Progression of apoptic signaling from mesenteric ischemia-reperfusion injury to lungs: correlation in the level of ER chaperones expression.

Multiple organ dysfunction syndrome (MODS) is characterized by the development of probably reversible, progressive dysfunction of vital systems in two or more organs, directly undamaged by surgery or other trauma. The organs which have the most common potential dysfunction are lungs, liver, kidneys, heart and gastrointestinal tract. The small intestine is the source of production of proinflammatory mediators leading and contributing to multiorgan failure. The endoplasmic reticulum (ER), after ischemia and post-ischemic reperfusion, is significantly involved in the activation of enterocyte apoptosis. The purpose of this study was to determine the stage of apoptosis in the lungs, initiated through inflammatory response from the small intestine. We analyzed changes in mRNA levels of pro-apoptotic genes Gadd153 (Chop) and anti-apoptotic genes Grp78 (Bip) in the small intestine wall and lung parenchyma. During experimental procedure the rats underwent 60 min of ischemia, caused by complete occlusion of the mesenteric arteria cranialis, with subsequent reperfusion and evaluation after 1 h, 24 h and 30 days (from R1, R24 to R30, respectively, each group n = 8). The gene expression levels were measured using RT-PCR followed by electrophoresis and visualization under UV. In the lungs we detected significantly lower level of expression Grp78 by 45 ± 6.9%. This suggests that ischemic attack and subsequent reperfusion did not promote ER stress in the lungs through induction of Gadd153 expression in the small intestine. There is still no effective approach to the treatment of affected ischemic intestine tissue, to stop the processes with could eventually lead to MODS. Therefore it is necessary to study changes in the damaged tissue at the molecular level and try to suggest possible therapeutic defined routes to the protection of tissue.

Effects of borneol and thymoquinone on TNBS-induced colitis in mice.

Components of plant essential oils have been reported to have health benefit properties, including antioxidative, anti-tumour, antimicrobial, anti-stress, and immunomodulative activities. We examined the anti-inflammatory effects of thymoquinone, the active ingredient in the volatile oil of Nigella sativa seeds, and borneol, the active component of Salvia officinalis essential oil, on TNBS-induced colitis in mice. Thymoquinone was added to the commercial diet at a concentration of 0.05 % and borneol at two concentrations (0.09% and 0.18%) and fed to ICR mice 5 days before induction of TNBS colitis. Seven days after TNBS administration the mice were killed and macroscopic and histological scores were evaluated. Cytokine mRNA expression in colonic tissue was assessed using quantitative realtime RT-PCR. We did not detect any significant changes in macroscopic and histological scores between experimental and control groups, but we observed a significant decrease in proinflammatory cytokine (IL-1beta and IL-6) mRNA expression in colon tissue in the 0.09% and 0.18% borneol-treated groups of mice in comparison to the control group. Surprisingly, we were not able to confirm anti-inflammatory effects of thymoquinone in TNBS colitis. In conclusion, our data show that borneol is able to significantly suppress proinflammatory cytokine mRNA expression in colonic inflammation, although no significant morphological changes are visible.

Renal activity of Akt kinase in experimental Type 1 diabetes.

Akt kinase regulates numerous cell functions including glucose metabolism, cell growth, survival, protein synthesis, and control of local hemodynamics. mTOR is one of down-stream effectors of Akt involved in the initiation of protein translation. However, renal Akt signaling in Type 1 diabetes (DM) in vivo, in particular under the conditions reflecting differences in metabolic control, has received less attention. Renal cortical activity and expression of Akt and mTOR (kinase assay, western blotting) were determined in streptozotocin-diabetic rats (D) with different levels of glycemic control (blood glucose 22.0+/-1.0, 13.4+/-1.5, 8.1+/-0.4 mmol/l, p<0.05 between the groups), achieved by varying insulin treatment (0, 4 and 12 IU/day), and in control rats with (C4) or without (C) chronic insulin administration. Renal Akt activity was reduced in D rats without insulin treatment and severe hyperglycemia (D-0, -62 %, p<0.01 vs. C), partially restored in moderately hyperglycemic rats (D-4, -30 %, p<0.05 vs. C), and normalized in D rats with intensive insulin and tight metabolic control (D-12). Expression of active mTOR paralleled Akt activity in D-0 (-51 %, p<0.01 vs. C), but not in D-4 and D-12 that demonstrated increases in active mTOR (+55 %, +80 % resp., p<0.05) as compared to C. Moreover, insulin activated renal Akt (+82 %, p<0.01), but not mTOR in C4. In conclusion, glycemic control and intensity of insulin treatment are important modulators of renal Akt and mTOR activity in diabetes. While Akt activity is reversible by tight metabolic control, combination of hyperglycemia and insulin treatment resulted in enhancement of mTOR activity. In addition to Akt, other signaling pathways likely contribute to regulation of renal mTOR activity in diabetes.

Cellular and subcellular localization of stathmin during oocyte and preimplantation embryo development.

Stathmin is a 19 kDa cytosolic phosphoprotein, proposed to act as a relay integrating diverse intracellular signaling pathways involved in regulation of cell proliferation, differentiation, and function. To gain further information about its significance during early development, we analyzed stathmin expression and subcellular localization in mouse oocytes and preimplantation embryos. RT-PCR analysis revealed a low expression of stathmin mRNA in unfertilized oocytes and a higher expression at the blastocyst stage. A fine cytoplasmic punctuate fluorescent immunoreactive stathmin pattern was detected in the oocyte, while it evolved toward an increasingly speckled pattern in the two-cell and later four- to eight-cell embryo, with even larger speckles at the morula stage. In blastocysts, stathmin immunoreactivity was fine and intense in inner cell mass cells, whereas it was low and variable in trophectodermal cells. Electron microscopic analysis allowed visualization with more detail of two types of stathmin immunolocalization: small clusters in the cytoplasm of oocytes and blastocyst cells, together with loosely arranged clusters around the outer membrane of cytoplasmic vesicles, corresponding to the immunofluorescent speckles in embryos until the morula stage. In conclusion, it appears from our results that maternal stathmin is accumulated in the oocyte and is relocalized within the oocyte and early preimplantation embryonic cell cytoplasm to interact with specific cytoplasmic membrane formations. Probably newly synthesized, embryonic stathmin is expressed in the blastocyst, where it is localized more uniformly in the cytoplasm mostly of inner cell mass (ICM) cells. These expression and localization patterns are probably related to the particular roles of stathmin at the successive steps of oocyte maturation and early embryonic development. They further support the proposed physiologic importance of stathmin in essential biologic regulation.

A modified method of intracytoplasmic sperm injection without the use of polyvinylpyrrolidone.

In a controlled study we compared the outcome of intracytoplasmic sperm injection (ICSI) performed by two different methods. The oocytes from 20 patients were equally divided into two groups and injected either by conventional ICSI using polyvinylpyrrolidone (PVP) or by a modified PVP-free ICSI procedure. While in the conventional ICSI method the spermatozoon is aspirated into the injection pipette, in the modified ICSI procedure the spermatozoon is attached to the end of the narrow micropipette by aspirating its tail. The sperm head is never drawn into the pipette. Accordingly, even a fast-moving spermatozoon can be 'caught' easily. As a result of such an aspiration the spermatozoon loses its motility. Therefore, PVP is required neither to slow down the movement of the spermatozoon nor to facilitate the movement of the solution in the injection pipette. A total of 230 mature oocytes were injected by both methods and the results were analysed. No differences were observed in survival rate between the two ICSI procedures (89% and 91%, respectively). However, the proportion of normally fertilized oocytes was significantly higher after microfertilization by modified ICSI (74%) when compared with the outcome of the conventional ICSI method (62%). The frequency of abnormal fertilization was not influenced by the method of ICSI used. The cleavage rate and quality of resulting embryos were also comparable. In conclusion, we have demonstrated a modified ICSI method which does not require the use of PVP. When compared with the conventional ICSI procedure, even better fertilization rates can be achieved. The proposed ICSI modification may provide an alternative procedure for elimination of the potentially harmful effects which may be associated with conventional ICSI.

Increased cell death in rat blastocysts exposed to maternal diabetes in utero and to high glucose or tumor necrosis factor-alpha in vitro.

The morphogenetic function of the transient phase of cell death that occurs during blastocyst maturation is not known but it is thought that its regulation results from a delicate balance between survival and lethal signals in the uterine milieu. In this paper, we show that blastocysts from diabetic rats have a higher incidence of dead cells than control embryos. Differential lineage staining indicated that increased nuclear fragmentation occurred mainly in the inner cell mass. In addition, terminal transferase-mediated dUTP nick end labeling (TUNEL) demonstrated an increase in the incidence of non-fragmented DNA-damaged nuclei in these blastocysts. Analysis of the expression of clusterin, a gene associated with apoptosis, by quantitative reverse transcription-polymerase chain reaction detected an increase in the steady-state level of its transcripts in blastocysts from diabetic rats. In situ hybridization revealed that about half the cells identified as expressing clusterin mRNA exhibited signs of nuclear fragmentation. In vitro experiments demonstrated that high D-glucose increased nuclear fragmentation, TUNEL labeling and clusterin transcription. Tumor necrosis factor-alpha (TNF-alpha), a cytokine whose synthesis is up-regulated in the diabetic uterus, did not induce nuclear fragmentation nor clusterin expression but increased the incidence of TUNEL-positive nuclei. The data suggest that excessive cell death in the blastocyst, most probably resulting from the overstimulation of a basal suicidal program by such inducers as glucose and TNF-alpha, may be a contributing factor of the early embryopathy associated with maternal diabetes.

Prevalence of psychopathology in patients suffering from breast and gastrointestinal cancer.

Prevalence of psychopathology in 107 in- and outpatients suffering from cancer was assessed by means of self-rating inventory (Symptom Check List (SCL-90)) and an interview. There were 86 women with breast cancer and 21 patients with gastrointestinal cancer (9 women and 12 men). Inventory was administered once after surgery. Psychopathology exceeding so called mean borderline values derived from control samples ranged from 2% to 33% of the patients in particular dimensions of the inventory. Maximal mean values of the psychopathology were found in the dimensions of somatisation, depression, anxiety, phobic anxiety, hostility and of items not included, reflecting mostly sleep and eating distortions. Mean values of the psychopathology were insignificantly lower in patients after mastectomy, compared with gastrointestinal cancer patients. No significant differences between initial (I and II) and advanced (III and IV) stages of the illness were achieved in the patients from both investigated samples. Screened patients are suitable for psychiatric or psychologic therapeutic intervention.

Neutralization of tumor necrosis factor alpha (TNF alpha) action on cell proliferation in rat blastocysts by antisense oligodeoxyribonucleotides directed against TNF alpha p60 receptor.

Antisense oligodeoxyribonucleotide inhibition of gene expression was used to test whether the p60 form of the tumor necrosis factor alpha (TNF alpha) receptor (Rp60) is responsible for mediating the negative effect of TNF alpha on the development of rat blastocysts in vitro. The antisense oligonucleotide was designed to overlap the translation initiation codon of the TNF alpha Rp60 mRNA. Preliminary experiments showed that concentrations of oligonucleotides above 10 microM in the culture medium were embryotoxic over 24 h. When used at nontoxic concentrations (8 microM), antisense oligonucleotides specifically decreased the abundance of intact TNF alpha Rp60 transcripts by 80% within 3 h of exposure. In contrast to results with control embryos, mRNA for the second form of TNF alpha receptor, TNF alpha Rp80, was detected in blastocysts exposed to antisense oligonucleotides to TNF alpha Rp60. Antisense oligonucleotides to TNF alpha Rp60 blocked the 25-30% decrease in cell proliferation induced by 50 ng/ml TNF alpha added to a standard culture medium and by 5 ng/ml TNF alpha added to a medium that had been conditioned by rat uterine cells. Sense oligonucleotides had no such protective effect. Because uterine cells from diabetic rats secrete higher levels of TNF alpha than those from control rats, antisense oligonucleotides were also tested in a medium that had been conditioned by diabetic uterine cells (cell-secreted TNF alpha concentration was 50 pg/ml in this medium, and no exogenous TNF alpha was added). Addition of antisense oligonucleotides to TNF alpha Rp60 improved the quality of this medium with respect to cell proliferation but failed to correct the high frequency of dead cells observed in the blastocysts.

Influence of 5-[2-(N,N-dimethylamino)ethoxy]-7-oxo-7H-benzo(C)fluorene hydrochloride (benflurone) on the activity of rat liver aspartate and alanine aminotransferases.

The influence of repeated s.c. administration of the cytostatic 5-[2-(N,N-dimethylamino)ethoxy]-7-oxo-7H-benzo(c)fluorene hydrochloride (benflurone, 25 mg/kg body weight daily) on the activities of aspartate and alanine aminotransferases (AST, ALT) per g of tissue, and protein concentration in the liver of adult male rats was studied. During the first week of benflurone administration, the activities of ALT and AST decreased by 2/3 and 1/3, respectively, in comparison with controls while the protein concentration did not show any substantial change. No in vitro influence of benflurone on AST and ALT was found even at the highest concentration tested (10(-4) M). The significance of the aminotransferase decrease after treatment with benflurone and possible participation of these changes in the side- or cytostatic effects of the compound are considered.