The Pharmacodynamic Characterization of an Antisense Oligonucleotide Against Monoamine Oxidase-B (MAO-B) in Rat Brain Striatal Tissue

1. The aim of our work was to pharmacodynamically characterize an antisense oligonucleotide sequence (5-GCC AAA CTT TTG CAT GAC-3) against MAO-B, using qualitative and quantitative analyses as assessment measures.2. Qualitative analysis using histochemical staining revealed that intracerebroventricular (ICV) administered antisense (100 picomoles twicedaily × 3.5 days) eliminated all visibly detectable histochemical staining for MAO-B throughout the striatum 1, 12, and 24 h after the last antisense treatment.3. Qualitative analysis using RT-PCR of the time course of MAO-B mRNA expression in the rat striatum following ICV administration of the antisense sequenceshowed that 12–24 h after the last administration there was a dramatic reduction in MAO-B mRNA expression in the striatum. The reverse and scrambled sequences generated no change in MAO-B mRNA at 1 or 24 h after the last treatment.4. Quantitative analysis using the MAO-B selective substrate 4-dimethylamino-phenethylamine (DMAPEA) showed that the antisense sequence reduced MAO-B activity by more than 40%, which was comparable to a single 2 mg/kg, ip dose of L-deprenyl.5. Quantitative analysis of neurotransmitter levels 24 h after the last treatment suggested that the antisense sequence did not produce any significant changes in neurotransmitter levels.6. Potential mechanisms for enhancing the antisense response and the speculated potential of an antisense against MAO-B for studying neurotoxicity, Parkinson's disease, and the aging process are also discussed.     

Red blood cell regulation of microvascular tone through adenosine triphosphate

The matching of blood flow with metabolic need requires a mechanism for sensing the needs of the tissue and communicating that need to the arterioles, the ultimate controllers of tissue perfusion. Despite significant strides in our understanding of blood flow regulation, the identity of the O(2) sensor has remained elusive. Recently, the red blood cell, the Hb-containing O(2) carrier, has been implicated as a potential O(2) sensor and contributor to this vascular control by virtue of its concomitant carriage of millimolar amounts of ATP, which it is able to release when exposed to a low-O(2) environment. To evaluate this possibility, we exposed perfused cerebral arterioles to low extraluminal O(2) in the absence and presence of red blood cells or 6% dextran and determined both vessel diameter and ATP in the vessel effluent. Only when the vessels were perfused with red blood cells did the vessels dilate in response to low extraluminal O(2). In addition, this response was accompanied by a significant increase in vessel effluent ATP. These findings support the hypothesis that the red blood cell itself serves a role in determining O(2) supply to tissue.     

Interrelationship of indices of body composition and zinc status in 11-yr-old New Zealand children

Serum zinc and hair zinc concentrations of some New Zealand children aged 11 yr, were examined in relation to selected anthropometric indices. Serum zinc concentrations (n=453) in boys and girls were similar and were unrelated to anthropometric indices and hair zinc concentrations. Mean hair zinc concentration (n=620) of the girls was higher than that for the boys (2.95±0.49 vs 2.46±0.47 μmol/g; p<0.001). Correlation analysis demonstrated that, for the boys, all the studied anthropometric indices, with the exception of height, were significantly related to hair zinc concentration and that the confounding effects of mid-parent height and the timing of the adolescent growth spurt was small. Results for the girls were similar but less significant. Dichotomizing the hair zinc results divided both the boys and girls into two groups: those with hair zinc <2.44 μmol/g were heavier (girls, 39.0 vs 35.2 kg; boys, 36.6 vs 34.7 kg) and fatter (mid-upper-arm fat area: girls, 15.2 vs 12.0 cm²; boys, 11.1 vs 9.5 cm²) compared to their counterparts with hair zinc >2.44 μmol/g. The results demonstrate that in these healthy New Zealand children, those with lower hair zinc concentrations are fatter and heavier than their high-hair-zinc counterparts.     

Intracellular Signal Triggered by Cholera Toxin in Saccharomyces boulardii and Saccharomyces cerevisiae

As is the case for Saccharomyces boulardii, Saccharomyces cerevisiae W303 protects Fisher rats against cholera toxin (CT). The addition of glucose or dinitrophenol to cells of S. boulardii grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for S. cerevisiae. The addition of CT to the same cells also resulted in trehalase activation. Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation. Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both S. boulardii and S. cerevisiae. These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade. The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells. This hypothesis was reinforced by experiments with ¹²⁵I-labeled CT showing specific binding of the toxin to yeast cells. The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts.     

Nitric Oxide Prevents Alveolar Senescence and Emphysema in a Mouse Model

N^ω-nitro-L-arginine methyl ester (L-NAME) treatment induces arteriosclerosis and vascular senescence. Here, we report that the systemic inhibition of nitric oxide (NO) production by L-NAME causes pulmonary emphysema. L-NAME-treated lungs exhibited both the structural (alveolar tissue destruction) and functional (increased compliance and reduced elastance) characteristics of emphysema development. Furthermore, we found that L-NAME-induced emphysema could be attenuated through both genetic deficiency and pharmacological inhibition of plasminogen activator inhibitor-1 (PAI-1). Because PAI-1 is an important contributor to the development of senescence both in vitro and in vivo, we investigated whether L-NAME-induced senescence led to the observed emphysematous changes. We found that L-NAME treatment was associated with molecular and cellular evidence of premature senescence in mice, and that PAI-1 inhibition attenuated these increases. These findings indicate that NO serves to protect and defend lung tissue from physiological aging.     

Developmental Block and Programmed Cell Death in Bos indicus Embryos: Effects of Protein Supplementation Source and Developmental Kinetics

The aims of this study were to determine if the protein source of the medium influences zebu embryo development and if developmental kinetics, developmental block and programmed cell death are related. The culture medium was supplemented with either fetal calf serum or bovine serum albumin. The embryos were classified as Fast (n = 1,235) or Slow (n = 485) based on the time required to reach the fourth cell cycle (48 h and 90 h post insemination - hpi -, respectively). The Slow group was further separated into two groups: those presenting exactly 4 cells at 48 hpi (Slow/4 cells) and those that reached the fourth cell cycle at 90 hpi (Slow). Blastocyst quality, DNA fragmentation, mitochondrial membrane potential and signs of apoptosis or necrosis were evaluated. The Slow group had higher incidence of developmental block than the Fast group. The embryos supplemented with fetal calf serum had lower quality. DNA fragmentation and mitochondrial membrane potential were absent in embryos at 48 hpi but present at 90 hpi. Early signs of apoptosis were more frequent in the Slow and Slow/4 cell groups than in the Fast group. We concluded that fetal calf serum reduces blastocyst development and quality, but the mechanism appears to be independent of DNA fragmentation. The apoptotic cells detected at 48 hpi reveal a possible mechanism of programmed cell death activation prior to genome activation. The apoptotic cells observed in the slow-developing embryos suggested a relationship between programmed cell death and embryonic developmental kinetics in zebu in vitro-produced embryos.     

Variation in Plasmodium falciparum Histidine-Rich Protein 2 (Pfhrp2) and Plasmodium falciparum Histidine-Rich Protein 3 (Pfhrp3) Gene Deletions in Guyana and Suriname

Guyana and Suriname have made important progress in reducing the burden of malaria. While both countries use microscopy as the primary tool for clinical diagnosis, malaria rapid diagnostic tests (RDTs) are useful in remote areas of the interior where laboratory support may be limited or unavailable. Recent reports indicate that histidine-rich protein 2 (PfHRP2)-based diagnostic tests specific for detection of P. falciparum may provide false negative results in some parts of South America due to the emergence of P. falciparum parasites that lack the pfhrp2 gene, and thus produce no PfHRP2 antigen. Pfhrp2 and pfhrp3 genes were amplified in parasite isolates collected from Guyana and Suriname to determine if there were circulating isolates with deletions in these genes. Pfhrp3 deletions were monitored because some monoclonal antibodies utilized in PfHRP2-based RDTs cross-react with the PfHRP3 protein. We found that all 97 isolates from Guyana that met the inclusion criteria were both pfhrp2- and pfhrp3-positive. In Suriname (N = 78), 14% of the samples tested were pfhrp2-negative while 4% were pfhrp3-negative. Furthermore, analysis of the genomic region proximal to pfhrp2 and pfhrp3 revealed that genomic deletions extended to the flanking genes. We also investigated the population substructure of the isolates collected to determine if the parasites that had deletions of pfhrp2 and pfhrp3 belonged to any genetic subtypes. Cluster analysis revealed that there was no predominant P. falciparum population substructure among the isolates from either country, an indication of genetic admixture among the parasite populations. Furthermore, the pfhrp2-deleted parasites from Suriname did not appear to share a single, unique genetic background.     

SERPINB11 Frameshift Variant Associated with Novel Hoof Specific Phenotype in Connemara Ponies

Horses belong to the order Perissodactyla and bear the majority of their weight on their third toe; therefore, tremendous force is applied to each hoof. An inherited disease characterized by a phenotype restricted to the dorsal hoof wall was identified in the Connemara pony. Hoof wall separation disease (HWSD) manifests clinically as separation of the dorsal hoof wall along the weight-bearing surface of the hoof during the first year of life. Parents of affected ponies appeared clinically normal, suggesting an autosomal recessive mode of inheritance. A case-control allelic genome wide association analysis was performed (n_cases = 15, n_controls = 24). Population stratification (λ = 1.48) was successfully improved by removing outliers (n_controls = 7) identified on a multidimensional scaling plot. A genome-wide significant association was detected on chromosome 8 (p_raw = 1.37x10^-10, p_genome = 1.92x10^-5). A homozygous region identified in affected ponies spanned from 79,936,024-81,676,900 bp and contained a family of 13 annotated SERPINB genes. Whole genome next-generation sequencing at 6x coverage of two cases and two controls revealed 9,758 SNVs and 1,230 indels within the ~1.7-Mb haplotype, of which 17 and 5, respectively, segregated with the disease and were located within or adjacent to genes. Additional genotyping of these 22 putative functional variants in 369 Connemara ponies (n_cases = 23, n_controls = 346) and 169 horses of other breeds revealed segregation of three putative variants adjacent or within four SERPIN genes. Two of the variants were non-coding and one was an insertion within SERPINB11 that introduced a frameshift resulting in a premature stop codon. Evaluation of mRNA levels at the proximal hoof capsule (n_cases = 4, n_controls = 4) revealed that SERPINB11 expression was significantly reduced in affected ponies (p<0.001). Carrier frequency was estimated at 14.8%. This study describes the first genetic variant associated with a hoof wall specific phenotype and suggests a role of SERPINB11 in maintaining hoof wall structure.