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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.)

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.

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: Hypertension (H) is associated with a large number of co-morbidities, including obesity. The correlation between two variables has been investigated., Objective: To analyze the correlation between the loss of body mass and blood pressure reduction in hypertensive patients undergoing exercising programs (EP)., Methods: One hundred eleven hypertensive patients with overweight or obesity were randomly divided into an experimental group (EG). Out of these, 57 (58 +/- 8.9 years old) participated in a three-month EP conducted three times a week in aerobic exercise sessions from 50% to 70% of VO2 peak for 30 to 60 minutes and resistance exercises; and a control group (CG) with 54 (60 +/- 7.7 years old) who did not participate in the EP. In the EG, blood pressure (BP) was measured before each session and the measurement of anthropometric variables (AV) at the beginning of the program and after three months. In the CG the BP and the VA were evaluated in the doctor's office at the beginning and at the end of the study. Data were expressed as mean +/- standard deviation (SD). Pearson correlation and t test were used. A value of p < 0.05 was considered significant., Results: In the CG there was no significant difference in AV and BP at the beginning and at the end of the study. In the EG, there was no significant alteration in the AV, however, there was blood pressure reduction of 12% in systolic BP (-17.5 mmHg, p = 0.001) and 9% in Diastolic BP (-8.1 mmHg, p = 0. 01) at the end of the study. There was no correlation between the AV and decrease in BP (r = 0.1)., Conclusion: The blood pressure reduction was not correlated with reduction of anthropometric measures after the exercising period.

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.

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.

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.

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.