Characterization of human DAAO variants potentially related to an increased risk of schizophrenia

Considering the key role of d-serine in N-methyl-d-aspartate receptor-mediated neurotransmission, it is highly relevant to define the role that enzymes play in d-serine synthesis and degradation. In particular, the details of regulation of the d-serine catabolic human enzyme d-amino acid oxidase (hDAAO) are unknown although different lines of evidence have shown it to be involved in schizophrenia susceptibility. Here we investigated the effect of three single nucleotide polymorphisms and known mutations in hDAAO, i.e., D31H, R279A, and G331V. A very low amount of soluble G331V hDAAO is produced in E. coli cells: the recombinant variant enzyme is fully active. Human U87 glioblastoma cells transiently transfected for G331V hDAAO show a low viability, a significant amount of protein aggregates, and augmented apoptosis. The recombinant D31H and R279A hDAAO variants do not show alterations in tertiary and quaternary structures, thermal stability, binding affinity for inhibitors, and the modulator pLG72, whereas the kinetic efficiency and the affinity for d-serine and for FAD were higher than for the wild-type enzyme. While these effects for the substitution at position 31 cannot be structurally explained, the R279A mutation might affect the hDAAO FAD-binding affinity by altering the “structurally ambivalent” peptide V47–L51. In agreement with the observed increased activity, expression of D31H and R279A hDAAO variants in U87 cells produces a higher decrease in cellular d/(d + l) serine ratio than the wild-type counterpart. In vivo, these substitutions could affect cellular d-serine concentration and its release at synapsis and thus might be relevant for schizophrenia susceptibility.     

Inflammatory cytokines and malnutrition as related to risk for cardiovascular disease in hemodialysis patients

Malnutrition and inflammation are associated with end-stage renal disease (ESRD). Interleukin (IL)-6 and tumor necrosis factor alpha (TNF-alpha) powerfully predict death from cardiovascular disease. The aim of our study was to establish an association between markers of inflammation and parameters of malnutrition in patients on hemodialysis. The study population consisted of 42 hemodialysis patients with different parameters of malnutrition. Blood samples were taken after an overnight fast, and plasma lipid profiles (total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides) were measured by using conventional enzymatic methods. Serum urea and creatinine levels were also measured by routine procedures. Plasma high-sensitivity C-reactive protein level (hs-CRP), TNF-alpha, and IL-6 were measured by enzyme-linked immunosorbent assay (ELISA). Standard Doppler echo examinations were used to determine plaque on carotid arteries, and end-diastolic diameter (EDD) and ejection fraction (EF) were measured by echocardiography. Malnourished patients exhibited significantly greater evidence of cardiovascular disease and carotid plaques. Factor (principal component) analysis indicated 6 latent factors with 67.5% of the variance explained within all investigated parameters. Cluster analysis was used to distinguish the inflammatory markers and the nutritional markers from other parameters and to visualize similarities between variables. In summary, this cross-sectional study in hemodialysis patients found a high prevalence of malnutrition, inflammation, carotid plaques, and cardiovascular disease. Malnourished dialysis patients are more often found with cardiovascular disease and carotid plaques. In addition, these patients have higher levels of inflammatory cytokines, which may partly explain the elevated risk for atherosclerotic vascular disease.     

Another smoking hazard: raised serum IgE concentration and increased risk of occupational allergy.

Individual smoking histories of a general population sample and of two groups of workers exposed to occupational allergens were related to serum IgE concentrations and results of radioallergosorbent and prick tests in the workers. The geometric mean IgE concentration was higher in smokers than in non-smokers. The distribution of serum IgE values in the two groups showed an apparent difference, with a bimodal appearance in the smokers. Evidence of sensitisation against occupational allergens was more common in workers who smoked. The adjuvant effect of smoking on IgE antibody production might be due to damage to airways mucosa and supports the mucosal theory of atopy.     

Perinatal development of laryngeal function

The resistance of the upper airway is strongly influenced by the action of opposing sets of laryngeal muscles. Expiratory airflow may be retarded by active adduction of the arytenoid cartilages or by a reduction in the activity of abductor muscles. In developing sheep the adductor muscles appear to represent the principal means by which lung recoil is opposed. This mechanism, which is most pronounced during non-rapid-eye-movement sleep, is regulated by afferent traffic from the lungs. In fetal sheep the laryngeal muscles are also influenced by breathing movements and sleep states. The adductor muscles are normally tonically active during non-rapid-eye-movement sleep when rhythmical breathing movements are absent. It is possible that this activity is at least partially responsible for elevated tracheal pressures and depressed flow of tracheal fluid during fetal apnea. This hypothesis has been tested by observing the effects of fetal paralysis and recurrent laryngeal nerve section. These experiments suggest that in the fetus near term the larynx makes a major contribution to upper airway resistance and hence to the maintenance of pulmonary expansion which has been shown to influence lung development.     

The future of antigen-specific immunotherapy of allergy

More than 25% of the population in industrialized countries suffers from immunoglobulin-E-mediated allergies. The antigen-specific immunotherapy that is in use at present involves the administration of allergen extracts to patients with the aim to cure allergic symptoms. However, the risk of therapy-induced side effects limits its broad application. Recent work indicates that the epitope complexity of natural allergen extracts can be recreated using recombinant allergens, and hypoallergenic derivatives of these can be engineered to increase treatment safety. It is proposed that these modified molecules will improve the current practice of specific immunotherapy and form a basis for prophylactic vaccination.     

Virally delivered cytokines alter the immune response to future lung infections

Respiratory syncytial virus (RSV) is an important cause of infant morbidity and mortality worldwide and is increasingly recognized to have a role in the development and exacerbation of chronic lung diseases. There is no effective vaccine, and we reasoned that it might be possible to skew the immune system towards beneficial nonpathogenic responses by selectively priming protective T-cell subsets. We therefore tested recombinant RSV (rRSV) candidates expressing prototypic murine Th1 (gamma interferon [IFN-γ]) or Th2 (interleukin-4 [IL-4]) cytokines, with detailed monitoring of responses to subsequent infections with RSV or (as a control) influenza A virus. Although priming with either recombinant vector reduced viral load during RSV challenge, enhanced weight loss and enhanced pulmonary influx of RSV-specific CD8⁺ T cells were observed after challenge in mice primed with rRSV/IFN-γ. By contrast, rRSV/IL-4-primed mice were protected against weight loss during secondary challenge but showed airway eosinophilia. When rRSV/IL-4-primed mice were challenged with influenza virus, weight loss was attenuated but was again accompanied by marked airway eosinophilia. Thus, immunization directed toward enhancement of Th1 responses reduces viral load but is not necessarily protective against disease. Counter to expectation, Th2-biased responses were more beneficial but also influenced the pathological effects of heterologous viral challenge.     

Activation-dependent adsorption of cytokines and toxins related to liver failure to carbon beads

In the course of severe pathological conditions, such as acute liver failure and sepsis, toxic metabolites and mediators of inflammation are released into the patient's circulation. One option for the supportive treatment of these conditions is plasmapheresis, in which plasma, after being separated from the cellular components of the blood, is cleansed by adsorption of harmful molecules on polymers or activated carbon. In this work, the adsorption characteristics of activated carbon beads with levels of activation ranging from 0 to 86% were assessed for both hydrophobic compounds accumulating in liver failure (bilirubin, cholic acid, phenol and tryptophan) and cytokines (tumor necrosis factor α and interleukin-6). Progressive activation resulted in significant gradual reduction of both bulk density and mean particle size, in an increase in the specific surface area, and to changes in pore size distribution with progressive broadening of micropores. These structural changes went hand in hand with enhanced adsorption of small adsorbates, such as IL-6 and cholic acid and, to a lesser extent, also of large molecules, such as TNF-α.     

Perinatal development of mammillotegmental connections in rats

Development of direct axonal connections of the hypothalamic mammillary bodies with ventral and dorsal tegmental nuclei of Gudden was studied on fixed rat brains from day 14 of embryonic development until day 10 of postnatal development using the method of diffusion of the lipophilic fluorescent carbocyanine tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate along the neuronal membranes. The tracer was inserted into the mammillary bodies or into the tegmentum and after incubation in a fixative fluorescent nerve cells and nerve fibers were visualized in the brain tissue. The mammillotegmental tract was found to start developing earlier than other conducting systems of the mammillary bodies. On days 14-15 of embryonic development, it was visualized as a bundle of axons running from the mammillary bodies caudally to the midbrain. A group of neurons in the midbrain tegmentum and their axons going to the mammillary bodies via the mammillary peduncle were first visualized on day 19 of embryonic development. The mammillotegmental tract and mammillary peduncle developed progressively from the moment of birth. Ventral and dorsal tegmental nuclei were formed in the midbrain by day 10 of the postnatal development. Thus, the formation of reciprocal connections of the mammillary bodies with midbrain tegmental nuclei was first described during perinatal development in rats.     

Perinatal development of mammillotegmental connections in rats

Development of direct axonal connections of the hypothalamic mammillary bodies with ventral and dorsal tegmental nuclei of Gudden was studied on fixed rat brains from day 14 of embryonic development until day 10 of postnatal development using the method of diffusion of the lipophilic fluorescent carbocyanine tracer 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate along the neuronal membranes. The tracer was inserted into the mammillary bodies or into the tegmentum and, after incubation in a fixative, fluorescent nerve cells and nerve fibers were visualized in the brain tissue. The mammillotegmental tract was found to start developing earlier than other projection systems of the mammillary bodies. On days 14–15 of embryonic development, it was visualized as a bundle of axons running from the mammillary bodies caudally to the midbrain. A group of neurons in the midbrain tegmentum and their axons going to the mammillary bodies via the mammillary peduncle were first visualized on day 19 of embryonic development. The mammillotegmental tract and mammillary peduncle developed progressively from the moment of birth. Ventral and dorsal tegmental nuclei were formed in the midbrain by day 10 of the postnatal development. Thus, the formation of reciprocal connections of the mammillary bodies with midbrain tegmental nuclei was first described during perinatal development in rats.     

Perinatal development of endothelial nitric oxide synthase-deficient mice

The purpose of this study was to evaluate the influence of endothelial nitric oxide synthase (eNOS) deficiency on fetal growth, perinatal survival, and limb development in a mouse model with a targeted mutagenesis of the Nos3 gene. Wild-type (Nos3+/+) and eNOS-deficient fetuses (Nos3-/-) were evaluated on Gestational Day (E)15 and E17, and newborn pups were observed on Day 1 of life (D1). The average term duration of pregnancy was 19 days. For the evaluation of postnatal development, a breeding scheme consisting of Nos3+/- x Nos3+/- and Nos3-/- x Nos3-/- mice was established, and offspring were observed for 3 wk. Southern blotting was used for genotyping. No significant differences in fetal weight, crown-rump lengths (CRL), and placental weight were seen between Nos3+/+ and Nos3-/- fetuses on E15. By E17, Nos3-/- fetuses showed significantly reduced fetal weights, CRL, and placental weights. This difference in body weight was also seen throughout the whole postnatal period. In pregnancies of Nos3-/- females, the average number of pups alive on D1 was significantly decreased compared to either E15 or E17. Placental histology revealed no abnormalities. On E15, E17, and D1, Nos3(-/-) fetuses demonstrated focal acute hemorrhages in the distal limbs in 0%, 2.6%, and 5.7%, respectively, of all mutant mice studied on the respective days. Bone measurements showed significantly shorter bones in the peripheral digits of hindpaws of Nos3-/- newborns. We conclude mice deficient for eNOS show characteristically abnormal prenatal and postnatal development including fetal growth restriction, reduced survival, and an increased rate of limb abnormalities. The development of this characteristic phenotype of eNOS-deficient mice dates back to the prenatal development during the late third trimester of pregnancy.     

Human papillomavirus-16 infection in advanced oral cavity cancer patients is related to an increased risk of distant metastases and poor survival

Background

Human papillomavirus (HPV) is an oncogenic virus causing oropharyngeal cancers and resulting in a favorable outcome after the treatment. The role of HPV in oral cavity squamous cell carcinoma (OSCC) remains ambiguous.

Objective

This study aimed to examine the effect of HPV infection on disease control among patients with OSCC following radical surgery with radiation-based adjuvant therapy.

Patients and Method

We prospectively followed 173 patients with advanced OSCC (96% were stage III/IV) who had undergone radical surgery and adjuvant therapy between 2004 and 2006. They were followed between surgery and death or up to 60 months. Surgical specimens were examined using a PCR-based HPV blot test. The primary endpoints were the risk of relapse and the time to relapse; the secondary endpoints were disease-free survival, disease-specific survival, and overall survival.

Results

The prevalence of HPV-positive OSCC was 22%; HPV-16 (9%) and HPV-18 (7%) were the genotypes most commonly encountered. Solitary HPV-16 infection was a poor predictor of 5-year distant metastases (hazard ratio, 3.4; 95% confidence interval, 1.4–8.0; P = 0.005), disease-free survival (P = 0.037), disease-specific survival (P = 0.006), and overall survival (P = 0.010), whereas HPV-18 infection had no impact on 5-year outcomes. The rate of 5-year distant metastases was significantly higher in the HPV-16 or level IV/V metastasis group compared with both the extracapsular spread or tumor depth ≥11-mm group and patients without risk factors (P<0.001).

Conclusions

HPV infections in advanced OSCC patients are not uncommon and clinically relevant. Compared with HPV-16-negative advanced OSCC patients, those with a single HPV-16 infection are at higher risk of distant metastases and poor survival despite undergoing radiation-based adjuvant therapy and require a more aggressive adjuvant treatment and a more thorough follow-up.     

Perinatal development of hepatic cholesterol synthesis in the rat

Rates of cholesterol synthesis and HMG CoA reductase activity in rat liver, have been reported to be high before and low after birth. The timing of the decline in perinatal rates of cholesterol synthesis, however, is uncertain. These studies, therefore, determined in vivo rates of cholesterol synthesis using [3H]water and hepatic reductase activity in vitro in perinatal rats. The lipid composition of the plasma, liver and its microsomal subfraction were also determined. Reductase activity increased during late gestation, remained high immediately after birth, then decreased with the commencement of suckling. Rates of cholesterol synthesis increased from gestation day 18 to 20, but in contrast to reductase activity, decreased on the day before birth. Plasma cholesterol and triacylglycerol levels increased to gestation day 19, then decreased to term. By the 6th h after birth, plasma and liver cholesterol and triacylglycerol levels had increased markedly. By 48 h after birth, the high hepatic cholesterol content was associated with an increase in the cholesteryl ester fraction. The microsomal cholesterol/phospholipid molar ratio decreased from gestation day 16 until 12 h after birth, then increased markedly from 36 to 48 h. There was an apparent inverse relationship between the change in microsomal cholesterol/phospholipid molar ratio and the fatty acid unsaturation index from gestation day 16 to 36 h after birth. The results suggest that in late gestation and before suckling, the low in vivo rate of hepatic cholesterol synthesis may not be due to low activity of HMG CoA reductase.     

Perinatal stress and increased fluctuating asymmetry of dental calcium in the laboratory rat

Recent studies have consistently reported an increased magnitude of fluctuating dental, long bone, and membranous bone asymmetry as a function of perinatal stress. The present study was designed to test the hypothesis that increases in the fluctuating asymmetry of calcium may be related to the metric changes in these calcium-dependent systems. Pregnant rats were exposed to noise stress from conception through weaning. Bilateral lower first molars were extracted from the neonates, and calcium levels were determined using a standard atomic absorption technique. Levels of fluctuating asymmetry of calcium were found to be significantly increased (p less than .01) in the audiogenic noise-stressed group compared to unstressed, normal controls. These results follow the pattern reported earlier for metric analysis of the dentition and support a stress-induced calcium-transport-disruption hypothesis.     

Morphological changes related to the climateric period of the adult Cuban women

The aim of the present study is to analyze the morphologic changes occurring along the period of a woman's life thatareknown as climacterium. Our sample consists of 648 women from different provinces of Cuba, but who lived in Havana for at least 15 years. Morphological variables such as height, weight, biliocristal and biachromial diameters and six subcutaneous fat skinfolds were measured. With regard to the menopausic condition, the sample was divided into three subpopulations: premenopausic, naturally menopausic and surgically menopausic (when the two ovaries had been removed). Age at menopause was calculated by both the “retrospective” (49.45±0.49) and “status quo” (48.83±0.02) methods. The results obtained point towards morphological differences between pre- and postmenopausic women. We observed greater accumulations of fatty tissue in the suprailiac regions of postmenopausal females. Lean body mass represents a bigger fraction of the total body weight in women who are still menstruating. In the case of surgically menopausic women, they are at a mid-distance between the premenopausic and naturally menopausic subpopulations. A discriminant analysis was carried out which confirmed the results previously observed. From this analysis it appeared the body mass index and the subcutaneous skinfolds are the variables that discriminante, that is, separate, the three subsamples.