Improving the activity of creatinase from Alcaligenes sp. KS-85 through semi-rational design / 生物工程学报

Creatinine levels in biological fluids are important indicators for the clinical evaluation of renal function. Creatinase (CRE, EC3.5.3.3) is one of the key enzymes in the enzymatic measurement of creatinine concentration, and it is also the rate-limiting enzyme in the whole enzymatic cascade system. The poor catalytic activity of CRE severely limits its clinical and industrial applications. To address this issue, a semi-rational design is applied to increase the activity of a creatinase from Alcaligenes sp. KS-85 (Al-CRE). By high-throughput screen of saturation mutagenesis libraries on the selected hotspot mutations, multiple variant enzymes with increased activity are obtained. The five-point best variant enzyme (I304L/F395V/K351V/Y63S/Q88A) were further obtained by recombine the improved mutations sites that to showed a 2.18-fold increased specific activity. Additionally, structure analysis is conducted to understand the mechanism of the activity change. This study paves the way for a better practical application of creatinase and may help further understand its catalytic mechanism.

Differentially expressed proteins in the precancerous stage of rat hepatocarcinogenesis induced by diethylnitrosamine / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To screen the differentially expressed proteins especially at the precancerous stage of diethylnitrosamine (DEN) induced hepatocarcinogenesis by comparative proteome research.</p><p><b>METHODS</b>Rats were divided into normal and DEN groups and sacrificed periodically. The liver samples were stained with gamma-glutamyl transpeptidase (GGT) and HE to distinguish the preneoplastic lesion (pre-HCC) from the normal and HCC tissues. The two-dimensional electrophoresis (2-DE) and mass spectrometry (MALDI-TOF-MS/MS) were then applied to analyze the differentially expressed protein between pre-HCC and normal tissues, pre-HCC and HCC, as well as HCC and normal tissues. A few of the candidate proteins such as laminin receptor 1 (67LR) and agmatinase were validated by Western blot and RT-PCR.</p><p><b>RESULTS</b>Totally, there were 82 proteins that differentially expressed two fold or more in one kind of tissues sample than the other, 47 of which occurred in the pre-HCC tissues. Eight proteins including 67LR were consistently up-regulated from normal tissue to pre-HCC and then to HCC tissues, while 22 proteins including agmatinase showed progressively down-regulated in these tissues samples.</p><p><b>CONCLUSION</b>The protein expression profiles are different during the process of hepatocarcinogenesis. Further study on the differentially expressed protein, especially these upregulated in the precancerous stage such as 67LR and agmatinase, might contribute to prevention and early diagnosis of human HCC.</p>

Clinical presentation and renal evaluation of human visceral leishmaniasis (kala-azar): a retrospective study of 57 patients in Brazil

Visceral leishmaniasis is an endemic disease caused by various species of Leishmania. We made a retrospective study of 57 consecutive patients with visceral leishmaniasis in Brazil. Patients with visceral leishmaniasis were identified using the registries of the São José Infectious Diseases Hospital. The sample was divided into two groups: patients with serum creatinine (Scr) <1.3mg/dL and Scr > 1.3mg/dL. We compared these two groups for differences in clinical manifestations and laboratory features. Patients' mean age was 28 ± 18 years old; 74 percent were male. The main clinical symptoms and signs presented in the initial evaluation were: fever (97 percent), splenomegaly (96.4 percent), weight loss (95.5 percent), pallor (93.6 percent), cough (89.7 percent), hepatomegaly (87.2 percent), asthenia (83.3 percent), anorexia (82.9 percent) and vomiting (73.9 percent). Acute renal failure was found in 15 patients (26.3 percent) and eight of these patients had ARF before amphotericin B administration. The mean age was higher in the group with Scr > 1.3mg/dL. Death occurred in three cases; all deaths occurred with Scr > 1.3mg/dL. There were no significant differences in the frequencies of the clinical symptoms and signs between the two groups. The laboratory data and demographic characteristics were significantly worse in the Scr > 1.3mg/dL group. Renal dysfunction is an important feature of this disease; it is associated with important morbidity and can increase mortality.

Identification of Proteins Differentially Expressed in the Conventional Renal Cell Carcinoma by Proteomic Analysis

Renal cell carcinoma (RCC) is one of the most malignant tumors in urology, and due to its insidious onset patients frequently have advanced disease at the time of clinical presentation. Thus, early detection is crucial in management of RCC. To identify tumor specific proteins of RCC, we employed proteomic analysis. We prepared proteins from conventional RCC and the corresponding normal kidney tissues from seven patients with conventional RCC. The expression of proteins was determined by silver stain after two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The overall protein expression patterns in the RCC and the normal kidney tissues were quite similar except some areas. Of 66 differentially expressed protein spots (p<0.05 by Student t-test), 8 different proteins from 11 spots were identified by MALDI-TOF-MS. The expression of the following proteins was repressed (p<0.05); aminoacylase-1, enoyl-CoA hydratase, aldehyde reductase, tropomyosin alpha-4 chain, agmatinase and ketohexokinase. Two proteins, vimentin and alpha-1 antitrypsin precursor, were dominantly expressed in RCC (p<0.05).