ricin conjugate, like many "physiological' ligands and viruses, is internalized by receptor-mediated endocytosis via the coated pit-endosomal pathway.     

Effects of cooling and warming conditions on post-thawed motility and fertility of cryopreserved buffalo spermatozoa.

The principal objective of this study was to derive an improved procedure for cryopreservation of swamp buffalo (Bubalus bubalis) spermatozoa. Experiments were conducted to determine effects of cooling rate, intermediate plunge temperature and warming rate on motility and acrosome integrity of spermatozoa. Spermatozoa were obtained from three bulls (three ejaculates/bull) and were subjected to nine cooling conditions before being frozen in liquid nitrogen: cooling at 10, 20, or 30 degrees C/min each to -40, -80, or -120 degrees C before being plunged into liquid nitrogen. The spermatozoa frozen under a given condition were then thawed either at 1000 or 200 degrees C/min. Cooling rate, intermediate temperature and warming rate significantly affected survival of spermatozoa obtained from the three bulls. Cooling spermatozoa from 4 to -120 degrees C either at 20 or 30 degrees C/min yielded better progressive motility compared to other cooling conditions (50 versus 30%). Rapid warming was superior to slow warming. In an additional study, motility and fertility of spermatozoa frozen after being cooled to -120 degrees C at 20 degrees C and 30 degrees C/min and those frozen by a standard protocol used routinely for semen processing were assessed. Progressive motility of cryopreserved spermatozoa cooled at 20 degrees C and 30 degrees C/min was 40%, while that of spermatozoa cryopreserved using a standard protocol was 25%. A total of 178 buffalo cows were inseminated with cryopreserved spermatozoa obtained from one bull, and their pregnancy status was assessed 60 days later by rectal palpation. Out of the 60, 26 (43%) and 23 of 58 (40%) cows inseminated with sperm cooled at 20 and 30 degrees C/min, respectively, became pregnant, whereas 17 of 60 (28%) cows inseminated with sperm frozen by a standard protocol became pregnant. This study demonstrates that an effective cryopreservation procedure for buffalo spermatozoa can be derived by systematic examination of various cryobiological factors.     

Factors affecting the cryosurvival of mouse two-cell embryos

A series of 4 experiments was conducted to examine factors affecting the survival of frozen-thawed 2-cell mouse embryos. Rapid addition of 1.5 M-DMSO (20 min equilibration at 25 degrees C) and immediate, rapid removal using 0.5 M-sucrose did not alter the frequency (mean +/- s.e.m.) of blastocyst development in vitro when compared to untreated controls (90.5 +/- 2.7% vs 95.3 +/- 2.8%). There was an interaction between the temperature at which slow cooling was terminated and thawing rate. Termination of slow cooling (-0.3 degrees C/min) at -40 degrees C with subsequent rapid thawing (approximately 1500 degrees C/min) resulted in a lower frequency of blastocyst development than did termination of slow cooling at -80 degrees C with subsequent slow thawing (+8 degrees C/min) (36.8 +/- 5.6% vs 63.9 +/- 5.7%). When slow cooling was terminated between -40 and -60 degrees C, higher survival rates were achieved with rapid thawing. When slow cooling was terminated below -60 degrees C, higher survival rates were obtained with slow thawing rates. In these comparisons absolute survival rates were highest among embryos cooled below -60 degrees C and thawed slowly. However, when slow cooling was terminated at -32 degrees C, with subsequent rapid warming, survival rates were not different from those obtained when embryos were cooled to -80 degrees C and thawed slowly (52.4 +/- 9.5%, 59.5 +/- 8.6%). These results suggest that optimal cryosurvival rates may be obtained from 2-cell mouse embryos by a rapid or slow thawing procedure, as has been found for mouse preimplantation embryos at later stages.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

温和气单孢菌YH311硫酸软骨素裂解酶的分离纯化与固定化

通过硫酸铵沉淀、QAESephadex-A50柱层析及Sephadex-G150凝胶过滤等纯化步骤,对源自温和气单孢菌YH311的ChSase进行了分离纯化。结果表明,ChSase经上述纯化步骤后被纯化了55倍,其最终纯度可达95%以上,比活为31.86u/mg。经SDSPAGE及IFE测定可知该酶的分子量约为80kD,等电点为4.3～4.8。将纯化后的ChSase用海藻酸钠或纤维素固定化后,ChSase的热稳定性及贮存稳定性均可得到大幅度的提高：固定化酶用80℃水浴处理120min或于4℃冰箱放置30d后仍可保留50%以上的相对活力;但固定化酶的收率较低,仅为18.56%和18.86%。     

Three dimensional visualisation of human facial exposure to solar ultraviolet.

A three dimensional computer model of the human face has been developed to represent solar ultraviolet exposures recorded by dosimeter measurements on a manikin headform under low cloud conditions and various solar zenith angles. Additionally, polysulfone dosimeters have been successfully miniaturised to provide the detailed measurements required across the face. The headform used in this research was scanned at 709 individual locations to make a wireframe mesh consisting of 18 vertical contours and 49 horizontal contours covering half the manikin's frontal facial topography. Additionally, the back of the headform and neck have also been scanned at 576 locations. Each scanned location has been used as a viable dosimeter position on the headform and represents a grid intersection point on the developed computer wireframe. A series of exposures recorded by dosimeters have been translated into three dimensional exposure ratio maps, representing ambient solar ultraviolet exposure. High dosimeter density has allowed for the development of individual topographic contour models which take into account complex variation in the face and improve upon previously employed techniques which utilise fewer dosimeters to interpolate exposure across facial contours. Exposure ratios for solar zenith angle ranges of 0 degrees -30 degrees, 30 degrees -50 degrees, and 50 degrees -80 degrees have been developed.     

Theoretical analysis of the ice crystal size distribution in frozen aqueous specimens.

To estimate theoretically how suited different freezing techniques are for freezing of freeze-etch specimens, it is necessary to know the relationship between specimen cooling rate and the resulting average ice crystal size. Using a somewhat simplified theoretical analysis, we have derived the approximate ice crystal size distribution of nonvitrified frozen aqueous specimens frozen at different cooling rates. The derived size distribution was used to calculate the relationship between relative change in average ice crystal size, (delta l/l), and relative change in specimen cooling rate delta (dT/dt)/(dT/dt). We found this relationship to be (delta l/l) = -k X delta (dT/dt)/(dT/dt) where k = 1.0 when specimen solidification takes place at about -6 degrees C, and k congruent to 1.3 when it takes place at about -40 degrees C.     

The mechanisms of reversible immobilization of fowl spermatozoa at body temperature

Intact fowl spermatozoa became almost immotile at 40 degrees C, but motility increased significantly at 30 degrees C. The oxygen consumption at both temperatures was 8-11 microliters O2/10(10) spermatozoa.min-1. The ATP concentration at 40 degrees C was higher than that at 30 degrees C but ADP concentration at 30 degrees C was higher than that at 40 degrees C. Consequently, the ATP/ADP ratio at 30 degrees C (1.9-2.2) increased to 3.5-3.7 at 40 degrees C. The motility of intact spermatozoa at 40 degrees C was effectively restored by 2 mM-Ca2+, 10% seminal plasma and 10% peritoneal fluid taken at the time of ovulation. In contrast, these effectors did not restore the motility of demembranated spermatozoa at 40 degrees C. Motility of demembranated spermatozoa was restored at 30 degrees C. These results suggest that the immobilization of fowl spermatozoa at 40 degrees C occurs due to a decrease in flagellar dynein ATPase activity. Furthermore, the action of effectors for motility such as Ca2+ may not be directly on the axoneme, but mediated by solubilized substances which have been removed by demembranation of the spermatozoa.     

Sweat ammonia excretion during submaximal cycling exercise

This study was undertaken to investigate whether part of the ammonia formed during muscular exercise was excreted with the sweat. Male medical students volunteered for the experiment. They exercised 30 min on a bicycle ergometer at 80 and 40% of the predetermined maximal O2 uptake (VO2max). Exercise at 80% VO2max was performed twice, at room temperature (20 degrees C) and in a cold room (0 degrees C), whereas exercise at 40% was performed only at room temperature (20 degrees C). Blood was collected from the antecubital vein immediately before and after exercise. Sweat was collected from the hypogastric region by use of gauze pads. It was shown that the plasma ammonia level was elevated after exercise at 80% VO2max and remained stable after exercise at 40% VO2max. The volume of sweat produced during exercise at 80% VO2max at 20 degrees C was 428 +/- 138 ml and at 0 degrees C 245 +/- 86 ml and during exercise at 40% VO2max was 183 +/- 69 ml. The ammonia concentration in the sweat after exercise at 80% VO2max at 20 degrees C was 7,140 mumol/l and at 0 degrees C 11,816 mumol/l. After exercise at 40% VO2max, it was 2,076 mumol/l. The total ammonia lost through the sweat during exercise at 80% VO2max was similar at both temperatures, despite the difference in the sweat volume (at 20 degrees C, 3,360 +/- 2,080 mumol; at 0 degrees C, 3,310 +/- 1,250 mumol). During exercise at 40% VO2max, it was 350 +/- 230 mumol. These results show that part of ammonia formed during exercise is lost with sweat. The amount lost increases with increased work rate and the plasma ammonia concentration.     

The Characteristics and Genetic Map Location of a Temperature Sensitive DNA Mutant of E. COLI K12

A temperature sensitive strain of E. coli K12 has been isolated in which residual DNA synthesis occurs at the 40 degrees C restrictive temperature; syntheses of RNA, protein and DNA precursors are not directly affected. The mutation has been designated dna-325 and is located at 89 min on the E. coli map in the same region where the dnaC locus is found. dnaC mutants are considered to be defective in DNA initiation. Some of the data are consistent with the view that the dna-325 mutation is temperature sensitive in the process of DNA initiation rather than DNA chain elongation: (1) more than two cell divisions occur after a shift to 40 degrees C; (2) upon a shift down to 30 degrees C, cell division occurs again only after the DNA content of the cells has doubled; (3) 80% more DNA is made at 30 degrees C in the presence of chloramphenicol after prior inhibition of DNA synthesis at 40 degrees C. These three observations indicate that rounds of DNA replication were completed at 40 degrees C. Also (4) infective lambda particles can be made at 40 degrees C long after bacterial DNA replication has ceased. It appears however that some DNA initiation can occur at 40 degrees C since (1) a limited amount of DNA synthesis does occur at 40 degrees C after prior alignment of the chromosomes by amino acid starvation at 30 degrees C, and (2) after incubation in bromouracil at the restrictive temperature, heavy DNA is found with both strands containing bromouracil.     

Characterization of goldfish heat shock protein-30 induced upon severe heat shock in cultured cells

Temperature-dependent changes of growth rate and protein components were investigated for primary cultured cells derived from goldfish caudal fin. When the culture temperature was shifted from 20 degrees C to 35 degrees C and 40 degrees C, the growth rate was increased at 35 degrees C as compared with that at 20 degrees C, but no cell growth was observed at 40 degrees C. The differential scanning calorimetry demonstrated the onset of the endothermic reaction for goldfish cellular components at 40 degrees C. Therefore, the temperature shift to 40 degrees C was found to be of severe heat shock for goldfish cultured cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that, although expression of 70-kDa components was slightly induced at 35 degrees C, the temperature shift to 40 degrees C markedly induced the expression of the 30-kDa component in addition to that of 70-kDa component. The N-terminal amino acid sequencing identified the 30- and 70-kDa components to be heat shock protein (Hsp)-30 and Hsp70, respectively. Northern blot analysis revealed that the enhanced Hsp30 messenger ribonucleic acid (mRNA) levels were only observed at 40 degrees C, whereas Hsp70 mRNA was slightly accumulated at 35 degrees C. These results indicated that Hsp30 might have important functions under severe heat stress condition.     

The influence of temperature on the biology of triatominae. IX. Rhodnius nasutus Stal, 1859 (Hemiptera, Reduviidae)

The influence of temperature on the biology of Rhodnius nasutus Stal, 1859 (Hemiptera, Reduviidae) was studied in order to obtain a larger number of triatominae reared in laboratory to use in xenodiagnosis and to be able to get information for their proper control. The experiments were performed in two climatized chambers at 25 +/- 0.5 degrees C and 30 +/- 1 degrees C, 70 +/- 5% of relative humidity and photoperiod of 12 hours. Data in relation to evolution cycle, period between emergency of adults and first oviposition, fecundity, fertility, amount of blood ingested and fast resistance, are presented. The mean duration of the evolutive cycle for males and females was 145.7 days at 25 degrees C and 114.2 days at 30 degrees C. The mean of blood ingested to complete the evolutive cycle was 304.3 mg and 350.3 mg, at 25 degrees C and 30 degrees C, respectively. The triatominae survival on conditions of absolute fast was significantly higher at 25 degrees C than at 30 degrees C (P less than 0.01).