Production and characterization of a secreted, C-terminally processed tyrosinase from the filamentous fungus Trichoderma reesei

A homology search of the genome database of the filamentous fungus Trichoderma reesei identified a new T. reesei tyrosinase gene tyr2, encoding a protein with a putative signal sequence. The gene was overexpressed in the native host under the strong cbh1 promoter, and the tyrosinase enzyme was secreted into the culture supernatant. This is the first report on a secreted fungal tyrosinase. Expression of TYR2 in T. reesei resulted in good yields, corresponding to approximately 0.3 and 1 g.L(-1) tyrosinase in shake flask cultures and laboratory-scale batch fermentation, respectively. T. reesei TYR2 was purified with a three-step purification procedure, consisting of desalting by gel filtration, cation exchange chromatography and size exclusion chromatography. The purified TYR2 protein had a significantly lower molecular mass (43.2 kDa) than that calculated from the putative amino acid sequence (61.151 kDa). According to N-terminal and C-terminal structural analyses by fragmentation, chromatography, MS and peptide sequencing, the mature protein is processed from the C-terminus by a cleavage of a peptide fragment of about 20 kDa. The T. reesei TYR2 polypeptide chain was found to be glycosylated at its only potential N-glycosylation site, with a glycan consisting of two N-acetylglucosamines and five mannoses. Also, low amounts of shorter glycan forms were detected at this site. T. reesei TYR2 showed the highest activity and stability within a neutral and alkaline pH range, having an optimum at pH 9. T. reesei tyrosinase retained its activity well at 30 degrees C, whereas at higher temperatures the enzyme started to lose its activity relatively quickly. T. reesei TYR2 was active on both l-tyrosine and l-dopa, and it showed broad substrate specificity.     

Is Leptin Concentration Associated with the Insulin Resistance Syndrome in Nondiabetic Men?

HAFFNER, STEVEN M., LEENA MYKKÄNEN, DAVID L. RAINWATER, PAULI KARHAPÄÄ, AND MARKU LAAKSO. Is leptin concentration associated with the insulin resistance syndrome in nondiabetic men? Obes Res. Objective Insulin resistance has been strongly associated with cardiovascular risk. Recently, leptin, a hormone that regulates appetite, has been associated with both obesity and insulin resistance. However, the possible relation of leptin to the insulin resistance syndrome has been controversial. Research Methods and Procedures To explore this issue, we examined the relation of leptin to blood pressure, lipid levels, low density lipoprotein (LDL) size, and glucose levels in 87 normoglycemic men. Results Leptin levels were significantly correlated with body mass index (BMI) (r = 0.494), fasting insulin (r = 0.576), whole-body glucose disposal rate (GDR) (r = ?0.566), fasting glucose (r = 0.510), total triglycerides (r= 0.294), apolipoprotein B 0 = 0.223), systolic blood pressure (r= O.223), and LDL size (r = ?0.244). After adjustment for BMI and GDR, leptin levels remained significantly correlated with fasting insulin, fasting glucose, triglyceride, apolipoprotein B, and systolic blood pressure. Leptin levels were also correlated with the number of metabolic risk factors (dyslipidemia, systolic blood pressure, and fasting glucose). Discussion We conclude that leptin concentrations may be associated with several cardiovascular risk factors related to insulin resistance syndrome. These associations are only partly explained by leptin's relationship with BMI and GDR.     

Rho‐kinase inhibitor Y‐27632 increases cellular proliferation and migration in human foreskin fibroblast cells

The idea of direct differentiation of somatic cells into other differentiated cell types has attracted a great interest recently. Rho‐kinase inhibitor Y‐27632 (ROCKi) is a potential drug molecule, which has been reported to support the gene expressions typical for the chondrocytes, thus restricting their phenotypic conversion to fibroblastic cells upon the cellular expansion. In this study, we have investigated the short‐term biological responses of ROCKi to human primary foreskin fibroblasts. The fibroblast cells were exposed to 1 and 10 μM ROCKi treatments. A proteomics analysis revealed expression changes of 56 proteins, and a further protein pathway analysis suggested their association with the cell morphology, the organization, and the increased cellular movement and the proliferation. These functional responses were confirmed by a Cell‐IQ time‐lapse imaging analysis. Rho‐kinase inhibitor treatment increased the cellular proliferation up to twofold during the first 12 h, and a wound model based migration assay showed 50% faster filling of the mechanically generated wound area. Additionally, significantly less vinculin‐associated focal adhesions were present in the ROCKi‐treated cells. Despite the marked changes in the cell behavior, ROCKi was not able to induce the expression of the chondrocyte‐specific genes, such as procollagen α₁(II) and aggrecan.     

Functional and Structural Properties of a Novel Protein and Virulence Factor (Protein sHIP) in Streptococcus pyogenes

Streptococcus pyogenes is a significant bacterial pathogen in the human population. The importance of virulence factors for the survival and colonization of S. pyogenes is well established, and many of these factors are exposed to the extracellular environment, enabling bacterial interactions with the host. In the present study, we quantitatively analyzed and compared S. pyogenes proteins in the growth medium of a strain that is virulent to mice with a non-virulent strain. Particularly, one of these proteins was present at significantly higher levels in stationary growth medium from the virulent strain. We determined the three-dimensional structure of the protein that showed a unique tetrameric organization composed of four helix-loop-helix motifs. Affinity pull-down mass spectrometry analysis in human plasma demonstrated that the protein interacts with histidine-rich glycoprotein (HRG), and the name sHIP (streptococcal histidine-rich glycoprotein-interacting protein) is therefore proposed. HRG has antibacterial activity, and when challenged by HRG, sHIP was found to rescue S. pyogenes bacteria. This and the finding that patients with invasive S. pyogenes infection respond with antibody production against sHIP suggest a role for the protein in S. pyogenes pathogenesis.     

Ten-Year Mortality and Cardiovascular Morbidity in the Finnish Diabetes Prevention Study—Secondary Analysis of the Randomized Trial

Background

The Finnish Diabetes Prevention Study (DPS) was a randomized controlled trial, which showed that it is possible to prevent type 2 diabetes by lifestyle changes. The aim of the present study was to examine whether the lifestyle intervention had an effect on the ten-year mortality and cardiovascular morbidity in the DPS participants originally randomized either into an intervention or control group. Furthermore, we compared these results with a population-based cohort comprising individuals of varying glucose tolerance states.

Methods and Findings

Middle-aged, overweight people with IGT (n = 522) were randomized into intensive intervention (including physical activity, weight reduction and dietary counseling), or control “mini-intervention” group. Median length of the intervention period was 4 years and the mean follow-up was 10.6 years. The population-based reference study cohort included 1881 individuals (1570 with normal glucose tolerance, 183 with IGT, 59 with screen-detected type 2 diabetes, 69 with previously known type 2 diabetes) with the mean follow-up of 13.8 years. Mortality and cardiovascular morbidity data were collected from the national Hospital Discharge Register and Causes of Death Register. Among the DPS participants who consented for register linkage (n = 505), total mortality (2.2 vs. 3.8 per 1000 person years, hazard ratio HR = 0.57, 95% CI 0.21–1.58) and cardiovascular morbidity (22.9 vs. 22.0 per 1000 person years, HR = 1.04, 95% CI 0.72–1.51) did not differ significantly between the intervention and control groups. Compared with the population-based cohort with impaired glucose tolerance, adjusted HRs were 0.21 (95% CI 0.09–0.52) and 0.39 (95% CI 0.20–0.79) for total mortality, and 0.89 (95% CI 0.62–1.27) and 0.87 (0.60–1.27) for cardiovascular morbidity in the intervention and control groups of the DPS, respectively. The risk of death in DPS combined cohort was markedly lower than in FINRISK IGT cohort (adjusted HR 0.30, 95% CI 0.17–0.54), but there was no significant difference in the risk of CVD (adjusted HR 0.88, 95% CI 0.64–1.21).

Conclusions

Lifestyle intervention among persons with IGT did not decrease cardiovascular morbidity during the first 10 years of follow-up. However, the statistical power may not be sufficient to detect small differences between the intervention and control groups. Low total mortality among participants of the DPS compared with individuals with IGT in the general population could be ascribed to a lower cardiovascular risk profile at baseline and regular follow-up.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00518167","term_id":"NCT00518167"}}NCT00518167     

Mannose inhibits hyaluronan synthesis by down-regulation of the cellular pool of UDP-N-acetylhexosamines

We found that d-mannose dose-dependently decreases hyaluronan synthesis in cultured epidermal keratinocytes to approximately 50%, whereas glucose, galactose, and fructose up to 20 mm concentration had no effect. The full inhibition occurred within 3 h following introduction of mannose and did not involve down-regulation of hyaluronan synthase (Has1-3) mRNA. Following introduction of mannose, there was an approximately 50% reduction in the cellular concentration of UDP-N-acetylhexosamines (UDP-HexNAc, i.e. UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine). On the other hand, 2 mm glucosamine in the culture medium increased UDP-HexNAc content, stimulated hyaluronan secretion, and negated the effect of mannose, supporting the notion that the inhibition by mannose on hyaluronan synthesis was because of down-regulated UDP-HexNAc content. The content of UDP-glucuronic acid, the other building block for hyaluronan synthesis, was not reduced by mannose but declined from 39 to 14% of controls by 0.2-1.0 mm 4-methylumbelliferone, another compound that inhibits hyaluronan synthesis. Applying 4-methylumbelliferone and mannose together produced the expected reductions in both UDP sugars but no additive reduction in hyaluronan production, indicating that the concentration of each substrate alone can limit hyaluronan synthesis. Mannose is a potentially useful tool in studies on hyaluronan-dependent cell functions, as demonstrated by reduced rates of keratinocyte proliferation and migration, functions known to depend on hyaluronan synthesis.     

Circulating RNAs as predictive markers for the progression of type 2 diabetes

Type 2 Diabetes Mellitus (T2DM) is the most prevalent form of diabetes in the USA, thus, the identification of biomarkers that could be used to predict the progression from prediabetes to T2DM would be greatly beneficial. Recently, circulating RNA including microRNAs (miRNAs) present in various body fluids have emerged as potential biomarkers for various health conditions, including T2DM. Whereas studies that examine the changes of miRNA spectra between healthy controls and T2DM individuals have been reported, the goal of this study is to conduct a baseline comparison of prediabetic individuals who either progress to T2DM, or remain prediabetic. Using an advanced small RNA sequencing library construction method that improves the detection of miRNA species, we identified 57 miRNAs that showed significant concentration differences between progressors (progress from prediabetes to T2DM) and non‐progressors. Among them, 26 have been previously reported to be associated with T2DM in either body fluids or tissue samples. Some of the miRNAs identified were also affected by obesity. Furthermore, we identified miRNA panels that are able to discriminate progressors from non‐progressors. These results suggest that upon further validation these miRNAs may be useful to predict the risk of conversion to T2DM from prediabetes.