Purification and characterization of the sunflower seed (Helianthus annuus L.) major aminopeptidase

The sunflower seed (Helianthus annuus L.) major peptidase was purified to molecular homogeneity. It is an 80 kDa enzyme with pI of 4.6 and optimal activity at pH 7.5–8.0 and 45–50°C. It is a thiol-dependent aminopeptidase hydrolyzing peptides in a step-by-step manner as cleaving after the N-terminal amino acid residue of the substrate. It requires substrate acyl parts with a free amino group in either α- or β-position and l-configuration of the adjacent carbon atom. The enzyme prefers amino acid residues with bulky hydrophobic side chains at P₁-position and its catalytic efficacy is affected by the structure of both P₁ and P₁′ parts of the substrate.     

The neuroprotective role of l-cysteine towards the effects of short-term exposure to lanthanum on the adult rat brain antioxidant status and the activities of acetylcholinesterase, (Na+,K+)- and Mg2+-ATPase

Lanthanum (La) is a rare earth element that is widely used for industrial, medical and agricultural purposes. Its neurotoxic effects are linked to its physical and chemical properties and its interaction with certain trace elements and membrane-bound enzymes. The aim of this study was to investigate the effects of short-term La-administration (as LaCl₃, 53 mg/kg) on the adult rat whole brain total antioxidant status (TAS) and the activities of acetylcholinesterase (AChE), Na⁺,K⁺-ATPase and Mg²⁺-ATPase, as well as the potential effect of the co-administration of the antioxidant l-cysteine (Cys, 7 mg/kg) on the above parameters. Twenty-eight male Wistar rats were divided into four groups: A (saline-treated control), B (La), C (Cys),and D (La and Cys). All rats were treated once daily with intraperitoneal injections of the tested compounds, for 1-week. Rats were sacrificed by decapitation and the above mentioned parameters were measured spectrophotometrically. Rats treated with La exhibited a significant reduction in brain TAS (−36%, P < 0.001, BvsA), that was partially limited by the co-administration of Cys (−13%, P < 0.01, DvsA), while Cys (group C) had no effect on TAS. The rat brain AChE activity was found significantly increased by both La (+23%, P < 0.001, BvsA) and Cys (+59%, P < 0.001, CvsA), while it was adjusted to control levels by the co-administration of La and Cys. The activity of rat brain Na⁺,K⁺-ATPase was significantly decreased by La-administration (−28%, P < 0.001, BvsA), while Cys supplementation could not reverse this decrease. The activity of Mg²⁺-ATPase exhibited a slight but statistically significant reduction due to La (−8%, P < 0.01, BvsA), that was further reduced by Cys co-administration (−25%, P < 0.001, DvsA). The above findings suggest that La short-term in vivo administration causes a statistically significant decrease in the rat brain TAS and an increase in AChE activity. Both effects can be, partially or totally, reversed into control levels by Cys co-administration, which could thus be considered for future applications as a neuroprotective agent against chronic exposure to La. The activities of Na⁺,K⁺- and Mg²⁺-ATPase that were inhibited by La, could not be reversed by Cys co-administration. A role for the already reported concentration-dependent interaction of La with Ca-binding sites (such as Ca²⁺-ATPase) might be considered for certain of the above phenomena.     

Health-related quality of life in the patients on maintenance hemodialysis: the analysis of demographic and clinical factors

Health-related quality of life (HRQoL) among hemodialysis (HD) patients recently became a nephrologist's focus of interest. HRQoL is an important predictor of outcome in HD patients and need to be regularly assessed. The aim of the present study was to compare the HRQoL of chronic HD patients with general population and to analyze influencing sociodemographic and clinical factors. We included 255 prevalent HD patients from four dialysis centers. HRQoL was measured with The Medical Outcomes Study Short Form 36 Health Survey Questionnaire (SF-36). This data were compared with control group (N = 132) from the general Croatian population. Comparisons of SF-36 scale scores of HD patients regarding demographic and clinical factors (age, gender, education level, dialysis vintage and diabetes) were also performed and analyzed with a multivariate regression analysis. HRQoL in prevalent HD patients was relatively low (mean Physical Component Summary, PCS = 33.7, mean Mental Component Summary, MCS = 43.0) and was lower compared to the control group from the general population in all HRQoL domains, PCS and MCS scores. Almost 53% of the HD patients had the critical score PCS < 43 + MCS < 51 as the predictor of death and hospitalization. Better HRQoL was revealed in the patients < 65 years old, males, patients with higher educational level and in the patients on maintenance HD less than one year. Age was the only statistically significant predictor of PCS and MCS. Developments of HD technology, treatment of comorbidities, continuous patients' education, social and psychological support and use of other renal replacement modalities, especially kidney transplantation, may improve the HRQoL in these patients.     

Differences in sexual functioning between patients with benign and malignant breast tumors

The aim of this study was to compare differences in sexual behavior between patients with benign and malignant breast tumors. A total of 187 patients treated for breast tumors (benign or malignant) at the General Hospital >Pozega<, Croatia, filled in the questionnaire between January 2001 and May 2003. Patients were asked to fill in the questionnaire one to ten years after treatment of breast tumor, while they were on their regular control visit. Deterioration in sexual life experienced 36.27% of patients with benign tumors and 51.76% of patients with malignant tumor (p<0.01). The main reason of sex life impairment in both groups was distortion of body image perception. Most of partners did not change their behavior toward women with breast tumors (48.72% for benign group and 41.82% or malignant group, p>0.05). A great amount of women in both groups felt certain change in her >body image<, but in greater extent in malignant group (41.18% vs. 25.49%), (p<0.05). From our results we can see that patients in this study do not recognize need for consultation with their physician regarding sex life after treatment of tumor (41.18% for benign and 35.29% in malignant group). It can be concluded that considerable amount of attention should be given to psychological aspects of recovery which can improve prognosis and quality of life in general.     

Comparative characteristics of chymotrypsin covalently bound on chemically and enzymatically oxidized agaroses

Summary The enzymatically activated agaroses compared with chemically activated show 25 fold lower amount of generated aldehyde groups, 33 fold lower binding capacity for chymotrypsin, 3 fold lower proteolytic as well as amidolytic activity toward AntAlaAlaPheNA of the corresponding fixed enzyme. Trans-cinnamoylimidazole titration data demonstrate 100% active bound enzyme in the case of enzymatically oxidized agaroses and 57% for chemically oxidized. The enzymic activation offers a small number of sites for ligand attachment in a unique microenvironment. The chemical activation yields a suitable matrix for effective chymotrypsin immobilization.     

p120RasGAP Is a Mediator of Rho Pathway Activation and Tumorigenicity in the DLD1 Colorectal Cancer Cell Line

KRAS is mutated in ∼40% of colorectal cancer (CRC), and there are limited effective treatments for advanced KRAS mutant CRC. Therefore, it is crucial that downstream mediators of oncogenic KRAS continue to be studied. We identified p190RhoGAP as being phosphorylated in the DLD1 CRC cell line, which expresses a heterozygous KRAS G13D allele, and not in DKO4 in which the mutant allele has been deleted by somatic recombination. We found that a ubiquitous binding partner of p190RhoGAP, p120RasGAP (RasGAP), is expressed in much lower levels in DKO4 cells compared to DLD1, and this expression is regulated by KRAS. Rescue of RasGAP expression in DKO4 rescued Rho pathway activation and partially rescued tumorigenicity in DKO4 cells, indicating that the combination of mutant KRAS and RasGAP expression is crucial to these phenotypes. We conclude that RasGAP is an important effector of mutant KRAS in CRC.     

Modified gateway system for double shRNA expression and Cre/lox based gene expression

Background  

The growing need for functional studies of genes has set the stage for the development of versatile tools for genetic manipulations.