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The preservation of genome integrity during sperm and egg development is vital for reproductive success. During meiosis, the tumor suppressor BRCA1/BRC-1 and structural maintenance of chromosomes 5/6 (SMC-5/6) complex genetically interact to promote high fidelity DNA double strand break (DSB) repair, but the specific DSB repair outcomes these proteins regulate remain unknown. Using genetic and cytological methods to monitor resolution of DSBs with different repair partners in Caenorhabditis elegans , we demonstrate that both BRC-1 and SMC-5 repress intersister crossover recombination events. Sequencing analysis of conversion tracts from homolog-independent DSB repair events further indicates that BRC-1 regulates intersister/intrachromatid noncrossover conversion tract length. Moreover, we find that BRC-1 specifically inhibits error prone repair of DSBs induced at mid-pachytene. Finally, we reveal functional interactions of BRC-1 and SMC-5/6 in regulating repair pathway engagement: BRC-1 is required for localization of recombinase proteins to DSBs in smc-5 mutants and enhances DSB repair defects in smc-5 mutants by repressing theta-mediated end joining (TMEJ). These results are consistent with a model in which some functions of BRC-1 act upstream of SMC-5/6 to promote recombination and inhibit error-prone DSB repair, while SMC-5/6 acts downstream of BRC-1 to regulate the formation or resolution of recombination intermediates. Taken together, our study illuminates the coordinated interplay of BRC-1 and SMC-5/6 to regulate DSB repair outcomes in the germline., Competing Interests: ET, AS, DA, JB, CR, NK, OR, DL No competing interests declared, (© 2024, Toraason et al.)

In sexually reproducing organisms, germ cells faithfully transmit the genome to the next generation by forming haploid gametes, such as eggs and sperm. Although most meiotic proteins are conserved between eggs and sperm, many aspects of meiosis are sexually dimorphic, including the regulation of recombination. The synaptonemal complex (SC), a large ladder-like structure that forms between homologous chromosomes, is essential for regulating meiotic chromosome organization and promoting recombination. To assess whether sex-specific differences in the SC underpin sexually dimorphic aspects of meiosis, we examined Caenorhabditis elegans SC central region proteins (known as SYP proteins) in oogenesis and spermatogenesis and uncovered sex-specific roles for the SYPs in regulating meiotic recombination. We find that SC composition, specifically SYP-2, SYP-3, SYP-5, and SYP-6, is regulated by sex-specific mechanisms throughout meiotic prophase I. During pachytene, both oocytes and spermatocytes differentially regulate the stability of SYP-2 and SYP-3 within an assembled SC. Further, we uncover that the relative amount of SYP-2 and SYP-3 within the SC is independently regulated in both a sex-specific and a recombination-dependent manner. Specifically, we find that SYP-2 regulates the early steps of recombination in both sexes, while SYP-3 controls the timing and positioning of crossover recombination events across the genomic landscape in only oocytes. Finally, we find that SYP-2 and SYP-3 dosage can influence the composition of the other SYPs in the SC via sex-specific mechanisms during pachytene. Taken together, we demonstrate dosage-dependent regulation of individual SC components with sex-specific functions in recombination. These sexual dimorphic features of the SC provide insights into how spermatogenesis and oogenesis adapted similar chromosome structures to differentially regulate and execute recombination., Competing Interests: CC, CR, AD, DL No competing interests declared, (© 2023, Cahoon et al.)

Lipid vesicles are known to undergo complex conformational transitions, but it remains challenging to systematically characterize nonequilibrium membrane dynamics in flow. Here, we report the direct observation of anisotropic vesicle relaxation from highly deformed shapes using a Stokes trap. Vesicle shape relaxation is described by two distinct characteristic timescales governed by the bending modulus and membrane tension. Interestingly, the fast double-mode timescale is found to depend on vesicle deflation or reduced volume. Experimental results are well described by a viscoelastic model of a deformed membrane. Overall, these results show that vesicle relaxation is governed by an interplay between membrane elastic moduli, surface tension, and vesicle deflation.

Lipid vesicles play a key role in fundamental biological processes. Despite recent progress, we lack a complete understanding of the non-equilibrium dynamics of vesicles due to challenges associated with long-time observation of shape fluctuations in strong flows. In this work, we present a flow-phase diagram for vesicle shape and conformational transitions in planar extensional flow using a Stokes trap, which enables control over the center-of-mass position of single or multiple vesicles in precisely defined flows [A. Shenoy, C. V. Rao and C. M. Schroeder, Proc. Natl. Acad. Sci. U. S. A., 2016, 113(15), 3976-3981]. In this way, we directly observe the non-equilibrium conformations of lipid vesicles as a function of reduced volume ν, capillary number Ca, and viscosity contrast λ. Our results show that vesicle dynamics in extensional flow are characterized by the emergence of three distinct shape transitions, including a tubular to symmetric dumbbell transition, a spheroid to asymmetric dumbbell transition, and quasi-spherical to ellipsoid transition. The experimental phase diagram is in good agreement with recent predictions from simulations [V. Narsimhan, A. P. Spann and E. S. Shaqfeh, J. Fluid Mech., 2014, 750, 144]. We further show that the phase boundary of vesicle shape transitions is independent of the viscosity contrast. Taken together, our results demonstrate the utility of the Stokes trap for the precise quantification of vesicle stretching dynamics in precisely defined flows.

Assembly of the endosomal sorting complex required for transport (ESCRT)-III executes the formation of intralumenal vesicles (ILVs) at endosomes. Repeated cycles of ESCRT-III function requires disassembly of the complex by Vps4, an ATPase with a microtubule interaction and trafficking (MIT) domain that binds MIT-interacting motifs (MIM1 or MIM2) in ESCRT-III subunits. We identified a putative MIT domain at the N-terminus of Doa4, which is the ubiquitin (Ub) hydrolase in Saccharomyces cerevisiae that deubiquitinates ILV cargo proteins. The Doa4 N-terminus is predicted to have the α-helical structure common to MIT domains, and it binds directly to a MIM1-like sequence in the Vps20 subunit of ESCRT-III. Disrupting this interaction does not prevent endosomal localization of Doa4 but enhances the defect in ILV cargo protein deubiquitination observed in cells lacking Bro1, which is an ESCRT-III effector protein that stimulates Doa4 catalytic activity. Deletion of the BRO1 gene (bro1Δ) blocks ILV budding, but ILV budding was rescued upon disrupting the interaction between Vps20 and Doa4. This rescue in ILV biogenesis requires Doa4 expression but is independent of its Ub hydrolase activity. Thus, binding of Vps20 to the Doa4 N-terminus inhibits a non-catalytic function of Doa4 that promotes ILV formation.

UBXD8 is a membrane-embedded recruitment factor for the p97/VCP segregase that has been previously linked to endoplasmic reticulum (ER)-associated degradation and to the control of triacylglycerol synthesis in the ER. UBXD8 also has been identified as a component of cytoplasmic lipid droplets (LDs), but neither the mechanisms that control its trafficking between the ER and LDs nor its functions in the latter organelle have been investigated previously. Here we report that association of UBXD8 with the ER-resident rhomboid pseudoprotease UBAC2 specifically restricts trafficking of UBXD8 to LDs, and that the steady-state partitioning of UBXD8 between the ER and LDs can be experimentally manipulated by controlling the relative expression of these two proteins. We exploit this interaction to show that UBXD8-mediated recruitment of p97/VCP to LDs increases LD size by inhibiting the activity of adipose triglyceride lipase (ATGL), the rate-limiting enzyme in triacylglycerol hydrolysis. Our findings show that UBXD8 binds directly to ATGL and promotes dissociation of its endogenous coactivator, CGI-58. These data indicate that UBXD8 and p97/VCP play central integrative roles in cellular energy homeostasis.

Background: Most classification tables of cardiorespiratory fitness (CRF) used in clinical practice are international and have not been validated for the Brazilian population. That can result in important discrepancies when that classification is extrapolated to our population., Objective: To assess the use of major CRF tables available in a Brazilian population sample of the Central High Plan of the state of Rio Grande do Sul (RS)., Methods: This study assessed the retrospective data of 2,930 individuals, living in 36 cities of the Central High Plan of the state of RS, and considered the following: presence of risk factors for cardiovascular disease and estimated maximum oxygen consumption (VO2peak) values obtained through exercise test with Bruce protocol. To classify CRF, the individuals were distributed according to sex, inserted in their respective age groups in the Cooper, American Heart Association (AHA) and Universidade Federal de São Paulo (Unifesp) tables, and classified according to their VO2peak., Results: Women had lower VO2peak values as compared with those of men (23.5 ± 8.5 vs. 31.7 ± 10.8 mL.kg-1.min-1, p < 0.001). Considering both sexes, VO2peak showed an inverse and moderate correlation with age (R = -0.48, p < 0.001). An important discrepancy in the CRF classification levels was observed between the tables, ranging from 49% (Cooper x AHA) to 75% (Unifesp x AHA)., Conclusion: Our findings indicate important discrepancy in the CRF classification levels of the tables assessed. Future studies could assess whether international tables could be used for the Brazilian population and populations of different regions of Brazil.

Proteins that fail to correctly fold or assemble into oligomeric complexes in the endoplasmic reticulum (ER) are degraded by a ubiquitin- and proteasome-dependent process known as ER-associated degradation (ERAD). Although many individual components of the ERAD system have been identified, how these proteins are organized into a functional network that coordinates recognition, ubiquitylation and dislocation of substrates across the ER membrane is not well understood. We have investigated the functional organization of the mammalian ERAD system using a systems-level strategy that integrates proteomics, functional genomics and the transcriptional response to ER stress. This analysis supports an adaptive organization for the mammalian ERAD machinery and reveals a number of metazoan-specific genes not previously linked to ERAD.

Background: Some normotensive sedentary individuals have an exaggerated increase in blood pressure (BP) during physical activity, which is a behavior that is called blood pressure hyper-reactivity., Objective: To investigate the effect of an aerobic exercise program (AEP) on blood pressure in individuals with blood pressure hyper-reactivity (BPH)., Methods: Ten male volunteers with BPH, aged 45 ± 10 years, referred to as the experimental group (EG), took part in an AEP on a treadmill, 3 times a week for two months. They were compared to 14 men with BPH, aged 48 ± 8 years, referred to as the control group (CG), who remained sedentary. The subjects were evaluated before and after the AEP by stress test for comparison purposes. We evaluated the initial, peak and test-end heart rates (HR), systolic blood pressure (SBP) and diastolic blood pressure (DBP)., Results: There a significant decrease in the initial SBP (-5%; p=0.01), initial DBP (-4.6%; p=0.01), peak SBP (-12.4%; p=0.001), peak DBP (-14.7%; p=0.03), final SBP (-4.6%, p=0.03) in the EG. The CG continued with its hyper-reactive behavior, which evolved to more exaggerated levels when the results before and after the study were compared (p<0.04). In the HR, there was an increase only in the final HR, of 11.3 bpm, after training (p=0.02). The test-peak VO2 increased by 4.4 ml.kg(-1) x min(-1) in the EG (p=0.01) and remained similar in the CG., Conclusion: The AEP normalized the behavior of the blood pressure hyper-reactivity in sedentary men.

Background: The effect of exercise on blood pressure (BP) is already known; however, the dose-response curve of the hypotensive effect of exercise in hypertensive individuals is yet to be clarified., Objective: To evaluate the dose-response curve of the number of sessions that are necessary to cause a hypotensive effect in hypertensive individuals., Methods: 88 individuals, aged 58 +/- 11 years, divided in Experimental group (EG), with 48 that participated in a physical exercise program (PEP), which consisted of 40 minutes of aerobic exercises performed 3x/week, for 3 months, at 70% of the VO2max, and muscular exercises at 40% of the maximal voluntary contraction (MVC) and Control Group (CG) with 40 individuals that did not participate in the PEP. The systolic (SAP) and diastolic (DAP) arterial pressures were measured before each of the 36 sessions in the EG and assessed by ambulatory blood pressure monitoring (ABPM) in the CG. Differences in BP, the variation rate (D%) and the maximum hypotensive effect (MHE%) were observed between sessions. The data were expressed as means +/- SD; the t test and correlation were used, with p<0.05 being considered significant., Results: There was no difference regarding BP values in the CG. The EG showed an important decrease of 15 mmHg in SAP and 7 mmHg in DAP, with a large part of this effect occurring as early as the first session and the majority up to the 5th session. There was a strong inverse correlation (R:-0.66) with the number of sessions., Conclusion: An important hypotensive effect was observed from the 1st session on and it was observed that the dose-response curve can be abrupt and decrescent, instead of flat.

The association between the peroxisome proliferator-activated receptor (PPAR)gamma2 Pro12Ala polymorphism and insulin resistance is reported to depend on low birth weight. Low birth weight itself has been linked to type 2 diabetes and cardiovascular diseases in adulthood. We assessed whether the PPARgamma2 Pro12Ala polymorphism determines body size at birth and whether metabolic differences between the genotypes are already detectable in the newborn. This study was conducted at the obstetrics department of the Charité, Berlin, Germany. One thousand nine hundred thirty white woman/child pairs were consecutively included and genotyped. The newborn's weight, length, and head circumference were measured. Total glycated hemoglobin in blood served as a surrogate of fetal insulin resistance and glucose use. We found that neither the fetal nor the maternal Pro12Ala genotype determined body size or total glycated hemoglobin at birth. The results suggest that the PPARgamma2 Pro12Ala polymorphism is not relevant for intrauterine growth. Previously reported effects of PPARgamma2 Pro12Ala on insulin resistance seem to arise later in life.

Endothelin (ET)-1 is a potent vasoconstrictor with profibrotic and proinflammatory effects. Increasing evidence suggests that ET-1 and its cognate receptors are involved in a variety of progressive renal disorders, including diabetes, hypertension and glomerulonephritis. Several laboratory studies have demonstrated elevated expression of ET-1, which colocalizes with glomerular and tubulointerstitial injury, in addition to enhanced urinary excretion. Moreover, ET-1 expression correlates with disease severity and renal function. With the availability of ET receptor antagonists, a pathogenetic role has been further corroborated in animal models, demonstrating both structural and functional improvement. Thus, antagonizing the ET system may be useful in major renal pathologies associated with glomerular and tubulointerstitial damage.

Background: The mechanisms responsible for the pathogeneses of gestational hypertension and preeclampsia are unclear. Tumor necrosis factor-alpha (TNF-alpha) is a pro-inflammatory Th(1)-type cytokine. TNFA gene is located in the human leukocyte antigen (HLA) class III region of the major histocompatibility complex (MHC) on chromosome 6. The high TNF-alpha mRNA expression may be associated with the TNF2 (A) allele, which is the polymorphism of TNF-alpha at position -308 in promoter region. This study assessed whether the TNF2 (A) allele at position -308 plays a role in the alteration of blood pressure (BP) and urinary protein excretion during pregnancy., Methods: The original prospective cohort study comprised 1623 pregnant women from January 2000 to October 2001. The G/A polymorphism was done by restriction fragment length polymorphism (RFLP) analysis with Nco I enzyme., Results: The distributions of the G/A polymorphism of TNF-alpha in the promoter region at position -308 were wild-type 72.4% and variant 27.6%, respectively. The frequency of TNF2 (A) allele was approximately 0.15 for Caucasian pregnant women in the study. It was not significantly different in the distributions of genotypes and G/A allele frequencies among the three groups of pregnant women with gestational hypertension, preexisting hypertension and normal blood pressure (P > 0.05). The maternal blood pressure in the third trimester was significantly higher in the group of women possessing the TNF2 (A) allele compared to homozygous for the TNF1 (G) allele (systolic BP, P < 0.01 and diastolic BP, P < 0.05). The elevated blood pressure in the TNF2 (A) group was accompanied by higher urinary protein excretion in the third trimester (P < 0.05). The blood pressure and urinary protein excretion did not change apparently between the two groups in the first and second trimesters (P > 0.05)., Conclusions: Maternal TNF2 (A) allele of TNF-alpha promoter region at position -308 could play a role in the alteration of blood pressures and/or enhancement of urinary protein excretion during pregnancy, and might play an important role in the development of both gestational hypertension and preeclampsia.

Background: Although endothelin I (ET-1) is a very potent vasoconstrictor, ET-1 transgenic (ET-1 tg) mice are not hypertensive. This might be due to higher bioavailability of nitric oxide (NO) in ET-1 tg, which counteracts the effect of vasoconstrictors. We hypothesized lower angiotensin II (Ang II) sensitivity of afferent arterioles in ET-1 tg., Methods: Afferent arterioles were manually dissected and microperfused. Changes of the luminal diameter due to application of vasoactive substances were used for assessment of the reactivity of afferent arterioles. We investigated the effect of L-NAME, an unspecific NO synthase inhibitor, on basal tone, and the sensitivity of afferent arterioles to Ang II with and without pre-treatment with L-NAME. The renin-angiotensin-system was characterized by expression analysis of angiotensin-receptors and renin at the mRNA level., Results: L-NAME reduced afferent arterioles diameters similarly in ET-1 tg and wild-types (WT). Ang II sensitivity determined by calculation of EC50 for Ang II was less in ET-1 tg compared with WT (P<0.05). Ang II reduced luminal diameters to a lesser extent in ET-1 tg compared to WT (P<0.05). After pre-treatment with L-NAME, Ang II sensitivity and maximum constriction of afferent arterioles were similar in ET-1 tg and WT. The expression of renin- and Ang II-receptor-mRNA in the kidney did not differ between either group., Conclusion: The loss of differences in the maximum constriction and Ang II sensitivity of afferent arterioles between ET-1 tg and WT in the absence of NO suggests pronounced NO effects in afferent arterioles of ET-1 tg. This might contribute to the maintenance of normal renal arteriolar tone in ET-1 tg mice.

Objective: Vitamin D may contribute to cardiovascular disease in the absence of hypercalcemia in patients with chronic kidney disease., Methods: We investigated the effects of long-term (6-week) treatment with 1,25(OH)2D3, at a non-hypercalcemic dosage (0.25 microg/kg per day per orally) in 5/6 nephrectomized rats: (i) vehicle-treated, sham-operated rats; (ii) 1,25(OH)2D3-treated, sham-operated rats; (iii) vehicle-treated, 5/6 nephrectomized rats; and (iv) 1,25(OH)2D3-treated, 5/6 nephrectomized rats., Results: Creatinine clearance after 6 weeks was significantly lower and parathyroid hormone levels were significantly higher in 1,25(OH)2D3-treated uremic rats, compared with uremic controls (P < 0.01). Serum calcium levels, as well as the calcium-phosphorus product, did not differ between both groups. Mean systolic blood pressure in 1,25(OH)2D3-treated animals was significantly increased, compared with vehicle (each P < 0.01). In addition, 1,25(OH)2D3-treated uremic animals showed left ventricular hypertrophy. Diffuse aortic calcification involving the intima and media layer occurred in 1,25(OH)2D3-treated uremic animals, but not in other groups. The mean aortic wall area and lumen area were increased two-fold in 1,25(OH)2D3-treated uremic animals compared with vehicle (P < 0.01), whereas the wall/lumen ratio remained unchanged, indicating fusiform aneurysm formation., Conclusions: Hypertension, left ventricular hypertrophy, aortic calcification, and aneurysm, without hypercalcemia, occurred in 1,25(OH)2D3-treated, 5/6 nephrectomized rats. These data indicate a permissive effect of uremia for cardiovascular damage induced by non-hypercalcemic doses of 1,25(OH)2D3.

Background: Rodents without a functional endothelin B (ETB) receptor develop salt-sensitive hypertension. The underlying mechanisms, however, are so far unknown. The ETB receptor is involved in endothelial function by modulating the activity of the endothelial nitric oxide synthesis as well as contributing to the control of endothelial prostacyclin synthesis. In the present study, we analysed whether salt alters endothelial function in rescued ETB receptor-deficient mice. We used mice with a rescue of the lethal phenotype of an ETB knockout. These mice were generated by crossbreeding ETB mice with dopamine-hydroxylase ETB transgenic mice., Methods: Adult rescued ETB-deficient mice were kept in parallel with wild-type control animals for 15 days on standard (0.2% NaCl) or salt-enriched (4% NaCl) chow, respectively. Systolic blood pressure was measured by the tail cuff method and endothelium-dependent and endothelium-independent vascular function was assessed in isolated aortic rings under isometric conditions., Results: Systolic blood pressure increased on salt-enriched chow in ETB receptor-deficient mice (166 +/- 12 mmHg), but neither in wild-type mice on high-salt diet (128 +/- 11 mmHg; P < 0.05) nor in ETB receptor-deficient mice on standard chow. The heart rate was similar in all groups at any point of time. Endothelium-dependent relaxation was impaired in ETB receptor-deficient mice (74 +/- 3 versus 96 +/- 5% of preconstriction for wild-type mice; P < 0.05) and was not significantly affected by a salt-enriched diet. Endothelium-independent relaxation was similar among all groups. Contractions to endothelin-1 were not significantly influenced by preincubation with the ETB receptor antagonist BQ-788, but were completely blunted by preincubation with the ETA receptor antagonist BQ-123 in all animals., Conclusion: Rescued ETB receptor-deficient mice develop salt-sensitive hypertension. Nevertheless, in this animal model of ETB receptor deficiency, endothelial function is impaired independent of salt-enriched diet or hypertension. This indicates that, in this model, salt-induced hypertension is not mediated by endothelial dysfunction.

Diabetic nephropathy is a serious complication of diabetes associated with a poor prognosis which deteriorates to end-stage renal disease. Increased urinary excretion of protein and albumin are early clinical markers for diabetic renal disease and increased risk of cardiovascular disease. Diabetes causes activation of the renal endothelin system inducing renal damage. We analyzed the effects of SLV306, an inhibitor of both neutral endopeptidase and endothelin-converting enzyme, on diabetes-induced alterations of kidney function and morphology in rats with streptozotocin-induced diabetes. The effects of SLV306 (30 mg/kg per day), captopril (10 mg/kg per day), and placebo on urinary protein and albumin excretion as well as on blood pressure were studied in diabetic rats in comparison to non-diabetic control rats. The rats were treated for 20 weeks. At the end of the study kidney morphology was also analyzed using computer-aided image analysis systems. Serum glucose and blood pressure were similar in all diabetic groups. No side-effects were observed with SLV306 and captopril treatment. Protein excretion was 17.3 +/- 3.0 mg/24 hours in untreated diabetic rats. Protein excretion decreased significantly in the SLV306 (4.8 +/- 0.9 mg/24 hours; P = 0.03 vs untreated diabetic rats) as well as in the captopril (5.1 +/- 1.0 mg/24 hours; P = 0.03 vs untreated diabetic rats) -treated diabetic rats. Albumin excretion was 0.51 +/- 0.12 mg/24 hours in the untreated diabetic group and decreased likewise in the SLV306-treated diabetic rats (0.09 +/- 0.03 mg/24 hours; P = 0.04 vs untreated diabetic rats). The captopril-treated diabetic rats showed a strong trend towards reduced albumin excretion (0.12 +/- 0.04 mg/24 hours; P = 0.06 vs untreated diabetic rats). Computer-aided image analysis revealed that renal interstitial matrix content was significantly decreased in diabetic rats treated with either the angiotensin-converting enzyme inhibitor or the neutral endopeptidase/endothelin-converting enzyme inhibitor as compared to untreated diabetic rats. It was found that SLV306 decreases renal matrix protein content as well as protein and albumin excretion in diabetic rats independent of blood pressure. These effects are comparable to those of angiotensinconverting enzyme inhibition.

Background: It has been shown that the macula densa participates in the regulation of increased renin expression in two-kidney one-clip (2K1C) renovascular hypertension. Prostaglandins might be one of the mediators of macula densa function, because the cyclooxygenase-2 (COX-2), one of the rate-limiting enzymes of the prostaglandin pathway, is upregulated in 2K1C renovascular hypertensive rats. We tested the effect of chronic COX-2 inhibition on blood pressure, urinary aldosterone excretion and kidney morphology, as well as kidney function., Methods: Four groups were established: two groups of 2K1C renovascular hypertensive rats treated with the specific COX-2 inhibitor Celecoxib (cele) (15 mg/kg per day) or placebo immediately after operation, and two sham-operated control groups fed with Celecoxib or placebo., Results: Long-term COX-2 inhibition in 2K1C renovascular hypertensive rats did not alter blood pressure at any point of time. Urinary aldosterone excretion was elevated by clipping the renal artery (2K1C, 8.1 +/- 1.9, versus controls, 3.6 +/- 0.5 ng/24 h; P = 0.05) but was not influenced by treatment with Celecoxib. Also, Celecoxib treatment did not alter glomerular filtration rate (GFR), serum sodium, serum creatinine, serum urea or proteinuria in 2K1C renovascular hypertensive rats. Interstitial fibrosis of the left clipped kidney was markedly reduced (2K1C, 6.19 +/- 0.83% versus 2K1C + cele 3.00 +/- 0.68% of total area; P = 0.012), whereas the interstitial fibrosis of the non-clipped kidney or the glomerulosclerosis of both kidneys were not affected by Celecoxib treatment., Conclusions: Celecoxib reduces the interstitial fibrosis of the clipped kidney. Blood pressure, urinary aldosterone excretion or whole kidney function were not affected in renal hypertensive rats.

Diabetic patients undergoing hemodialysis demonstrate much worse survival rates than do nondiabetic patients undergoing hemodialysis. To search for risk predictors, a prospective cohort study was performed with 245 hemodialysis patients, including 84 with diabetes mellitus, for 2 yr. C-reactive protein, troponin T (TnT), total, HDL, LDL, and lipoprotein(a) cholesterol, apoA2, apoB, triglyceride, fibrinogen, D-dimer, albumin, and creatinine levels and clinical characteristics at the time of entry were recorded. Survival rates were compared with Kaplan-Meier and Cox regression analyses. Forty-three diabetic patients and 30 nondiabetic patients died. Among diabetic patients, oliguria (<200 ml/d) (relative risk, 3.24; 95% confidence interval, 1.63 to 6.41; P = 0.001), elevated C-reactive protein levels (relative risk, 2.57; 95% confidence interval, 1.06 to 6.18; P = 0.035), and elevated D-dimer levels (relative risk, 2.36; 95% confidence interval, 1.11 to 5.01; P = 0.025) predicted all-cause mortality rates. Oliguria was by far the most important predictor, particularly for infectious disease-related death (relative risk, 23.35; 95% confidence interval, 2.60 to 209.97; P = 0.005). Among nondiabetic patients, elevated TnT levels (relative risk, 4.00; 95% confidence interval, 1.58 to 10.10; P = 0.003), elevated D-dimer levels (relative risk, 3.45; 95% confidence interval, 1.27 to 9.33; P = 0.015), and low cholesterol levels (relative risk, 3.61; 95% confidence interval, 1.34 to 9.71; P = 0.011) predicted all-cause mortality rates. Subdivision of the causes of death among nondiabetic patients revealed that TnT levels predicted cardiovascular mortality rates (relative risk, 5.38; 95% confidence interval, 1.11 to 26.10; P = 0.037) and infectious disease-related mortality rates (relative risk, 12.02; 95% confidence interval, 1.42 to 191.96; P = 0.023). In conclusion, mortality predictors among patients undergoing hemodialysis differed substantially between diabetic and nondiabetic patients. Strategies to reduce mortality rates should consider these differences.

Human ECE-1 is expressed in four isoforms with different tissue distribution and its mRNA and protein levels are altered under certain pathophysiological conditions. To investigate the transcriptional regulation of ECE-1, we studied the regulatory region of ECE-1c, the major ECE-1 isoform. A genomic clone comprising the complete human ECE-1 gene including the putative ECE-1c-specific promoter was obtained. Up to 968 bp upstream of the putative c-specific translation initiation start codon and several serial deletion mutants were subcloned into a reporter vector and transfected into endothelial (BAEC, EA.hy926, ECV304) and epithelial (MDA MB435S, MCF7) cells, showing very strong promoter activity in comparison to the SV40 promoter and to the previously described ECE-1a and 1b promoters. Transfection of serial deletion mutants indicated two positive regulatory regions within the promoter (-142/-240 and -240/490) likely involved in binding GATA and ETS transcription factors. RNase protection assay (RPA) and 5'-RACE revealed multiple transcriptional start sites located at about -110, -140 and -350 bp. Site-directed mutagenesis demonstrated a crucial role for the E2F cis-element for basal ECE-1c promoter activity. Additionally, we found a correlation between isoform-specific ECE-1 mRNA levels and corresponding ECE-1a, 1b, 1c promoter activities.

Endothelin-converting enzyme-1 (ECE-1) mRNA is expressed in three isoforms, termed a, b, and c, originating from alternative promoters. In cultured bovine aortic endothelial cells, we detected mRNA isoform expression of ECE-1a and ECE-1b/c, respectively. Investigating transcriptional mechanisms of bovine endothelial ECE-1a expression in more detail, we identified multiple transcription start sites localized 120-415 nucleotides upstream from the presumptive translation start codon by RNase protection assay and 5' RACE. Using luciferase reporter gene assays we found that 1.4 kb of the 5' untranslated region showed strong promoter activity in endothelial cells. Sequence analysis revealed 71% overall homology of the bovine ECE-1a promoter with its human homologue. The proximal 680 base pair promoter region was shown to contain cis elements that are sufficient for basal and serum-induced transcriptional activation.

The endothelins, a family of closely related vasoactive and mitogenic peptides, are thought to play an important role in cardiovascular pathophysiology. The conversion of the inactive precursor "big endothelin" to the biologically active peptide is catalyzed in vitro and in vivo by endothelin-converting enzymes (ECE). Recently the cDNA cloning of two homologous proteins, termed ECE-1 and ECE-2, has been reported. ECE-1 may play a key role in the activation and regulation of the cardiovascular endothelin proteolytic cascade. ECE-1 mRNA is expressed in two isoforms, termed alpha and beta, which are identical except for the 5'-terminal regions. To investigate the transcriptional regulation of isoform-specific ECE-1 mRNA expression we isolated phage clones from a human genomic library and identified the alpha- and beta-specific exons of ECE-1. The exon/intron organization of the 5'-terminal region of the human ECE-1 gene in conjunction with putative transcription initiation start sites suggests the existence of two alternative promoters, each directing the expression of either isoform. A reverse transcription/polymerase chain reaction assay indicated differential mRNA expression of ECE-1 isoforms. Using a luciferase reporter gene assay, we found that the genomic region upstream of exon 1 alpha confers strong promoter activity in the human endothelial cell line ECV 304, which was previously shown to express predominantly ECE-1 alpha mRNA. Transfection of serial deletion mutants in ECV304 cells indicated the existence of three positive and also one negative regulating element within 2 kb of the alpha-promoter region. Luciferase reporter gene studies also revealed that the genomic region upstream of exon 3, which encodes the putative ECE-1 beta specific N-terminus, was able to direct luciferase expression in primary cultured bovine aortic endothelial cells, indicating the existence of an alternative promoter. Transfection of nested deletions spanning 1.2 kb upstream of the putative translation initiation codon of ECE-1 beta suggested the existence of three positive regulating regions within the beta-specific promoter. Both ECE-1 promoters lack TATA or CAAT boxes, and the two show different patterns of consensus sequences for transcription factors, suggesting a differential transcriptional regulation of isoform-specific ECE-1 mRNA expression.

The free amino acid content in needles of Norway spruce trees (about 45-year-old) was determined by means of HPLC. The studied trees have been growing at a forestry site in the Black Forest which is characterized by a high impact of ozone and magnesium deficiency. Measurements were carried out on visibly healthy and on damaged trees on several dates during two vegetation periods and during the course of a day. The amino acids occurring at the highest concentrations were glutamate, arginine, aspartate, and glutamine. The typical seasonal changes in the content of free amino acids, with their minimum during summer, were disturbed in the needles of the damaged trees. Particularly in summer and autumn the damaged trees showed increased levels of total amino acids and of most single amino acids. During the course of a day, the needles of the damaged trees showed a reduced amplitude in diurnal variations with the absolute level almost always higher in comparison to that of the undamaged trees. The results suggest that a shift in protein turnover towards enhanced degradation has taken place.

The effect of the lipid lowering drug etofibrate was investigated on lipid peroxidation as well as on cholesterol level. Rabbits were given a 0.1% cholesterol containing diet. Total cholesterol, LDL-cholesterol, triglycerides and lipid peroxidation, expressed as thiobarbituric acid reactive products, were determined. Treatment with etofibrate led to a marked decrease in total cholesterol and LDL-cholesterol. Furthermore, Cu(2+)-induced lipid peroxide formation was reduced in etofibrate treated rabbits. These results could be confirmed in a human study when patients with moderate hypercholesterolaemia were treated with etofibrate (2 x 500 mg/day) for a period of eight weeks. It could be shown that the onset of lipid peroxidation was remarkably increased, an effect which was completely reversible. Thus, etofibrate is effective not only in lowering plasma cholesterol but also in rendering LDL less susceptible to oxidation.

The effect of U-50,488H, a selective kappa opioid receptor agonist on the intake of food in food-deprived and non-deprived spontaneously hypertensive and normotensive Wistar-Kyoto rats was determined. In food deprived Wistar-Kyoto rats, intraperitoneal administration of U-50,488H, produced a bell-shaped curve on the intake of food, consistent with earlier reports in the literature. Thus, at a dose of 0.1 mg/kg there was a significant increase in the intake of food and at 10 mg/kg it caused a decrease in the intake of food, at 2, 4 and 6 hr after the treatment with drug. The amount of food consumed at each dose and the time interval was greater in hypertensive rats than in Wistar-Kyoto rats. Similar effects were produced when the animals were not deprived of food. The basal intake of food in the two strains of rats did not differ. The results indicate a greater sensitivity of spontaneously hypertensive rats to kappa opioid receptor agonists and further the previous observations that compared to Wistar-Kyoto rats, the hypertensive rats have a greater density of central kappa opioid receptors.