Evaluation of anthelminthic and antiprotozoal activity of myrrh (Commiphora myrrha) methanolic extract.

To treat and control parasitic infections, traditional medical remedies using plant products are utilized as antiparasitic agents rather than standard synthetic chemicals due to drug resistance. Myrrh, a resinous exudate of Commiphora myrrha (Burseraceae), is a powerful antioxidant with a variety of medicinal uses. This study aimed to investigate the effect of the myrrh methanolic extract (MyE) of three concentrations (100, 50, and 25 mg/ml) on the sporulation of oocysts and as an anthelminthic effector via in vitro study. Characterization of the plant was done by Fourier-transform infrared spectroscopy (FT-IR). The earthworm, Eisenia fetida, is used as a model worm to evaluate the anthelminthic activity of MyE. Eimeria labbeana-like oocysts are used as a model protozoan parasite in anticoccidial assays. The sporulation and inhibition (%) of E. labbeana-like were assessed by MyE compared to other chemical substances. FT-IR revealed the presence of twelve active compounds. Our results showed that paralysis and death of earthworms at MyE (100 mg/ml) were 7.88 ± 0.37 and 9.24 ± 0.60 min, respectively, which is more potency when compared to mebendazole (reference drug). In all treated worms, microscopic examinations revealed obvious surface architecture abnormality. This study shows that MyE affects oocysts sporulation in a dose-dependent manner. At 24 and 36 hr, a high concentration of MyE (100 mg/ml) inhibits sporulation by 90.95 and 87.17 %. At 36 hr, other concentrations of MyE (50 and 25 mg/ml), as well as amprolium, DettolTM, and phenol inhibits oocyst sporulation by 40.17 %, 29.34 %, 45.09 %, 85.11 %, and 61.58 %, respectively. According to our research, the MyE extract had powerful anthelmintic and anticoccidial properties.

Morphological Re-description of Camallanus polypteri) Nematoda: Camallanidae) Infecting the African Sharptooth Catfish Clarias gariepinus (Clariidae).

Fish represents one of the major sources of animal proteins, and different species of fish are susceptible to infections with parasites which cause severe tissue damage and cell destruction of the infected organ. Therefore, in 2019, this parasitological study was conducted to assess the helminth parasites infecting the African sharptooth catfish Clarias gariepinus that were collected from Lake Manzala, Egypt. Only nematode parasite was reported as a prevalent infection from the fish stomach with an infection rate of 7.5%. Depending on the seasonal prevalence, the extent of the infection was analyzed. It was indicated that parasite infection was only reported as 15% in the winter season. Morphological and morphometric analyses of the present parasite species revealed that it possesses all the characteristics of the Camallanus genus, whereas it is closely related to Camallanus polypteri described previously. It is characterized by the presence of a buccal capsule with longitudinal internal ridges, some of which are very short and ranged from 8-14 in males and 8-9 in females. The esophagus consisted of muscular and glandular portions, the middle position of the excretory pore to the muscular esophagus, the anterior location of deirids to the nerve ring, posterior end of males with two unequal spicules and caudal papillae; nonetheless, it is smooth and straight in females. In addition, some morphology and measurement differences for the different body parts were identified with other Camallanus species. Therefore, the present study can provide a full morphologically re-description of Camallanus polypteri with a new geographical location in the Egyptian freshwater.

Detection of Raillietina saudiae from the domestic pigeon in Saudi Arabia through 18S and 28S rDNA genes.

Raillietina saudiae is a well-studied avian gastrointestinal parasite belonging to the family Davaineidae and is the most prevalent cyclophyllid tapeworm infecting pigeon in Saudi Arabia. The present study considered as a complementary analysis of Al-Quraishy et al. (2019; Parasitol Int 71, 59-72) with molecular studies for two ribosomal DNA genes employed for precise recognition of this Raillietina species. The annotated partial 18S and 28S rDNA gene regions were found to be 888 and 900 bp long that utilized further to elucidate their genetic relationships at species level using maximum likelihood method. The query sequence of R. saudiae is well aligned and placed within the Davaineidae family, with the same clade of all species of Raillietina that well separated from other cyclophyllidean cestodes especially taeniid and hymenolepid species. Sequence data recorded the monophyly of Raillietina species. The current phylogeny supports the usage of the partial 18S and 28S rDNA genes as reliable markers for phylogenetic reconstructions.

Molecular phylogeny of Sarcocystis fayeri (Apicomplexa: Sarcocystidae) from the domestic horse Equus caballus based on 18S rRNA gene sequences and its prevalence.

Sarcocystosis is a parasitic disease caused by an intracellular protozoan parasite Sarcocystis belonging to the phylum Apicomplexa. These parasites have a requisite two-host life cycle. Recently, there are many Sarcocystis species that identified morphologically. In the present study, diaphragmatic muscle samples from the domestic horse (Equus caballus) were examined for Sarcocystis infection. The natural infection with sarcocysts was recorded to be 62·5% for only microcysts in the infected muscles. Molecular analysis using the 18S rRNA gene was conducted to swiftly and accurately identify the recovered species. Studies on the expression of the 18S rRNA gene have confirmed that the present parasite isolates belong to the Sarcocystis genus. The sequence data showed significant identities (>80%) with archived gene sequences from species within the Sarcocystidae family, and a dendrogram showing the phylogenetic relationship was constructed. The most closely related species were the previously described Sarcocystis fayeri and Sarcocystis bertrami. The current data showed that the present species was identified as S. fayeri and deposited in GenBank (accession number MF614956.1). This study highlights the importance of the genetic data in the exact taxonomy within sarcocystid species.

New Host and Locality Record of Parapharyngodon Japonicus (Nematoda: Oxyuroidea) from the Egyptian Changeable Lizard Agama Mutabilis (Agamidae): A Light and Scanning Electron Microscopy.

Parapharyngodon (Oxyurida) is a lizard gastrointestinal nematode parasite with a life cycle including lizards as main hosts. However, some species are known to parasitize anurans. In the present study, P. japonicus isolated from the large intestine of the Egyptian changeable lizard, Agama mutabilis was described and illustrated. Forty five specimens of these animals were collected from south Sinai desert, Egypt during the period from May to September 2017. After necropsy, the body was opened by a longitudinal incision from vent to throat, and the gastrointestinal tract was removed. The esophagus, stomach, small and large intestines were examined separately for helminthes. The recovered nematodes were examined by light and scanning electron microscopy. Thirty six specimens (80.0 %) were found to be naturally infected. The parasite was robust with prominent cuticular transverse annulations. Mouth surrounded by three bilobed lips, each with tiny labial papillae. Three pairs of caudal papillae were observed in male worms; 1 pair precloacal, 1 pair sublateral in cloacal opening line, 1 pair in proximal region of caudal appendage on its narrowed point. The posterior extremity beard dorsally directed caudal appendages. Females were with a conical posterior end terminated at a terminal spike. Ovaries reached esophageal isthmus but not wrapped around corpus. The parasite recorded was compared morphologically and morphometrically with the most similar species, it was found that it was most similar to P. japonicus with new host and locality records.

The role of microalbuminuria as a predictor of subclinical cardiovascular events in rheumatoid arthritis patients and its relation to disease activity.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that affects many body tissues and leads to major morbidity and mortality. Renal disease in RA is clinically important because it restricts the management of primary disease and increases mortality. The objectives of this study are to (1) investigate the difference between RA patients with and without microalbuminuria (MAU) and (2) find out the relation between MAU and disease activity as well as subclinical cardiovascular effects. Ninety RA patients were divided into two groups according to the presence of MAU, in addition to 30 healthy volunteers. ESR, hs-CRP, RF, lipid profile, urinary microalbumin, GFR, renal function tests, carotid intima media thickness (cIMT), flow-mediated dilatation of the brachial artery (FMD), ECG, and echocardiographic examination were performed for patients and controls. MAU positive RA patients revealed significantly higher lipid profile, ESR, hs-CRP, DAS 28, cIMT, and lower FMD as well as ECG and echocardiographic abnormalities compared to MAU negative RA patients. Moreover, there was significant positive correlation between MAU and DAS28, hs-CRP, LDL, cIMT as well as negative correlation with FMD%. In our study, all RA patients with MAU had a normal serum creatinine concentration and gave a negative result with Albustix. MAU is significantly correlated with ESR, hs-CRP, lipid profile, cIMT, and FMD% in RA patients; therefore, it can be used as an index to measure disease activity as well as subclinical cardiovascular affection in RA patients.

Interactions between the intestinal cestode Polyonchobothrium clarias (Pseudophyllidea: Ptychobothriidae) from the African sharptooth catfish Clarias gariepinus and heavy metal pollutants in an aquatic environment in Egypt.

In an aquatic environment, there is a profound and inverse relationship between environmental quality and disease status of fish. Parasites are one of the most serious limiting factors in aquaculture. Therefore, the present investigation was carried out during the period of February-December 2014 to determine the parasitic infections in the African sharptooth catfish Clarias gariepinus, relative to the capability of internal parasites to accumulate heavy metals. Up to 100 catfish were examined for gastrointestinal helminths and 38% of fish were found to be infected with the cestode Polyonchobothrium clarias. The morphology of this parasite species, based on light and scanning electron microscopy, revealed that the adult worm was characterized by a rectangular scolex measuring 0.43-0.58 (0.49 ± 0.1) mm long and 0.15-0.21 (0.19 ± 0.1) mm wide, with a flat to slightly raised rostellum armed with a crown with two semicircles each bearing 13-15 hooks, followed by immature, mature and gravid proglottids which were about 29-55 (45), 16-30 (24) and 15-39 (28) in number, respectively. The mature proglottid contained a single set of genitalia in which medullary testes measured 0.09-0.13 (0.11 ± 0.01) mm long and 0.05-0.08 (0.06 ± 0.01) mm wide; a bi-lobed ovary was situated near the posterior margin of the proglottid, extending laterally up to the longitudinal excretory canals; the tubular uterus arose from the ootype up to the anterior margin of the proglottid; and vitelline follicles were cortical. The greater portion of the gravid proglottid was occupied by a uterus filled with unoperculate and embryonated eggs. Chemical analysis confirmed that the concentrations of heavy metals (Zn, Cu, Mn, Cd, Ni and Pb) accumulated in P. clarias were higher than in fish tissues and values recommended by FAO/WHO, with the exception of Zn, which was found to be higher in fish kidneys than in the cestode. This supports the hypothesis that cestodes of fish can be regarded as useful bioindicators when evaluating the environmental pollution of aquatic ecosystems by heavy metals.

RUNX2 and WWOX genes as molecular biomarkers and candidates for targeted therapy in Egyptian patients with primary conventional osteosarcoma

Background: The conventional osteosarcoma (OS) is the commonest primary malignant, bone tumor with complex genomic profiles and poor survival. Runt-related transcription factor 2 (RUNX2) and WW domain containing oxidoreductase (WWOX) genes are implicated in normal osteogenesis as well as in the development of primary conventional OS. Methods: We retrospectively assessed protein and RNA expression of the RUNX2 and WWOX genes by quantitative real time PCR (qPCR) and immunohistochemistry (IHC) in 80 cases of primary OS and 20 normal control (NC) subjects. Proteins and RNA expression levels of both genes were correlated to clinico-pathological features of the patients, progression free and overall survival (PFS& OS) rates. Results: In OS, RUNX2 protein was detected in 72/80 (90%) cases compared to 4/20 (20%) NC samples (p. < 0.001) and RUNX2-RNA was up regulated (up to 103.2 folds) in 60/80 (75%) (p = 0.01). WWOX protein and RNA (up to 7.2 folds) were detected in all NC samples but in 24/80 (30%) and 20/80 (20%) OS cases; respectively (p. < 0.001 for each). The concordance between the RNA and protein expressions for RUNX2 and WWOX was significantly high (X_trend

Improved diagnostics targeting c-MET in non-small cell lung cancer: expression, amplification and activation?

BACKGROUND: Several c-MET targeting inhibitory molecules have already shown promising results in the treatment of patients with Non-small Cell Lung Cancer (NSCLC). Combination of EGFR- and c-MET-specific molecules may overcome EGFR tyrosine kinase inhibitor (TKI) resistance. The aim of this study was to allow for the identification of patients who might benefit from TKI treatments targeting MET and to narrow in on the diagnostic assessment of MET. METHODS: 222 tumor tissues of patients with NSCLC were analyzed concerning c-MET expression and activation in terms of phosphorylation (Y1234/1235 and Y1349) using a microarray format employing immunohistochemistry (IHC). Furthermore, protein expression and MET activation was correlated with the amplification status by Fluorescence in Situ Hybridization (FISH). RESULTS: Correlation was observed between phosphorylation of c-MET at Y1234/1235 and Y1349 (spearman correlation coefficient rs = 0.41; p < 0.0001). No significant correlation was shown between MET expression and phosphorylation (p > 0.05). c-MET gene amplification was detected in eight of 214 patients (3.7%). No significant association was observed between c-MET amplification, c-MET protein expression and phosphorylation. CONCLUSION: Our data indicate, that neither expression of c-MET nor the gene amplification status might be the best way to select patients for MET targeting therapies, since no correlation with the activation status of MET was observed. We propose to take into account analyzing the phosphorylation status of MET by IHC to select patients for MET targeting therapies. Signaling of the receptor and the activation of downstream molecules might be more crucial for the benefit of therapeutics targeting MET receptor tyrosine kinases than expression levels alone.

Efficacy of a sun protection workbook for kidney transplant recipients: a randomized controlled trial of a culturally sensitive educational intervention.

A culturally sensitive educational intervention that encouraged sun protection behaviors among kidney transplant recipients (KTRs) was developed and the short-term efficacy was evaluated. Non-Hispanic White, Hispanic/Latino and non-Hispanic Black patients, who received a transplant 2-24 months prior to the study, were randomized into two study groups: intervention versus standard of care. Electronic reminders tailored to the weather conditions were sent every 2 weeks by text message or email. Self-reported surveys and biologic measurements were obtained prior to the intervention and 6 weeks later. Among the 101 study participants, there was a statistically significant increase in knowledge, recognition of personal risk of developing skin cancer, willingness to change sun protection behavior and self-reported performance of sun protection in participants receiving the intervention in comparison with those receiving standard of care (p < 0.05). The pigment darkening of the sun-exposed forearm and sun damage of the forearm and sunburns/skin irritation from the sun were significantly less in participants receiving the intervention (p < 0.05). Providing sun protection education at the beginning of summer with reminders tailored to weather conditions helped KTRs adopt sun protection practices. This sun protection program for KTRs may be incorporated into the care provided by the nephrologist or transplant surgeon.

Relations among glycemic control, circulating endothelial cells, nitric oxide, and flow mediated dilation in patients with type 2 diabetes mellitus.

OBJECTIVES: Circulating endothelial cells (CEC) have been identified as a surrogate marker of endothelial dysfunction. The aim of this study was to determine the association of glycemic control with CEC and endothelial function in patients with type 2 diabetes mellitus (DM). METHODS: We studied 30 patients with type 2 DM and 20 age and sex matched healthy controls (HC). Number of circulating endothelial cells was measured by flow cytometry. Endothelial function was studied by measuring flow mediated vasodilation (FMD%) in the brachial artery and serum level of nitric oxide (NO). RESULTS: CEC count was significantly elevated in patients with DM, than HC (35.3±15.1 vs. 7.3±2.4, p<0.001) and in patients with HbA1c>7 than patients with HbA1c≤7 (47.4±5.5 vs. 19.5±5.7, p<0.001). FMD% and NO were lower in DM patients than HC (3.5±0.85 vs. 9.5±3.1, p<0.001 and 37.8±6.1 vs. 64.1±5.7, p<0.001 respectively). FMD% and NO were lower in patients with HbA1c>7 as compared to patients with HA1c≤7 (2.8±0.4 vs. 4.3±0.4, p<0.001 and 33.1±2.9 vs. 43.9±2.8, respectively, p<0.001). HbA1c correlated negatively with FMD% and NO levels and positively with CEC. CEC count correlated negatively with FMD% and NO. There was a significant positive correlation between CEC count and HBA1c (p<0.001 for all correlations). CONCLUSION: CEC is associated with markers of endothelial dysfunction and disease control in patients with type 2 DM. These findings suggest a potential role of CEC in the pathophysiology of cardiovascular disease in type 2 diabetic and raise the importance of tight glycemic control.

Robust reporter system based on chalcone synthase rppA gene from Saccharopolyspora erythraea.

Industrial overproducing strains present unique hosts for expression of heterologous gene clusters encoding secondary metabolite biosynthesis. For this purpose, efficient gene expression tools and methods are needed. A robust and versatile reporter system based on the rppA gene from Saccharopolyspora erythraea is presented as the method of choice when studying gene expression in actinomycete hosts. The method is easily scalable to accommodate high-throughput procedure, and collected samples can be easily stored and re-tested when needed. The product of RppA is an inert 1,3,6,8-tetrahydroxynaphthalene which spontaneously oxidises to a dark-red quinone flaviolin providing a qualitative visual assessment of gene expression on an agar plate as well as a quantitative spectrophotometric measurement in liquid broth without the need for invasive procedures or external substrate addition. The applicability of the reporter system has been demonstrated by expressing the rppA gene under the control of the heterologous promoters actII-ORF4/PactI, ermE and its upregulated variant ermE*. The model streptomycete Streptomyces coelicolor, and three industrially important species, Streptomyces tsukubaensis (FK506), Streptomyces cinnamonensis (monensin) and Streptomyces rimosus (oxytetracycline) were used as hosts. The reporter system has shown its utility independently of cultivation conditions or composition of growth medium, from simple laboratory to complex industrial media. The simplicity and robustness of the system, demonstrated even in industrial settings, shows great potential for wider use in different microbial hosts and applications, and may thus represent a new generic and versatile tool useful to a wider scientific community.

Amino acid sensing by Ssy1.

Saccharomyces cerevisiae senses extracellular amino acids using two members of the family of amino acid transporters, Gap1 or Ssy1; aspects of the latter are reviewed here. Despite resemblance with bona fide transporters, Ssy1 appears unable to facilitate transport. Exposure of yeast to amino acids results in Ssy1-dependent transcriptional induction of several genes, in particular some encoding amino acid transporters. Amino acids differ strongly in their potency, leucine being the most potent one known. Using a selection system in which potassium uptake was made dependent on amino acid signalling, our laboratory has obtained and described gain-of-function mutations in SSY1. Some alleles conferred inducer-independent signalling; others increased apparent affinity for inducers. These results revealed that amino acid transport is not required for signalling and support the notion that sensing by Ssy1 occurs via its direct interaction with extracellular amino acids. Current work includes development of quantitative assays of sensing. We use the finding by Per Ljungdahl's laboratory that the signal transduction from Ssy1 involves proteolytic removal of an inhibitory part of the transcriptional activator Stp1. Protein-A Z-domain fused to the C-terminus of Stp1 and Western analysis using antibody against horseradish peroxidase allow quantification of sensing.

A point mutation in PTPRC is associated with the development of multiple sclerosis.

Multiple sclerosis (MS) is the most common demyelinating disease of the central nervous system. It is widely accepted that a dysregulated immune response against brain resident antigens is central to its yet unknown pathogenesis. Although there is evidence that the development of MS has a genetic component, specific genetic factors are largely unknown. Here we investigated the role of a point mutation in the gene (PTPRC) encoding protein-tyrosine phosphatase, receptor-type C (also known as CD45) in the heterozygous state in the development of MS. The nucleotide transition in exon 4 of the gene locus interferes with mRNA splicing and results in altered expression of CD45 isoforms on immune cells. In three of four independent case-control studies, we demonstrated an association of the mutation with MS. We found the PTPRC mutation to be linked to and associated with the disease in three MS nuclear families. In one additional family, we found the same variant CD45 phenotype, with an as-yet-unknown origin, among the members affected with MS. Our findings suggest an association of the mutation in PTPRC with the development of MS in some families.

Rapid identification of local T cell expansion in inflammatory organ diseases by flow cytometric T cell receptor Vbeta analysis.

Oligoclonal expansion of antigen-specific T cells occurs frequently during inflammatory diseases. These cells may persist for a long time at high frequency in the body and be enriched in the affected tissues. As a screening test for expanded cell T cell populations at sites of inflammation, we developed an optimized methodology for flow-cytometry-based quantification of T cell receptor Vbeta (TCRBV) expression. We first validated the specificity of a TCRBV-specific monoclonal antibody set by direct comparison with PCR-based analysis of mono- and polyclonal T cell samples. This monoclonal antibody (mAb) panel recognized approximately two thirds of the T cell receptor alpha/beta repertoire in a group of 64 healthy donors and allowed defining TCR usage in the CD4+ and CD8+ subsets. The reliable detection of expanded Vbeta gene families in T cell populations was confirmed in experiments on superantigen-stimulated T cells. Through differential TCR analysis on T cell subpopulations in cerebrospinal fluid and blood in patients with acute encephalitis, we were able to identify locally expanded CD8+ T cells. The power of this approach affords not only high-throughput comparative TCR analysis for immunological studies in vitro, but also rapid ex vivo identification of cell populations enriched in organ compartments during inflammatory diseases.

The presumed potassium carrier Trk2p in Saccharomyces cerevisiae determines an H+-dependent, K+-independent current.

Ionic currents related to the major potassium uptake systems in Saccharomyces cerevisiae were examined by whole cell patch-clamping, under K+ replete conditions. Those currents have the following properties. They (1) are inward under all conditions investigated, (2) arise instantaneously with appropriate voltage steps, (3) depend solely upon the moderate affinity transporter Trk2p, not upon the high affinity transporter Trk1p. They (4) appear to be independent of the extracellular K+ concentration, (5) are also independent of extracellular Ca2+, Mg2+ and Cl- but (6) are strongly dependent on extracellular pH, being large at low pH (up to several hundred pA at -200 mV and pH 4) and near zero at high pH (above 7.5). They (7) increase in proportion to log[H+]o, rather than directly in proportion to the proton concentration and (8) behave kinetically as if each transporter cycle moved one proton plus one (high pH) or two (low pH) other ions, as yet unidentified. In view of background knowledge on K+ transport related to Trk2p, the new results suggest that the K+ status of yeast cells modulates both the kinetics of Trk2p-mediated transport and the identity of ions involved. That modulation could act either on the Trk2 protein itself or on interactions of Trk2 with other proteins in a hypothetical transporter complex. Structural considerations suggest a strong analogy to the KtrAB system in Vibrio alginolyticus and/or the TrkH system in Escherichia coli.

Cns1 is an essential protein associated with the hsp90 chaperone complex in Saccharomyces cerevisiae that can restore cyclophilin 40-dependent functions in cpr7Delta cells.

Saccharomyces cerevisiae harbors two cyclophilin 40-type enzymes, Cpr6 and Cpr7, which are components of the Hsp90 molecular chaperone machinery. Cpr7 is required for normal growth and is required for maximal activity of heterologous Hsp90-dependent substrates, including glucocorticoid receptor (GR) and the oncogenic tyrosine kinase pp60(v-src). In addition, it has recently been shown that Cpr7 plays a major role in negative regulation of the S. cerevisiae heat shock transcription factor (HSF). To better understand functions associated with Cpr7, a search was undertaken for multicopy suppressors of the cpr7Delta slow-growth phenotype. The screen identified a single gene, designated CNS1 (for cyclophilin seven suppressor), capable of suppressing the cpr7Delta growth defect. Overexpression of CNS1 in cpr7Delta cells also largely restored GR activity and negative regulation of HSF. In vitro protein retention experiments in which Hsp90 heterocomplexes were precipitated resulted in coprecipitation of Cns1. Interaction between Cns1 and the carboxy terminus of Hsp90 was also shown by two-hybrid analysis. The functional consequences of CNS1 overexpression and its physical association with the Hsp90 machinery indicate that Cns1 is a previously unidentified component of molecular chaperone complexes. Thus far, Cns1 is the only tetratricopeptide repeat-containing component of Hsp90 heterocomplexes found to be essential for cell viability under all conditions tested.

Requirement for Hsp90 and a CyP-40-type cyclophilin in negative regulation of the heat shock response.

The heat shock response is a highly conserved mechanism that allows cells to withstand a variety of stress conditions. Activation of this response is characterized by increased synthesis of heat shock proteins (HSPs), which protect cellular proteins from stress-induced denaturation. Heat shock transcription factors (HSFs) are required for increased expression of HSPs during stress conditions and can be found in complexes containing components of the Hsp90 molecular chaperone machinery, raising the possibility that Hsp90 is involved in regulation of the heat shock response. To test this, we have assessed the effects of mutations that impair activity of the Hsp90 machinery on heat shock related events in Saccharomyces cerevisiae. Mutations that either reduce the level of Hsp90 protein or eliminate Cpr7, a CyP-40-type cyclophilin required for full Hsp90 function, resulted in increased HSF-dependent activities. Genetic tests also revealed that Hsp90 and Cpr7 function synergistically to repress gene expression from HSF-dependent promoters. Conditional loss of Hsp90 activity resulted in both increased HSF-dependent gene expression and acquisition of a thermotolerant phenotype. Our results reveal that Hsp90 and Cpr7 are required for negative regulation of the heat shock response under both stress and nonstress conditions and establish a specific endogenous role for the Hsp90 machinery in S. cerevisiae.

The peptidyl-prolyl isomerase domain of the CyP-40 cyclophilin homolog Cpr7 is not required to support growth or glucocorticoid receptor activity in Saccharomyces cerevisiae.

CyP-40 cyclophilins are found in association with molecular chaperone Hsp90.steroid receptor complexes. The amino-terminal portion of these cyclophilins harbors the characteristic peptidyl-prolyl isomerase (PPIase) domain, whereas three copies of the tetratricopeptide (TPR) motif, a structure shown to be involved in protein-protein interactions, and a putative calmodulin-binding domain are located in the carboxyl-terminal half of the protein. The TPR domains mediate binding to Hsp90, but a requirement for the PPIase domain has not been established. To address this, we have investigated the effects of mutations that alter the PPIase domain of the Saccharomyces cerevisiae CyP-40 homolog, Cpr7. Because Cpr7 is required for rapid growth and full Hsp90 activity, a functional assessment of the PPIase domain could be performed in vivo. A mutation in the catalytic domain altering a conserved site predicted to be essential for isomerase activity did not compromise Cpr7 function. Furthermore, deletion of the entire PPIase domain did not significantly affect growth or Hsp90-mediated steroid receptor activity. These results indicate that the TPR-containing carboxyl terminus of Cpr7 is sufficient for fundamental Cpr7-dependent activity.