Evaluation of cystine transport in cultured human kidney cells and establishment of cystinuria type I phenotype by antisense technology

Cystinuria is a rare hereditary disease resulting in recurrent stone formation and the need for repeated invasive interventions. So far, two responsible genes have been identified which encode the two transporters, rBAT and b^0,+AT forming a heterodimer to transport cystine in proximal tubular cells (PTC) and whose defect results in increased excretion of cystine. A human cell line mimicing the phenotype of cystinuria in vitro is yet to be developed. Human kidney (HK)-2 is a PTC line derived from normal HK. After determining the presence of rBAT gene by RT-PCR and Western blot analysis, radioactively labeled cystine (S³⁵) was used to evaluate the functional presence of the amino acid transport in HK-2 cells when cultured in vitro. To achieve a cystinuria type I phenotype in HK-2 cells, the rBAT gene was silenced using antisense oligonucleotides complimentary to human rBAT mRNA. The reduced transport activity of cystine was then determined by radiolabeled cystine uptake measurements. RT-PCR and Western blot confirmed the expression of the rBAT gene in HK-2 cells. Considerable transport of the radio labeled cystine was observed in HK-2 cells and was linearly dependent on the incubation time with the amino acid. The cystine transport in rBAT knockdown cells after incubation with antisense oligonucleotides was significantly lower compared to control (0.76 vs. 0.98%; P = 0.0008), proving a transient knock-down of the rBAT gene. This study demonstrates the presence of the b^0,+ amino acid transport system in human proximal tubular HK-2 cells when cultured in vitro. Inhibition of this transport system is possible by using antisense technology. A permanent inhibition of the cystine transport, based on our model, would be useful for the development and evaluation gene therapeutic approaches. Gunnar Wendt-Nordahl, Sreedhar Sagi contributed equally to this work.     

Effect of adrenergic stimulation on cutaneous microcirculation immediately after surgical adventitiectomy in a rat skin flap model

Chronic sympathetic denervation leads to the development of supersentivity to adrenergic agents. Free flap surgery results in the disruption of the autonomic nerve fibers running along the anastomosed vessels. We therefore investigated the early effect of surgical sympathectomy on the reactivity of cutaneous microcirculation challenged to adrenergic agents. Two epigastric flaps were elevated and exposed in 15 rats. On the right flap (Side A), a circular adventitiectomy of the feeder vessels was realized to provide surgical sympathectomy. On the left flap (Side N), vessels were kept intact. The following drugs were then given intravenously successively: phenylephrine (10 and 15 microg kg(-1)), norepinephrine (10 microg kg(-1)), prazocin (1 mg kg(-1)) followed by norepinephrine (10 microg kg(-1)). Cutaneous microcirculation was assessed using Laser-Doppler Flowmeters simultaneously on the two flaps after each drug administration. Mean arterial pressure was also measured. On side N, phenylephrine and norepinephrine resulted in a transient increase in cutaneous microcirculation followed by a more prolonged reduction. On side A, only the initial increase was observed, which was greater and longer as compared with side N, and paralleled the increase in mean arterial pressure. After prazocin pre-treatment, norepinephrine produced a transient increase in cutaneous microcirculation similar on both sides, and parallel to the changes in arterial pressure. No decrease in cutaneous microcirculation was observed. Immediately after surgical adventitiectomy, the vasoconstriction produced by alpha-adrenergic agents is prevented. No denervation-induced hypersentivity is observed. Surgical sympathectomy might protect cutaneous flaps from vasoconstriction induced by endogenous catecholamines release.     

Dietary cooked navy beans and their fractions attenuate colon carcinogenesis in azoxymethane-induced ob/ob mice

Based on the protective effects of cooked dry bean consumption in a human intervention study, we evaluated which fraction of cooked dry beans is responsible for its cancer-preventive effects. Cooked navy beans (whole beans), the insoluble fraction (bean residue) or soluble fraction of the 60% (vol:vol) ethanol extract of cooked navy beans (bean extract), or a modified AIN-93G diet (16.6% fat including 12.9% lard) as control diet were fed to 160 male obese ob/ob mice after 2 azoxymethane injections. In comparison to control-fed mice, dysplasia, adenomas, or adenocarcinomas were detected in fewer mice on either bean fraction diet (percent reduction from control: whole beans 54%, P=0.10; bean residue 81%, P=0.003; bean extract 91%, P=0.007), and any type of colon lesions, including focal hyperplasia, were found in fewer mice on each of the 3 bean diets percent reduction from control: whole bean 56%, P=0.04; bean residue 67%, P=0.01; bean extract 87%, P=0.0003. These results suggest that both the soluble and the insoluble fraction of the extract contribute to the cancer-protective effect of cooked navy beans.     

Violence risks in nursing--results from the European 'NEXT' Study

BACKGROUND: Recent research suggests that violence in health care is increasing and that it strongly influences the recruitment and retention of nurses as well as sick leave and burnout levels. AIMS: To identify the prevalence of violence in nursing and to provide a basis for appropriate interventions. METHODS: Nurses from 10 European countries answered to a questionnaire and to a follow-up assessment. Stepwise adjusted multiple logistic regression was used to assess the association between frequency of violence, factors related to teamwork and other work-related factors and outcomes, such as burnout, intention to leave nursing and intention to change institution. RESULTS: A total of 39,894 nurses responded to the baseline questionnaire (51% response rate). After adjustment for age, gender and other risk factors, quality of teamwork appeared to be a major factor with odds ratio (OR) 1.35 (1.24-1.48) for medium quality and 1.52 (1.33-1.74) for low quality. Uncertainty regarding patients' treatments was linked with violence, with a clear gradient (OR 1.59, 1.47-1.72 for medium uncertainty and 2.13, 1.88-2.41 for high uncertainty). Working only night shift was at high risk (OR 2.17, 1.76-2.67). High levels of time pressure and physical load were associated with violence OR 1.45 (1.24-1.69) and 1.84 (1.66-2.04), respectively. High and medium frequency of violence was associated with higher levels of burnout, intent to leave nursing and intent to change institution. A 1-year follow-up assessment indicated stability in the relationships between outcomes. CONCLUSION: This study supports efforts aimed at improving teamwork-related factors as they are associated with a decrease in violence against nurses.     

Follow-up of women after a first episode of postmenopausal bleeding and endometrial thickness greater than 4 millimeters

OBJECTIVE: To estimate the incidence of recurrent postmenopausal bleeding among women who were diagnosed with an endometrial thickness greater than 4 mm. METHODS: We designed a prospective cohort study and included consecutive women not using hormone replacement therapy, presenting with a first episode of postmenopausal bleeding. We evaluated patients who had an endometrial thickness greater than 4 mm at transvaginal ultrasonography and benign endometrial sampling; presence of carcinoma was ruled out by office endometrial sampling, hysteroscopy, and/or dilation and curettage. Time until recurrent bleeding was measured, and diagnosis at recurrent bleeding was recorded. RESULTS: Among 318 patients who had an endometrial thickness greater than 4 mm, 222 patients had benign histology results and were available for follow-up. During follow-up, 47 (21%, 95% confidence interval 16-27%) patients had recurrent bleeding, with a median time to recurrent bleeding of 49 weeks (interquartile range 18 to 86 weeks). There was no difference with respect to recurrence rate between patients with polyp removal, patients with a normal hysteroscopy, and patients with office endometrial sampling alone at the initial workup. Two patients were diagnosed with atypical endometrial hyperplasia upon recurrent bleeding. CONCLUSION: The recurrence rate of postmenopausal bleeding in women with endometrial thickness greater than 4 mm is 20%. This recurrence rate is not related to incorporation of hysteroscopy or polyp removal at the initial workup. LEVEL OF EVIDENCE: II.     

Up-regulation of the CYP1 family in rat and human liver by the aliphatic isothiocyanates erucin and sulforaphane

On the basis of animal studies, the chemopreventive activity of isothiocyanates has been linked to their ability to modulate carcinogen-metabolising enzyme systems, including cytochrome P450. However, the potential of isothiocyanates to influence these enzyme systems in human liver has not been investigated. We have evaluated the modulation of cytochrome P450 expression in two human liver samples by erucin and sulforaphane, in comparison to rat, following the incubation of precision-cut human and rat liver slices with the two isothiocyanates. Both compounds failed to influence cytochrome P450 activity, as exemplified by the dealkylations of methoxy-, ethoxy- and pentoxyresorufin, and benzyloxyquinoline, in either human or rat liver. Impairment of activity was, however, observed in some activities at high concentrations (50microM), which was attributed to toxicity. At the apoprotein level, however, both compounds markedly elevated CYP1A2/1B1 levels in rat liver, but in human liver only a modest increase was evident, and only in one of the livers. CYP3A2 apoprotein levels were modestly elevated in rat liver by both isothiocyanates both of which, however, failed to influence CYP3A4 expression in human liver. Neither isothiocyanate, in either rat or human liver, modulated CYP2B apoprotein levels. It may be inferred that (a) human and rat liver differ in their response to erucin and sulforaphane, (b) erucin and sulforaphane, despite being small molecular weight aliphatic compounds, up-regulate the CYP1 family but no increase in activity is observed as a result of mechanism-based inhibition, and (c) the chemopreventive effect of isothiocyanates, at dietary levels of intake, is unlikely to be due to inhibition of the cytochrome P450-mediated bioactivation of carcinogens.     

Melatonin receptors,heterodimerization, signal transduction and binding sites: what's new?

Melatonin is a neurohormone that has been claimed to be involved in a wide range of physiological functions. Nevertheless, for most of its effects, the mechanism of action is not really known. In mammals, two melatonin receptors, MT1 and MT2, have been cloned. They belong to the G-protein-coupled receptor (GPCR) superfamily. They share some specific short amino-acid sequences, which suggest that they represent a specific subfamily. Another receptor from the same subfamily, the melatonin-related receptor has been cloned in different species including humans. This orphan receptor also named GPR50 does not bind melatonin and its endogenous ligand is still unknown. Nevertheless, this receptor has been shown to behave as an antagonist of the MT1 receptor, which opens new pharmacological perspectives for GPR50 despite the lack of endogenous or synthetic ligands. Moreover, MT1 and MT2 interact together through the formation of heterodimers at least in cells transfected with the cDNA of these two receptors. Lastly, signalling complexes associated with MT1 and MT2 receptors are starting to be deciphered. A third melatonin-binding site has been purified and characterized as the enzyme quinone reductase 2 (QR2). Inhibition of QR2 by melatonin may explain melatonin's protective effect that has been reported in different animal models and that is generally associated with its well-documented antioxidant properties.