A mouse model for mitochondrial myopathy and cardiomyopathy resulting from a deficiency in the heart/muscle isoform of the adenine nucleotide translocator.

In an attempt to create an animal model of tissue-specific mitochondrial disease, we generated 'knockout' mice deficient in the heart/muscle isoform of the adenine nucleotide translocator (Ant1). Histological and ultrastructural examination of skeletal muscle from Ant1 null mutants revealed ragged-red muscle fibers and a dramatic proliferation of mitochondria, while examination of the heart revealed cardiac hypertrophy with mitochondrial proliferation. Mitochondria isolated from mutant skeletal muscle exhibited a severe defect in coupled respiration. Ant1 mutant adults also had a resting serum lactate level fourfold higher than that of controls, indicative of metabolic acidosis. Significantly, mutant adults manifested severe exercise intolerance. Therefore, Ant1 mutant mice have the biochemical, histological, metabolic and physiological characteristics of mitochondrial myopathy and cardiomyopathy.

Mice lacking tissue non-specific alkaline phosphatase die from seizures due to defective metabolism of vitamin B-6.

In humans, deficiency of the tissue non-specific alkaline phosphatase (TNAP) gene is associated with defective skeletal mineralization. In contrast, mice lacking TNAP generated by homologous recombination using embryonic stem (ES) cells have normal skeletal development. However, at approximately two weeks after birth, homozygous mutant mice develop seizures which are subsequently fatal. Defective metabolism of pyridoxal 5'-phosphate (PLP), characterized by elevated serum PLP levels, results in reduced levels of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in the brain. The mutant seizure phenotype can be rescued by the administration of pyridoxal and a semi-solid diet. Rescued animals subsequently develop defective dentition. This study reveals essential physiological functions of TNAP in the mouse.

Testicular degeneration in Bclw-deficient mice.

To identify genes required for mammalian spermatogenesis, we screened lines of mutant mice created using a retroviral gene-trap system for male infertility. Homozygous ROSA41 male mice exhibit sterility associated with progressive testicular degeneration. Germ-cell defects are first observed at 19 days post-natal (p19). Spermatogenesis is blocked during late spermiogenesis in young adults. Gradual depletion of all stages of germ cells results in a Sertoli-cell-only phenotype by approximately six months of age. Subsequently, almost all Sertoli cells are lost from the seminiferous tubules and the Leydig cell population is reduced. Molecular analysis indicates that the gene mutated is Bclw, a death-protecting member of the Bcl2 family. The mutant allele of Bclw in ROSA41 does not produce a Bclw polypeptide. Expression of Bclw in the testis appears to be restricted to elongating spermatids and Sertoli cells. Potential roles for Bclw in testicular function are discussed.

Potassium softens vascular endothelium and increases nitric oxide release.

In the presence of aldosterone, plasma sodium in the high physiological range stiffens endothelial cells and reduces the release of nitric oxide. We now demonstrate effects of extracellular potassium on stiffness of individual cultured bovine aortic endothelial cells by using the tip of an atomic force microscope as a mechanical nanosensor. An acute increase of potassium in the physiological range swells and softens the endothelial cell and increases the release of nitric oxide. A high physiological sodium concentration, in the presence of aldosterone, prevents these changes. We propose that the potassium effects are caused by submembranous cortical fluidization because cortical actin depolymerization induced by cytochalasin D mimics the effect of high potassium. In contrast, a low dose of trypsin, known to activate sodium influx through epithelial sodium channels, stiffens the submembranous cell cortex. Obviously, the cortical actin cytoskeleton switches from gelation to solation depending on the ambient sodium and potassium concentrations, whereas the center of the cell is not involved. Such a mechanism would control endothelial deformability and nitric oxide release, and thus influence systemic blood pressure.

Dietary Sodium 'Controversy'-Issues and Potential Solutions.

PURPOSE OF REVIEW: High dietary sodium is estimated to be the leading dietary risk for death attributed to 1.8 million deaths in 2019. There are uniform recommendations to reduce sodium consumption based on evidence that increased dietary sodium is responsible for approximately a third of the prevalence of hypertension, and meta-analyses of randomized controlled trials show that sodium reduction lowers blood pressure, cardiovascular disease, and total mortality. Nevertheless, there is a perception that the beneficial effect of reducing dietary sodium is controversial. We provide experiential evidence relating to some sources of the controversy and propose potential solutions. RECENT FINDINGS: Inappropriate research methodology, lack of rigor in research, conflicts of interest and commercial bias, questions of professional conduct, and lack of policies to protect public interests are likely to contribute to the controversy about reducing dietary sodium. There is a failure to protect policies to reduce dietary sodium from nonscientific threats. Significant efforts need to be made to ensure the integrity of nutritional research and maintain public trust.

Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death.

Multiple death signals influence mitochondria during apoptosis, yet the critical initiating event for mitochondrial dysfunction in vivo has been unclear. tBID, the caspase-activated form of a "BH3-domain-only" BCL-2 family member, triggers the homooligomerization of "multidomain" conserved proapoptotic family members BAK or BAX, resulting in the release of cytochrome c from mitochondria. We find that cells lacking both Bax and Bak, but not cells lacking only one of these components, are completely resistant to tBID-induced cytochrome c release and apoptosis. Moreover, doubly deficient cells are resistant to multiple apoptotic stimuli that act through disruption of mitochondrial function: staurosporine, ultraviolet radiation, growth factor deprivation, etoposide, and the endoplasmic reticulum stress stimuli thapsigargin and tunicamycin. Thus, activation of a "multidomain" proapoptotic member, BAX or BAK, appears to be an essential gateway to mitochondrial dysfunction required for cell death in response to diverse stimuli.

Cognitive impairment and white matter damage in hypertension: a pilot study.

OBJECTIVES: Hypertension has been associated with impaired cognition. Diffusion tensor imaging (DTI) and magnetic resonance spectroscopy were applied to assess white matter abnormalities in treated vs untreated hypertension and if these correlated with neuropsychological performance. METHODS: Subjects were 40 patients with medically treated hypertension (mean age 69.3 years), 10 patients with untreated hypertension (mean age 57.6 years) and 30 normotensive controls (mean age 68.2 years). Hypertension was defined as a previous diagnosis and taking hypertensive medication, or a resting blood pressure of >140/90 mmHg on the day of assessment. RESULTS: Patients with treated hypertension performed worse on immediate (P = 0.037) as well as delayed memory tasks (P = 0.024) compared with normotensive controls. Cognitive performance was worse in untreated compared with treated hypertension on executive functions (P = 0.041) and psychomotor speed (P = 0.003). There was no significant correlation between cognition and any of the imaging parameters in treated hypertension. However, in untreated hypertension the results revealed a positive correlation between an executive functioning and attention composite score and DTI mean diffusivity values (P = 0.016) and between psychomotor speed and spectroscopy NAA/tCr levels (P = 0.015). CONCLUSIONS: These results suggest there is cognitive impairment in hypertension. Treated hypertension was associated with deficits in memory while untreated hypertension revealed a more 'subcortical' pattern of cognitive impairment.

Salt and blood pressure in children and adolescents.

To study the relationship between salt intake and blood pressure in children and adolescents, we analysed the data of a large cross-sectional study (the National Diet and Nutrition Survey for young people), which was carried out in Great Britain in 1997 in a nationally representative sample of children aged between 4 and 18 years. A total of 1658 participants had both salt intake and blood pressure recorded. Salt intake was assessed by a 7-day dietary record. The average salt intake, which did not include salt added in cooking or at the table, was 4.7+/-0.2 g/day at the age of 4 years. With increasing age, there was an increase in salt intake, and by the age of 18 years, salt intake was 6.8+/-0.2 g/day. There was a significant association of salt intake with systolic blood pressure as well as with pulse pressure after adjusting for age, sex, body mass index and dietary potassium intake. An increase of 1 g/day in salt intake was related to an increase of 0.4 mm Hg in systolic and 0.6 mm Hg in pulse pressure. The magnitude of the association with systolic blood pressure is very similar to that observed in a recent meta-analysis of controlled trials where salt intake was reduced. The consistent finding of our present analysis of a random sample of free-living individuals with that from controlled salt reduction trials provides further support for a reduction in salt intake in children and adolescents.

Identification and characterisation of Staphylococcus aureus on low cost screen printed carbon electrodes using impedance spectroscopy.

Staphylococcus aureus infections are a cause of significant morbidity and mortality, in addition to representing a considerable economic burden. The aim of this study was to explore a low cost screen printed electrode as a sensor for the detection of S. aureus using impedance spectroscopy. S. aureus was incubated in chambers containing the electrodes and the results analysed using a novel normalisation approach. These results show that it is possible to detect the presence of S. aureus in LB media after 30 min incubation of a 1% growth culture, in addition to being able to see immediate cell concentration dependant changes in 0.9% NaCl. These observations imply that a number of electrochemical mechanisms cause a change in the impedance as a result of the presence of S. aureus, including adsorption to the electrode surface and the metabolism of the bacteria during growth. The study suggests that this detection approach would be useful in a number of clinical scenarios where S. aureus leads to difficult to treat infections.

Abnormalities of nasal potential difference measurement in Liddle's syndrome.

In Liddle's syndrome, a rare inherited form of hypertension, epithelial sodium channel mutations appear to cause high blood pressure by increasing sodium reabsorption through sodium channels in the renal distal tubule. This increase in channel activity has not been confirmed previously by in vivo measurement. We have made transnasal potential difference measurements (effective in detection of increased sodium channel activity in cystic fibrosis) in three brothers with genetically proven Liddle's syndrome, their unaffected sister, and 40 normotensive controls. Maximum potential difference after 2 wk off treatment in the affected brothers was -30.4+/-1.2 mV (values mean+/-SD, lumen-negative with respect to submucosa) and was significantly more lumen-negative than that of the control group (-18.6+/-6.8 mV, P = 0.0228) or the unaffected sister (-18.25 mV, P < 0.01). The change in potential difference after topical application of 10(-)4 M amiloride was greater in the Liddle's patients, 14.0+/-2.1 mV, than in controls (7.9+/-3.9 mV, P = 0.0126) or the unaffected sister (5.5 mV, P < 0.05). This is the first in vivo demonstration of increased sodium channel activity in Liddle's syndrome. If these results are confirmed in other kindreds with this condition, then nasal potential difference measurements could provide a simple clinical test for Liddle's syndrome.

Expression of human adenosine deaminase in murine hematopoietic cells.

Multiple replication-defective retrovirus vectors were tested for their ability to transfer and express human adenosine deaminase in vitro and in vivo in a mouse bone marrow transplantation model. High-titer virus production was obtained from vectors by using both a retrovirus long terminal repeat promoter and internal transcriptional units with human c-fos and herpes virus thymidine kinase promoters. After infection of primary murine bone marrow with one of these vectors, human adenosine deaminase was detected in 60 to 85% of spleen colony-forming units and in the blood of 14 of 14 syngeneic marrow transplant recipients. This system offers the opportunity to assess methods for increasing efficiency of gene transfer, for regulation of expression of foreign genes in hematopoietic progenitors, and for long-term measurement of the stability of expression in these cells.

Increased consumption of fruit and vegetables is related to a reduced risk of coronary heart disease: meta-analysis of cohort studies.

Increased consumption of fruit and vegetables has been shown to be associated with a reduced risk of coronary heart disease (CHD) in many epidemiological studies, however, the extent of the association is uncertain. We quantitatively assessed the relation between fruit and vegetable intake and incidence of CHD by carrying out a meta-analysis of cohort studies. Studies were included if they reported relative risks (RRs) and corresponding 95% confidence interval (CI) of CHD with respect to frequency of fruit and vegetable intake. Twelve studies, consisting of 13 independent cohorts, met the inclusion criteria. There were 278,459 individuals (9143 CHD events) with a median follow-up of 11 years. Compared with individuals who had less than 3 servings/day of fruit and vegetables, the pooled RR of CHD was 0.93 (95% CI: 0.86-1.00, P=0.06) for those with 3-5 servings/day and 0.83 (0.77-0.89, P<0.0001) for those with more than 5 servings/day. Subgroup analyses showed that both fruits and vegetables had a significant protective effect on CHD. Our meta-analysis of prospective cohort studies demonstrates that increased consumption of fruit and vegetables from less than 3 to more than 5 servings/day is related to a 17% reduction in CHD risk, whereas increased intake to 3-5 servings/day is associated with a smaller and borderline significant reduction in CHD risk. These results provide strong support for the recommendations to consume more than 5 servings/day of fruit and vegetables.

Phase I Studies of Acebilustat: Biomarker Response and Safety in Patients with Cystic Fibrosis.

There is a significant unmet need for safe and effective anti-inflammatory treatment for cystic fibrosis. The aim of this study was to evaluate the safety of acebilustat, a leukotriene A4 hydrolase inhibitor, and its effect on inflammation biomarkers in patients with cystic fibrosis. Seventeen patients with mild to moderate cystic fibrosis were enrolled and randomized into groups receiving placebo or doses of 50 mg or 100 mg acebilustat administered orally, once daily for 15 days. Sputum neutrophil counts were reduced by 65% over baseline values in patients treated with 100 mg acebilustat. A modestly significant 58% reduction vs. placebo in sputum elastase was observed with acebilustat treatment. Favorable trends were observed for reduction of serum C-reactive protein and sputum neutrophil DNA in acebilustat-treated patients. No changes in pulmonary function were observed. Acebilustat was safe and well tolerated. The results of this study support further clinical development of acebilustat for treatment of cystic fibrosis.

Extracellular ATP inhibits the small-conductance K channel on the apical membrane of the cortical collecting duct from mouse kidney.

We have used the patch-clamp technique to study the effects of changing extracellular ATP concentration on the activity of the small-conductance potassium channel (SK) on the apical membrane of the mouse cortical collecting duct. In cell-attached patches, the channel conductance and kinetics were similar to its rat homologue. Addition of ATP to the bathing solution of split-open single cortical collecting ducts inhibited SK activity. The inhibition of the channel by ATP was reversible, concentration dependent (K(i) = 64 microM), and could be completely prevented by pretreatment with suramin, a specific purinergic receptor (P(2)) blocker. Ranking of the inhibitory potency of several nucleotides showed strong inhibition by ATP, UTP, and ATP-gamma-S, whereas alpha, beta-Me ATP, and 2-Mes ATP failed to affect channel activity. This nucleotide sensitivity is consistent with P(2)Y(2) purinergic receptors mediating the inhibition of SK by ATP. Single channel analysis further demonstrated that the inhibitory effects of ATP could be elicited through activation of apical receptors. Moreover, the observation that fluoride mimicked the inhibitory action of ATP suggests the activation of G proteins during purinergic receptor stimulation. Channel inhibition by ATP was not affected by blocking phospholipase C and protein kinase C. However, whereas cAMP prevented channel blocking by ATP, blocking protein kinase A failed to abolish the inhibitory effects of ATP. The reduction of K channel activity by ATP could be prevented by okadaic acid, an inhibitor of protein phosphatases, and KT5823, an agent that blocks protein kinase G. Moreover, the effect of ATP was mimicked by cGMP and blocked by L-NAME (N(G)-nitro-l-arginine methyl ester). We conclude that the inhibitory effect of ATP on the apical K channel is mediated by stimulation of P(2)Y(2) receptors and results from increasing dephosphorylation by enhancing PKG-sensitive phosphatase activity.

A systematic molecular genetic approach to study mammalian germline development.

It is difficult to study gene expression in mammalian embryonic germ cells as PGCs constitute only a minor proportion of the mouse embryo. We have overcome this problem by using a novel combination of established molecular and transgenic approaches. A line of mice has been generated in which the cells of the germ lineage express the beta-galactosidase reporter gene during embryogenesis. Using this line, germ cells have been purified to near homogeneity from embryos at discrete stages during germline development by use of a stain for beta-gal activity and a fluorescence activated cell sorter. Subsequently, cDNA libraries have been constructed from each germ cell population using a modified lone-linker PCR strategy. These combined cDNA libraries represent genes expressed in PGCs during mammalian germline development. To facilitate a molecular genetic approach to studying mammalian germline development, these cDNA libraries will be pooled to form an arrayed, addressed reference embryonic germ cell cDNA library. In parallel with large-scale cDNA sequencing efforts; genes that are differentially expressed in germ cells will be identified by screening the reference library with probes generated by subtractive hybridization. Complementary DNAs identified using this approach will be analyzed by sequencing, database comparison, genomic mapping and in situ hybridization to ascertain the potential functional importance of each gene to germline development. In addition to providing a wealth of novel information regarding patterns of gene expression during mammalian germline development, these results will form the basis for future experiments to determine the function of these genes in this process.

Alkaline phosphatase knock-out mice recapitulate the metabolic and skeletal defects of infantile hypophosphatasia.

Hypophosphatasia is an inborn error of metabolism characterized by deficient activity of the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP) and skeletal disease due to impaired mineralization of cartilage and bone matrix. We investigated two independently generated TNSALP gene knock-out mouse strains as potential models for hypophosphatasia. Homozygous mice (-/-) had < 1% of wild-type plasma TNSALP activity; heterozygotes had the predicted mean of approximately 50%. Phosphoethanolamine, inorganic pyrophosphate, and pyridoxal 5'-phosphate are putative natural substrates for TNSALP and all were increased endogenously in the knock-out mice. Skeletal disease first appeared radiographically at approximately 10 days of age and featured worsening rachitic changes, osteopenia, and fracture. Histologic studies revealed developmental arrest of chondrocyte differentiation in epiphyses and in growth plates with diminished or absent hypertrophic zones. Progressive osteoidosis from defective skeletal matrix mineralization was noted but not associated with features of secondary hyperparathyroidism. Plasma and urine calcium and phosphate levels were unremarkable. Our findings demonstrate that TNSALP knock-out mice are a good model for the infantile form of hypophosphatasia and provide compelling evidence for an important role for TNSALP in postnatal development and mineralization of the murine skeleton.

Sodium is more important than calcium in essential hypertension.

There is much circumstantial and some direct evidence in humans to suggest that a high consumption of salt predisposes communities and individuals to the development of essential hypertension. Recent work has suggested possible mechanisms whereby a high salt intake could cause a rise in blood pressure in genetically susceptible persons. Restriction of salt intake in the diet lowers blood pressure in many subjects with high blood pressure and this fall in blood pressure is mediated in part by a diminished renin response to sodium restriction as hypertension develops. The effect of sodium restriction, like diuretics, is additive to most blood pressure lowering drugs, particularly those that inhibit the renin system such as beta-blockers and angiotensin converting enzyme inhibitors. Claims that a slight reduction in calcium intake may be important in the development of high blood pressure are disputed. Furthermore, no satisfactory hypothesis has been put forward to explain how a small reduction in dietary calcium intake could cause high blood pressure. Large increases in calcium intake have been reported to lower blood pressure in both normotensive and hypertensive humans. The three published studies, however, are not in agreement.

Sodium and potassium intake and blood pressure.

There is increasing circumstantial evidence that the very high sodium diet combined with low potassium intake that most Western communities now eat may be, at least in part, responsible for the prevalence of high blood pressure. This circumstantial evidence combined with animal evidence has been considered sufficient in some countries to make a general recommendation to reduce sodium intake. If high sodium intake is an important cause of high blood pressure, it is not clear at the present time how it may do so. In this report, evidence is reviewed for one hypothesis suggesting an inherited defect in the kidney's ability to excrete sodium in patients who are going to develop essential hypertension, together with evidence for a raised concentration of an inhibitor of sodium transport. In patients with established hypertension, moderate restriction of sodium intake appears to lower blood pressure and moderate potassium supplementation to also lower blood pressure. While further evidence is required, particularly long-term studies, it would seem prudent to recommend to patients with essential hypertension or a strong family history of hypertension that they restrict dietary sodium intake moderately and increase dietary potassium intake by the consumption of more fruits and vegetables and, perhaps, the use of a potassium-based salt substitute. This regimen could obviate or reduce the need for drug treatment in some patients with mild to moderate hypertension.