Circulation dynamics of Salmonella enterica subsp. enterica ser. Gallinarum biovar Gallinarum in a poultry farm infested by Dermanyssus gallinae.

Dermanyssus gallinae (Mesostigmata: Dermanyssidae, De Geer, 1778) is an ectoparasite of poultry, suspected to play a role as a vector of Salmonella enterica subsp. enterica ser. Gallinarum. Despite an association between them being reported, the actual dynamics in field remain unclear. Therefore, the present study aimed to confirm the interactions among mites, pathogen and chickens. The study was carried out in an industrial poultry farm infested by D. gallinae, during an outbreak of fowl typhoid. The presence of S. Gallinarum in mites was assessed and quantified by a semi-nested polymerase chain reaction (PCR) and real-time PCR, respectively, in mites collected during two subsequent productive cycles and the sanitary break. The anti-group D Salmonella antibodies were quantified by an enzyme-linked immunosorbent assay. During the outbreak and the sanitary break, S. Gallinarum was constantly present in mites. In the second cycle, scattered positivity was observed, although hens did not exhibit signs of fowl typhoid, as a result of the vaccination with BIO-VAC SGP695 (Fatro, Ozzano Emilia Bo, Italy). The data strongly suggest that D. gallinae acts as reservoir of S. Gallinarum, thus allowing the pathogen to persist in farms. Furthermore, the present study has highlighted the interactions among D. gallinae, S. Gallinarum and hens with respect to enhancing the mite-mediated circulation of S. Gallinarum in an infested poultry farm.

Large diameter fully covered self-expanding metal stent placement for palliation of proximal malignant esophageal strictures.

In patients with malignant esophageal strictures within 6 cm from the upper esophageal sphincter, self-expanding metal stents placement represents a challenge because there is an increased risk of complications. The aim of this study was to assess the safety and effectiveness of large-diameter WallFlex(®) fully covered self-expanding metal stents for palliation of patients with proximal malignant esophageal strictures. From March 2010 to December 2012, 12 patients with proximal strictures (4-6 cm from the upper esophageal sphincter) and six with very proximal strictures (<4 cm from the upper esophageal sphincter) were palliated with this fully covered self-expanding metal stent and included in the study. Technical success was 100% and clinical success was 94%. The mean baseline dysphagia score was 3.2, and 1 week after stenting it improved significantly to 1.3 (P < 0.001). Early complications occurred in four patients, more frequently in patients with very proximal strictures as compared with patients with proximal strictures (P = 0.02). Late complications occurred in five patients, and there were no differences between patients with very proximal strictures or proximal strictures (P = 0.245). The mean survival after stent placement was 119 days, and no differences between patients with very proximal strictures versus proximal strictures were found (P = 0.851). There was no stent-related mortality or 30-day mortality. Our results suggested that a large-diameter fully covered self-expanding metal stent is an effective and secure device for palliation of patients with proximal malignant esophageal strictures.

Liver steatosis in celiac disease: the open door.

Non-alcoholic fatty liver disease (NAFLD) is a common disease of unknown origin characterized by histological features similar to alcoholic-like liver injury but in the absence of significant alcohol intake. Non-alcoholic fatty liver disease refers to a spectrum of diseases of the liver ranging from simple steatosis (i.e., fatty infiltration of the liver) to nonalcoholic steatohepatitis (i.e., steatosis with inflammation and hepatocyte necrosis) to cirrhosis. Non-alcoholic fatty liver disease is frequently associated with disorders such as insulin resistance, obesity, type 2 diabetes mellitus, hyperlipidemia and protein-calorie malnutrition. However, in a subgroup of NAFLD patients, the true relevant cause remains undetermined. Celiac disease (CD) is a common immune-mediated disorder and develops in genetically susceptible subjects after the ingestion of gluten proteins. Celiac disease has been found in about 10% of patients with unexplained abnormal liver tests, and in about 3.5% of patients with NAFLD as the only manifestation of the disease. The frequency of subclinical or silent presentations in older children and adults highlights the importance of CD screening in patients with unexplained chronic abnormal liver function tests and NAFLD without any specific etiology. The pathogenesis of liver steatosis in CD is uncertain. The aims of this review are to describe the possible mechanisms involved in the occurrence and progression of liver steatosis in CD patients.

Zn[Formula: see text]Ni[Formula: see text]Te semiconductor nanocrystals in transparent glass for optoelectronic device applications.

Doping glass with semiconductors, particularly with nanostructured semiconductors, has attracted attention due to the large optical absorption cross-sections of the latter. Based on this property, Ni[Formula: see text] (5 wt%) doped phosphate glass and Zn[Formula: see text]Ni[Formula: see text]Te (x = 0.5, 1.0, 5.0 and 10.0 wt% of Ni[Formula: see text]) nanocrystals (NCs) doped phosphate glasses (GCs) were prepared by fusion method and subsequent heat treatment. Influence of Ni[Formula: see text] on structural, thermo-optical and third-order nonlinear optical properties have been analysed through various spectroscopic characterizations. The XRD pattern of the glass (G) exhibits the amorphous nature of the host material while GCs exhibit not only amorphous halo but also the presence of quantum dots (QDs) or nanocrystals (NCs) phases. TEM analysis of the studied GCs samples confirm the presence of quantum dots (QDs) and bulk NCs with an average diameter of approximately 4.2 [Formula: see text] 0.3 nm and 13.4 [Formula: see text] 0.2 nm, respectively. Several phosphate groups were observed and reported from Raman and FTIR-ATR spectra. The absorption band positions confirmed that Ni[Formula: see text] ions resemble to the octahedral symmetry. The intensity of absorption band around 1352 nm ([Formula: see text]T[Formula: see text](F) [Formula: see text] [Formula: see text]A[Formula: see text](F)) increased with the increase of Ni[Formula: see text] in GCs which is an indicative of the [Formula: see text]Ni[Formula: see text] coordination. The emission properties such as emission cross-sections ([Formula: see text]) full width at half maxima (FWHM) for the [Formula: see text]T[Formula: see text](D) [Formula: see text] [Formula: see text]T[Formula: see text](F) (visible) and [Formula: see text]A[Formula: see text](F) [Formula: see text] [Formula: see text]T[Formula: see text](F) (near-infrared) emission transitions were reported. Among the glass-containing semiconductor nanocrystals (GCs), the emission cross-sections in GC4 sample (x = 10% of Ni[Formula: see text]) are the largest for both the visible (11.88 [Formula: see text] 10[Formula: see text] cm[Formula: see text]) and infrared (0.98 [Formula: see text] 10[Formula: see text] cm[Formula: see text]) transitions. Thermal diffusivity (D), thermal conductivity (K) and temperature dependent optical path length change (ds/dT) were obtained through time-resolved thermal lens (TL) and thermal relaxation (TR) methods. The D and K parameters do not change significantly with increase of Ni[Formula: see text] ions (0.5-5%) in GCs. Nonlinear-refractive index and nonlinear absorption of the studied samples were also obtained using femtosecond Z-scan technique. The increase of nonlinear absorption coefficient ([Formula: see text]) is observed from GC2 (2.53 [Formula: see text] 10[Formula: see text] cm/W) to GC4 (7.98 [Formula: see text] 10[Formula: see text] cm/W). The GC4, sample with 10 wt% of Ni[Formula: see text], showed the lowest ds/dT (1.22 [Formula: see text] 10[Formula: see text] K[Formula: see text]) with good lasing (FOM and emission cross-sections) and nonlinear absorption properties suggesting that it can be a good candidate for visible-red emission light conversion in LED technology.

Cold sensitivity in rice (Oryza sativa L.) is strongly correlated with a naturally occurring I99V mutation in the multifunctional glutathione transferase isoenzyme GSTZ2.

GSTZs [Zeta class GSTs (glutathione transferases)] are multifunctional enzymes that belong to a highly conserved subfamily of soluble GSTs found in species ranging from fungi and plants to animals. GSTZs are known to function as MAAIs [MAA (maleylacetoacetate) isomerases], which play a role in tyrosine catabolism by catalysing the isomerization of MAA to FAA (fumarylacetoacetate). As tyrosine metabolism in plants differs from animals, the significance of GSTZ/MAAI is unclear. In rice (Oryza sativa L.), a major QTL (quantitative trait locus) for seedling cold tolerance has been fine mapped to a region containing the genes OsGSTZ1 and OsGSTZ2. Sequencing of tolerant (ssp. japonica cv. M-202) and sensitive (ssp. indica cv. IR50) cultivars revealed two SNPs (single nucleotide polymorphisms) in OsGSTZ2 that result in amino acid differences (I99V and N184I). Recombinant OsGSTZ2 containing the Val99 residue found in IR50 had significantly reduced activity on MAA and DCA (dichloroacetic acid), but the Ile184 residue had no effect. The distribution of the SNP (c.295A>G) among various rice accessions indicates a significant association with chilling sensitivity in rice seedlings. The results of the present study show that naturally occurring OsGSTZ2 isoforms differ in their enzymatic properties, which may contribute to the differential response to chilling stress generally exhibited by the two major rice subspecies.

The septins: roles in cytokinesis and other processes.

The septins are a novel family of proteins that were first recognized in yeast as proteins associated with the neck filaments. Recent work has shown that septins are also present in other fungi, insects, and vertebrates. Despite the apparent differences in modes of cytokinesis amongst species, septins appear to be essential for this process in both fungal and animal cells. The septins also appear to be involved in various other aspects of the organization of the cell surface.

Effects of smoking and different BMI cutoff points on surgical site infection after elective open ventral hernia repair.

PURPOSE: BMI > 40 kg/m2 and smoking have been identified as risk factors for surgical site infections (SSIs) after ventral hernia repair, however, the relationship with lower BMI values has not been described. The objective of this study was to analyze the relationship between different BMI thresholds, smoking, and SSI after open ventral hernia repair. METHODS: All patients who underwent an elective non-emergent open ventral hernia repair with mesh were extracted from the 2011 to 2016 NSQIP database. Bivariate, multivariate logistic regression, and Classification and Regression Tree (CART) analyses were used. RESULTS: Of 55,240 patients, 2,620 (4.7%) developed SSIs (superficial: 58.5%, deep:27%, organ-space: 16%). BMI (OR: 1.035; 95% CI:1.03-1.04; p < 0.001) and smoking (OR:1.51; 95% CI:1.37-1.67; p < 0.001) were identified in logistic regression analysis as the two most modifiable risk factors independently associated with SSIs. CART analysis demonstrated that the lowest SSI rate belonged to non-smokers with BMI < 24.2 kg/m2 (1.9%), and the highest SSI to smokers with BMI > 42.3 kg/m2 (12%). Between these values, there was a stepwise increase in SSI rate as BMI increased, while smoking added additional risk in each group. CONCLUSION: Following open hernia repair, the association between SSI and being overweight starts at a BMI of 24.2, a threshold lower than previously described. The risk of SSI increases in a stepwise fashion as BMI increases and is augmented by smoking. Future studies are needed to determine if SSI reduction can be achieved with a combination of smoking cessation and weight loss using these BMI thresholds.

Outcomes of concomitant mesh placement and intestinal procedures during open ventral hernia repair.

PURPOSE: The concept of mesh use during open ventral hernia repair with a concomitant intestinal procedure remains controversial and it remains unclear whether the SSI profile of contaminated fields in this setting more closely resembles clean-contaminated or dirty wounds. METHODS: Patients who underwent an open ventral hernia repair and intestinal procedures were extracted from the ACS-NSQIP database. Data analysis was performed for mesh versus no mesh groups in aggregate and matched cohorts. The 30-day outcomes including mortality, morbidity, surgical site infections (SSI), readmission, reoperation, and length of stay were compared between the groups. RESULTS: Of 5104 patients in the mesh group, 3297 patients were matched 1:1 with patients without mesh. Mesh placement was associated with higher overall morbidity (35.1% vs. 29.8%; p < 0.001), overall SSI (27.1% vs. 18%; p < 0.001), deep SSI (5.9% vs. 4.3%; p = 0.003), organ-space SSI (6.8% vs. 5.5%; p = 0.027), reoperation (9.9% vs. 8.2%; p = 0.016), readmission (16.7% vs. 12.7%; p < 0.001), and longer hospital stays (10.9 ± 15.2 vs. 9.7 ± 10.7; p < 0.001). When mesh was used, the SSI profile of contaminated fields was similar to dirty wounds (26.4% vs. 27.5%; p = 0.702), rather than clean-contaminated fields (26.4% vs. 19.2%; p < 0.001). CONCLUSION: Concomitant bowel procedure and mesh placement at the time of open ventral hernia repair was associated with worse outcomes. The SSI profile of the contaminated fields was more similar to dirty wounds. When deciding whether to place mesh during ventral hernia repairs with concomitant bowel procedures, strong consideration should be given to the increased risk of short-term complications versus the potential long-term benefits.

A septin-based hierarchy of proteins required for localized deposition of chitin in the Saccharomyces cerevisiae cell wall.

Just before bud emergence, a Saccharomyces cerevisiae cell forms a ring of chitin in its cell wall; this ring remains at the base of the bud as the bud grows and ultimately forms part of the bud scar marking the division site on the mother cell. The chitin ring seems to be formed largely or entirely by chitin synthase III, one of the three known chitin synthases in S. cerevisiae. The chitin ring does not form normally in temperature-sensitive mutants defective in any of four septins, a family of proteins that are constituents of the "neck filaments" that lie immediately subjacent to the plasma membrane in the mother-bud neck. In addition, a synthetic-lethal interaction was found between cdc12-5, a temperature-sensitive septin mutation, and a mutant allele of CHS4, which encodes an activator of chitin synthase III. Two-hybrid analysis revealed no direct interaction between the septins and Chs4p but identified a novel gene, BNI4, whose product interacts both with Chs4p and Cdc10p and with one of the septins, Cdc10p; this analysis also revealed an interaction between Chs4p and Chs3p, the catalytic subunit of chitin synthase III. Bni4p has no known homologues; it contains a predicted coiled-coil domain, but no other recognizable motifs. Deletion of BNI4 is not lethal, but causes delocalization of chitin deposition and aberrant cellular morphology. Overexpression of Bni4p also causes delocalization of chitin deposition and produces a cellular morphology similar to that of septin mutants. Immunolocalization experiments show that Bni4p localizes to a ring at the mother-bud neck that lies predominantly on the mother-cell side (corresponding to the predominant site of chitin deposition). This localization depends on the septins but not on Chs4p or Chs3p. A GFP-Chs4p fusion protein also localizes to a ring at the mother-bud neck on the mother-cell side. This localization is dependent on the septins, Bni4p, and Chs3p. Chs3p, whose normal localization is similar to that of Chs4p, does not localize properly in bni4, chs4, or septin mutant strains or in strains that accumulate excess Bni4p. In contrast, localization of the septins is essentially normal in bni4, chs4, and chs3 mutant strains and in strains that accumulate excess Bni4p. Taken together, these results suggest that the normal localization of chitin synthase III activity is achieved by assembly of a complex in which Chs3p is linked to the septins via Chs4p and Bni4p.

Antioxidant, Cytotoxicity, and Antiophidian Potential of Alstonia macrophylla Bark.

The objective of this research was to find the possible pharmacognosy of the bark of the Philippine Alstonia macrophylla Wall. ex G.Don (AM). Gas chromatographic-mass spectral (GC-EI-MS) characterization and energy dispersive X-ray spectroscopy (EDX) were performed to detect the bioactive constituents. EDX analysis of AM bark displayed a high content of potassium (3.26%) and calcium (2.96%). Eight constituents were detected in AM crude dichloromethane (DCM) extracts, which consisted of a long-chain unsaturated fatty acid (17:0) and fatty acid esters such as ethyl hexadecanoate and methyl hexadecanoate. Extraction of AM bark using methanol and dimethyl sulfoxide (MeOH/DMSO) solvents resulted in the identification of 17 constituents, principally alkaloids (alstonerine, 34.38%; strictamin, 5.23%; rauvomitin, 4.29%; and brucine, 3.66%) and triterpenoids (γ-sitosterol, 3.85%; lupeol, 3.00%; 24-methylenecycloartanol, 2.81%; campesterol, 2.71%; ß-amyrin, 2.30%; and stigmasterol, 2.13%). MeOH/DMSO samples of AM were used in the selected bioassays. The samples exhibited efficient free radical scavenging activity (IC50 = 0.71 mg/mL) and were noncytotoxic to normal HDFn (IC50 > 100 µg/mL) and neoplastic THP-1 cell lines (IC50 = 67.22 µg/mL) while highly degenerative to MCF-7 (IC50 = 6.34 µg/mL), H69PR (IC50 = 7.05 µg/mL), and HT-29 (IC50 = 9.10 µg/mL). Most interestingly, the AM samples inhibited the northern Philippine Cobra's (Naja philippinensis Taylor) venom (IC50 = 297.27 ± 9.33 µg/mL) through a secretory phospholipase A2 assay.

Genetic variation and possible origins of weedy rice found in California.

Control of weeds in cultivated crops is a pivotal component in successful crop production allowing higher yield and higher quality. In rice-growing regions worldwide, weedy rice (Oryza sativa f. spontanea Rosh.) is a weed related to cultivated rice which infests rice fields. With populations across the globe evolving a suite of phenotypic traits characteristic of weeds and of cultivated rice, varying hypotheses exist on the origin of weedy rice. Here, we investigated the genetic diversity and possible origin of weedy rice in California using 98 simple sequence repeat (SSR) markers and an Rc gene-specific marker. By employing phylogenetic clustering analysis, we show that four to five genetically distinct biotypes of weedy rice exist in California. Analysis of population structure and genetic distance among individuals reveals diverse evolutionary origins of California weedy rice biotypes, with ancestry derived from indica, aus, and japonica cultivated rice as well as possible contributions from weedy rice from the southern United States and wild rice. Because this diverse parentage primarily consists of weedy, wild, and cultivated rice not found in California, most existing weedy rice biotypes likely originated outside California.

Infective complications in renal allograft recipients: epidemiology and outcome.

INTRODUCTION: Successful renal transplantation strictly depends on good control of rejection and better prevention and treatment of infections, which remain serious threats. METHODS: This retrospective, observational study of 245 renal allograft recipients who underwent transplantation between January 2002 and December 2005 included a 21+/-10 months follow-up. RESULTS: A total of 110 (44.9%) patients developed an infective process during the posttransplantation period, namely, 232 infective processes. Eighty patients developed at least 1 episode of urinary tract infection (UTI) 11 patients (4%) had a wound infection, and 30 patients (12%) had pneumonia. We diagnosed 35 cases of bacteremia (35%), whereas cytomegalovirus (CMV) infection was demonstrated in 40 patients (16%). CONCLUSIONS: Immunosuppressive therapy, necessary to avoid acute and chronic rejection, exposes patients to a higher rate of infectious complications. The immunosuppressive protocols led to a relatively low incidence of infectious complications, mainly of little clinical significance. The highest incidence was evident by the sixth month after transplantation, when the immunosuppressive regimen exercised its most depressive effects on patient immune systems.

New-onset diabetes mellitus after kidney transplantation: the role of immunosuppression.

BACKGROUND: Complications related to posttransplantation immunosuppressive therapy remain common. New-onset diabetes mellitus after transplantation (PTDM) is a well-recognized complication associated with reduced graft and patient survival. The type of immunosuppression may be responsible for more than two thirds of PTDM. We retrospectively reviewed our experience in a population of 284 kidney transplant recipients, evaluating the incidence of PTDM with regard to the type of immunosuppression. PATIENTS AND METHODS: From January 2001 to December 2005, 284 kidney transplantations were performed using tacrolimus-based (TAC) immunosuppression in 192 patients and a cyclosporine-based (CyA) regimen in 62 patients, whereas 30 patients received sirolimus-based immunosuppression. RESULTS: The overall incidence of PTDM was 4.9%. Among the immunosuppression protocols, 8 patients (4.1%) received TAC and 6 patients (9.6%) received CyA, whereas no patients treated with sirolimus developed PTDM. Graft and patient survival rates were 93% and 100%, respectively. CONCLUSIONS: The overall risk of PTDM with recent immunosuppressive protocols is low, but it is increased among calcineurin inhibitor (CNI)-treated kidney transplant recipients. Sirolimus did not increase the risk of PTDM, allowing potential clinical application in diabetic recipients and in patients affected by PTDM.

Cell growth control: little eukaryotes make big contributions.

The story of rapamycin is a pharmaceutical fairytale. Discovered as an antifungal activity in a soil sample collected on Easter Island, this macrocyclic lactone and its derivatives are now billion dollar drugs, used in, and being evaluated for, a number of clinical applications. Taking advantage of its antifungal property, the molecular Target Of Rapamycin, TOR, was first described in the budding yeast Saccharomyces cerevisiae. TORs encode large, Ser/Thr protein kinases that reside in two distinct, structurally and functionally conserved, multi-protein complexes. In yeast, these complexes coordinate many different aspects of cell growth. TOR complex 1, TORC1, promotes protein synthesis and other anabolic processes, while inhibiting macroautophagy and other catabolic and stress-response processes. TORC2 primarily regulates cell polarity, although additional readouts of this complex are beginning to be characterized. TORC1 appears to be activated by nutrient cues and inhibited by stresses and rapamycin; however, detailed mechanisms are not known. In contrast, TORC2 is insensitive to rapamycin and physiological regulators of this complex have yet to be defined. Given the unsurpassed resources available to yeast researchers, this simple eukaryote continues to contribute to our understanding of eukaryotic cell growth in general and TOR function in particular.

Bud8p and Bud9p, proteins that may mark the sites for bipolar budding in yeast.

The bipolar budding pattern of a/alpha Saccharomyces cerevisiae cells appears to depend on persistent spatial markers in the cell cortex at the two poles of the cell. Previous analysis of mutants with specific defects in bipolar budding identified BUD8 and BUD9 as potentially encoding components of the markers at the poles distal and proximal to the birth scar, respectively. Further genetic analysis reported here supports this hypothesis. Mutants deleted for BUD8 or BUD9 grow normally but bud exclusively from the proximal and distal poles, respectively, and the double-mutant phenotype suggests that the bipolar budding pathway has been totally disabled. Moreover, overexpression of these genes can cause either an increased bias for budding at the distal (BUD8) or proximal (BUD9) pole or a randomization of bud position, depending on the level of expression. The structures and localizations of Bud8p and Bud9p are also consistent with their postulated roles as cortical markers. Both proteins appear to be integral membrane proteins of the plasma membrane, and they have very similar overall structures, with long N-terminal domains that are both N- and O-glycosylated followed by a pair of putative transmembrane domains surrounding a short hydrophilic domain that is presumably cytoplasmic. The putative transmembrane and cytoplasmic domains of the two proteins are very similar in sequence. When Bud8p and Bud9p were localized by immunofluorescence and tagging with GFP, each protein was found predominantly in the expected location, with Bud8p at presumptive bud sites, bud tips, and the distal poles of daughter cells and Bud9p at the necks of large-budded cells and the proximal poles of daughter cells. Bud8p localized approximately normally in several mutants in which daughter cells are competent to form their first buds at the distal pole, but it was not detected in a bni1 mutant, in which such distal-pole budding is lost. Surprisingly, Bud8p localization to the presumptive bud site and bud tip also depends on actin but is independent of the septins.

IntegraEPI: a Grid-based epidemic surveillance system.

The proposal of new analytical techniques has guided innovative methodological developments in public health interventions. The goal of this work is show advances in the development of a large scale system for space-time visualization, monitoring, modeling and analysis of epidemic data using a Grid platform. The resulting virtual laboratory, dubbed IntegraEPI, is expected to provide better epidemic forecasting and to define better strategies to fight the spread of a disease, in which new population-level interventions could be evaluated and iteratively refined using computational simulations, with tangible benefits for real-world epidemic prevention and control efforts.

Heat shock induces enzymes of trehalose metabolism, trehalose accumulation, and thermotolerance in Schizosaccharomyces pombe, even in the presence of cycloheximide.

Exponentially growing cells of the fission yeast, Schizosaccharomyces pombe, contained virtually no trehalose at 27 degrees C but rapidly accumulated large quantities during heat shock at 40 degrees C. Activities of trehalose-6-phosphate synthase and trehalase also increased upon heat shock. Thermotolerance of the cells, measured as survival at 52 degrees C, increased in parallel to trehalose accumulation and decreased in parallel to the trehalose levels when cells were shifted back to 27 degrees C. Trehalose levels, activities of enzymes of trehalose metabolism and thermotolerance strongly increased upon heat shock even in the presence of cycloheximide, indicating that none of these effects requires protein synthesis. The data support the hypothesis that trehalose acts as a thermoprotectant in Schizosaccharomyces pombe.

A method to study the rapid phosphorylation-related modulation of neutral trehalase activity by temperature shifts in yeast.

Heat shock enhanced the synthesis of neutral trehalase in growing cells of Saccharomyces cerevisiae, as detected by immunological methods. The activity of the enzyme was measured in extracts obtained by two methods: cells were either harvested by filtration and subsequent disruption with glass beads at 0-4 degrees C or immediately frozen with liquid nitrogen in the presence of Triton X-100, followed by thawing at 30 degrees C. The first procedure yielded artificially high activities of neutral trehalase in heat-shocked cells due to rapid (less than 1 min) activation during handling at 4 degrees C before homogenization. Activity of the enzyme in these homogenates decreased 75-90% upon a treatment with alkaline phosphatase, indicating that activation was due to phosphorylation. The second procedure yielded low trehalase activities for heat-shock treated cells, much higher activities for cells shifted back for some seconds to 27 degrees C, and very low activities again for cells shifted from 27 to 40 degrees C for a second time. Thus, permeabilization of cells following rapid freezing in Triton X-100 is a method of choice to study post-translational modulation of the neutral trehalase of S. cerevisiae by phosphorylation and dephosphorylation.

Acquisition of thermotolerance in Saccharomyces cerevisiae without heat shock protein hsp 104 and in the absence of protein synthesis.

Acquisition of thermotolerance in response to a preconditioning heat treatment at 40 degrees C was studied in mutants of the yeast Saccharomyces cerevisiae lacking a specific heat shock protein or the ability to synthesize proteins at 40 degrees C. A mutant carrying a deletion of heat shock protein hsp 104 and the corresponding wildtype strain were both highly sensitive to heat stress at 50.4 degrees C without preconditioning but both acquired almost the same level of thermotolerance after 60 min of preconditioning. Both strains showed equal induction of trehalose-6-phosphate synthase and accumulated equal levels of trehalose during the treatment. The conditional mutant ts--187 synthesized no proteins during the preconditioning heat treatment but nevertheless acquired thermotolerance, albeit to a lesser degree than the corresponding wildtype strain. Induction of trehalose-6-phosphate synthase and accumulation of trehalose were reduced to a similar extent. These results show that acquisition of thermotolerance and accumulation of trehalose are closely correlated during heat preconditioning and are modulated by protein synthesis but do not require it.