Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease

BACKGROUND: Candida albicans, a commensal organism, is a part of the normal flora of healthy individuals. However, once the host immunity is compromised, C. albicans opportunistically causes recurrent superficial or fatal systemic candidiasis. Secreted aspartic proteases (Sap), encoded by 10 types of SAP genes, have been suggested to contribute to various virulence processes. Thus, it is important to elucidate their biochemical properties for better understanding of the molecular mechanisms that how Sap isozymes damage host tissues. METHODOLOGY/PRINCIPAL FINDINGS: The SAP7 gene was cloned from C. albicans SC5314 and heterogeneously produced by Pichia pastoris. Measurement of Sap7 proteolytic activity using the FRETS-25Ala library showed that Sap7 was a pepstatin A-insensitive protease. To understand why Sap7 was insensitive to pepstatin A, alanine substitution mutants of Sap7 were constructed. We found that M242A and T467A mutants had normal proteolytic activity and sensitivity to pepstatin A. M242 and T467 were located in close proximity to the entrance to an active site, and alanine substitution at these positions widened the entrance. Our results suggest that this alteration might allow increased accessibility of pepstatin A to the active site. This inference was supported by the observation that the T467A mutant has stronger proteolytic activity than the wild type. CONCLUSIONS/SIGNIFICANCE: We found that Sap7 was a pepstatin A-insensitive protease, and that M242 and T467 restricted the accessibility of pepstatin A to the active site. This finding will lead to the development of a novel protease inhibitor beyond pepstatin A. Such a novel inhibitor will be an important research tool as well as pharmaceutical agent for patients suffering from candidiasis

Dental therapy before and after radiotherapy–an evaluation on patients with head and neck malignancies

The present investigation evaluates the dental care situation of patients with head and neck cancer before and after radiotherapy. The situations of these patients in 1993 and 2005 were compared to detect similarities, differences and developments. In the years 1993 and 2005, 37 and 36 patients, respectively, with head and neck cancer treated by the local departments of otorhinolaryngology and of radiotherapy were examined consecutively according to their aftercare appointments. Time points of radiotherapy treatment of the patients evaluated in 1993 varied from 1984 to 1993. The patients evaluated in 2005 had received radiotherapy between 1998 and 2005. Therefore the applied radiotherapeutic regimen differed not only between the two groups of patients, but also within each group. The information for these investigations was provided anonymously. It was evaluated with descriptive statistics. The evaluation of the data shows distinct differences with respect to preventive and therapeutic dental care measures. In 2005, 35 out of 36 patients (97.2%) had a dental consultation before radiotherapy (1993, 65%). All 27 dentate patients (100%) obtained a splint for fluoride application (1993, none). 29% fewer edentulous patients were seen than in 1993. The number of teeth destroyed decreased from 19.2% (1993) to 7.8% in 2005. Mycoses due to Candida spp. and chronic failures in wound healing were rare (5.5%). In the course of the 12 years, prophylactic measures, such as the application of splints for fluoride treatment, were intensified. However, concepts for the dental care of patients undergoing radiotherapy, especially following the radiation, should be widened to avoid ruined teeth and long delayed wound healings

Circuit-based interrogation of sleep control.

Sleep is a fundamental biological process observed widely in the animal kingdom, but the neural circuits generating sleep remain poorly understood. Understanding the brain mechanisms controlling sleep requires the identification of key neurons in the control circuits and mapping of their synaptic connections. Technical innovations over the past decade have greatly facilitated dissection of the sleep circuits. This has set the stage for understanding how a variety of environmental and physiological factors influence sleep. The ability to initiate and terminate sleep on command will also help us to elucidate its functions within and beyond the brain

Review & meta-analysis: isopropanolic black cohosh extract iCR for menopausal symptoms - an update on the evidence

A systematic literature search revealed 35 clinical studies and one meta-analysis comprising 43,759 women, of which 13,096 were treated with isopropanolic Cimicifuga racemosa extract (iCR). Compared to placebo, iCR was significantly superior for treating neurovegetative and psychological menopausal symptoms, with a standardized mean difference of 0.694 in favor of iCR (p<0.0001). Effect sizes were larger when higher dosages of iCR as monotherapy or in combination with St. John's wort (Hypericum perforatum [HP]) were given ( 1.020 and 0.999, respectively), suggesting a dose-depend- ency. For psychological symptoms, the iCRþHP combination was superior to iCR monotherapy. Efficacy of iCR was comparable to low-dose transdermal estradiol or tibolone. Yet, due to its better tol- erability, iCR had a significantly better benefit-risk profile than tibolone. Treatment with iCR/iCRþHP was well tolerated with few minor adverse events, with a frequency comparable to placebo. The clin- ical data did not reveal any evidence of hepatotoxicity. Hormone levels remained unchanged and estrogen-sensitive tissues (e.g. breast, endometrium) were unaffected by iCR treatment. As benefits clearly outweigh risks, iCR/iCRþHP should be recommended as an evidence-based treatment option for natural climacteric symptoms. With its good safety profile in general and at estrogen-sensitive organs, iCR as a non-hormonal herbal therapy can also be used in patients with hormone-dependent diseases who suffer from iatrogenic climacteric symptoms

Secreted Aspartic Proteinase Family of Candida tropicalis

Medically important yeasts of the genus Candida secrete aspartic proteinases (Saps), which are of particular interest as virulence factors. Like Candida albicans, Candida tropicalis secretes in vitro one dominant Sap (Sapt1p) in a medium containing bovine serum albumin (BSA) as the sole source of nitrogen. Using the gene SAPT1 as a probe and under low-stringency hybridization conditions, three new closely related gene sequences, SAPT2 to SAPT4, encoding secreted proteinases were cloned from a C. tropicalis λEMBL3 genomic library. All bands identified by Southern blotting of EcoRI-digested C. tropicalis genomic DNA with SAPT1 could be assigned to a specific SAP gene. Therefore, the SAPT gene family of C. tropicalis is likely to contain only four members. Interestingly, the SAPT2 and SAPT3 gene products, Sapt2p and Sapt3p, which have not yet been detected in C. tropicalis cultures in vitro, were produced as active recombinant enzymes with the methylotrophic yeast Pichia pastoris as an expression system. As expected, reverse transcriptase PCR experiments revealed a strong SAPT1 signal with RNA extracted from cells grown in BSA medium. However, a weak signal was obtained with all other SAPT genes under several conditions tested, showing that these SAPT genes could be expressed at a basic level. Together, these experiments suggest that the gene products Sapt2p, Sapt3p, and Sapt4p could be produced under conditions yet to be described in vitro or during infection

Loss of Heterozygosity at an Unlinked Genomic Locus Is Responsible for the Phenotype of a Candida albicans sap4Δ sap5Δ sap6Δ Mutant ▿

The diploid genome of the pathogenic yeast Candida albicans exhibits a high degree of heterozygosity. Genomic alterations that result in a loss of heterozygosity at specific loci may affect phenotypes and confer a selective advantage under certain conditions. Such genomic rearrangements can also occur during the construction of C. albicans mutants and remain undetected. The SAP2 gene on chromosome R encodes a secreted aspartic protease that is induced and required for growth of C. albicans when proteins are the only available nitrogen source. In strain SC5314, the two SAP2 alleles are functionally divergent because of differences in their regulation. Basal expression of the SAP2-2 allele, but not the SAP2-1 allele, provides the proteolytic degradation products that serve as inducers for full SAP2 induction. A triple mutant lacking the SAP4, SAP5, and SAP6 genes, which are located on chromosome 6, has previously been reported to have a growth defect on proteins, suggesting that one of the encoded proteases is required for SAP2 expression. Here we show that this sap4Δ sap5Δ sap6Δ mutant has become homozygous for chromosome R and lost the SAP2-2 allele. Replacement of one of the SAP2-1 copies in this strain by SAP2-2 and its regulatory region restored the ability of the sap4Δ sap5Δ sap6Δ mutant to utilize proteins as the sole nitrogen source. This is an illustrative example of how loss of heterozygosity at a different genomic locus can cause the mutant phenotype attributed to targeted deletion of a specific gene in C. albicans