Potent and selective oxytocin receptor agonists without disulfide bridges

Oxytocin (OT) is a neuropeptide involved in a wide variety of physiological actions, both peripherally and centrally. Many human studies have revealed the potential of OT to treat autism spectrum disorders and schizophrenia. OT interacts with the OT receptor (OTR) as well as vasopressin 1a and 1b receptors (V_1aR, V_1bR) as an agonist, and agonistic activity for V_1aR and V_1bR may have a negative impact on the therapeutic effects of OTR agonism in the CNS. An OTR-selective agonistic peptide, FE 202767, in which the structural differences from OT are a sulfide bond instead of a disulfide bond, and N-alkylglycine replacement for Pro at position 7, was reported. However, the effects of amino acid substitutions in OT have not been comprehensively investigated to compare OTR, V_1aR, and V_1bR activities. This led us to obtain a new OTR-selective analog by comprehensive amino acid substitutions of OT and replacement of the disulfide bond. A systematic amino acid scanning (Ala, Leu, Phe, Ser, Glu, or Arg) of desamino OT (dOT) at positions 2, 3, 4, 5, 7, and 8 revealed the tolerability for the substitution at positions 7 and 8. Further detailed study showed that trans-4-hydroxyproline (trans-Hyp) at position 7 and γ-methylleucine [Leu(Me)] at position 8 were markedly effective for improving receptor selectivity without decreasing the potency at the OTR. Subsequently, a combination of these amino acid substitutions with the replacement of the disulfide bond of dOT analogs with a sulfide bond (carba analog) or an amide bond (lactam analog) yielded several promising analogs, including carba-1-[trans-Hyp⁷,Leu(Me)⁸]dOT (14) with a higher potency (7.2 pM) at OTR than that of OT and marked selectivity (>10,000-fold) over V_1aR and V_1bR. Hence, we investigated comprehensive modification of OT and obtained new OT analogs that exhibited high potency at OTR with marked selectivity. These OTR-selective agonists could be useful to investigate OTR-mediated effects on psychiatric disorders.     

NeuN immunoreactivity in the brain of Xenopus laevis

Neuronal nuclear antigen (NeuN), discovered in mice brain cell nuclei by Mullen et al. (1992), is used as an excellent marker of post-mitotic neurons in vertebrates. In this study, the expression pattern of NeuN was examined in the Xenopus brain to explore phylogenetic differences in NeuN expression. Anti-NeuN antibody showed selective staining in mouse and Xenopus brain extracts, but the number and molecular weight of the bands differed in Western blotting analysis. In immunostaining, anti-NeuN antibody showed selective staining of neurons, but not glial cells, in the Xenopus brain. Most neurons, including olfactory bulb mitral cells and cerebellar Purkinjie cells, which show no immunoreactivity in birds/mammals, showed NeuN immunoreactivity in Xenopus. This study revealed that anti-NeuN antibody is a useful marker of post-mitotic neurons in amphibians, but it also stains neurons that show no reactivity in more derived animals.     

Genetic diversity and conservation units in wild and captive populations of endangered freshwater fishes: a case of <Emphasis Type="Italic">Hemigrammocypris rasborella</Emphasis> in Shizuoka,Japan

Population structure and genetic diversity were examined using partial mitochondrial cytochrome b gene sequences of four wild, one reintroduced, and five captive populations of the endangered cyprinid Hemigrammocypris rasborella from three river systems in the easternmost region of the species’ range in Shizuoka Prefecture, central Honshu, Japan. We detected loss of genetic diversity from portions of the wild and captive populations, as well as suspected nonindigenous haplotypes in some captive, reintroduced, and even wild populations. Given the population structure revealed, we suggest that the populations should be managed with consideration for both the endemism and viability (avoidance of inbreeding depression) of the local populations.     

Mitogenic and adjuvant activities of polysaccharides from the cellular slime mold,Dictyostelium discoideum NC-4

Cell surface and extracellular polysaccharide fractions obtained from Dictyostelium discoideum NC-4 cultured in bacteria-free medium showed strong B-cell mitogenic activities. Upon periodate treatment of the extracellular polysaccharide fraction this activity completely disappeared. The extracellular polysaccharide fraction could also enhance the antibody response in vitro against sheep red blood cells.     

Two novel gene mutations (Glu174→Lys,Phe383→Tyr) causing the “hepatic” form of carnitine palmitoyltransferase II deficiency

Carnitine palmitoyltransferase II (CPT II) deficiency has two different clinical forms, one with “hepatic” and the other with “muscular” symptoms. We studied the molecular basis of the “hepatic” form in two Japanese siblings. Their CPT II activity in lymphoblasts was reduced to 3% of the level observed in normal controls. cDNA analysis showed that the proband was a compound heterozygote. One allele carried a new mutation, G621→A (Glu174→Lys). The other carried three single-base substitutions; a new mutation, T1249→A (Phe383→Tyr), and two previously reported polymorphisms. The brother had the same four substitutions. Neither of the two new mutations in this study was detected in the 60 alleles of 30 Japanese control subjects. Secondary structure prediction analysis of the mutated CPT II protein was different from that of the normal protein. We concluded that these mutations caused the “hepatic” form of CPT II deficiency in the probands. Received: 16 October 1995     

Guidepost neurons for the lateral olfactory tract: Expression of metabotropic glutamate receptor 1 and innervation by glutamatergic olfactory bulb axons

The guidepost neurons for the lateral olfactory tract, which are called lot cells, are the earliest‐generated neurons in the neocortex. They migrate tangentially and ventrally further down this tract, and provide scaffolding for the olfactory bulb axons projecting into this pathway. The molecular profiles of the lot cells are largely uncharacterized. We found that lot cells specifically express metabotropic glutamate receptor subtype‐1 at a very early stage of development. This receptor is functionally competent and responds to a metabotropic glutamate receptor agonist with a transient increase in the intracellular calcium ion concentration. When the glutamatergic olfactory bulb axons were electrically stimulated, lot cells responded to the stimulation with a calcium increase mainly via ionotropic glutamate receptors, suggesting potential neurotransmission between the axons and lot cells during early development. Together with the finding that lot cells themselves are glutamatergic excitatory neurons, our results provide another notable example of precocious interactions between the projecting axons and their intermediate targets. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

Dewatering of microalgae suspensions by cake filtration with filter cloths

Journal of Applied Phycology - Microalgae are a promising bioresource because they produce various materials that can be used as fuels, nutraceuticals, cosmetics, and pharmaceuticals. Additionally,...     

Heterogeneity of Hippo signalling activity in different histopathologic subtypes of renal cell carcinoma

This study aimed to reveal the prognostic role of the Hippo pathway in different histopathological subtypes of renal cell carcinoma (RCC). The TCGA-KIRC (n = 537), TCGA-KIRP (n = 291) and TCGA-KICH (n = 113), which contain data about clear cell (ccRCC), papillary (pRCC) and chromophobe RCC (chRCC), respectively, were investigated. Gene Set Variation Analysis was used to compare the activity of many pathways within a single sample. Oncogenic pathway-related expression differed between cases of ccRCC involving low and high Hippo pathway activity. There were two subsets of ccRCC, in which the cancer exhibited lower and higher Hippo signalling activity, respectively, compared with normal tissue. In the ccRCC cohort, lower Hippo pathway activity was associated with a higher clinical stage (p < 0.001). The Hippo pathway (HR = 0.29; 95% CI = 0.17–0.50, p < 0.001), apoptosis (HR = 6.02; 95% CI = 1.47–24.61; p = 0.013) and the p53 pathway (HR = 0.09; 95% CI = 0.02–0.36; p < 0.001) were identified as independent prognostic factors for ccRCC. The 5-year overall survival of the ccRCC patients with low and high Hippo pathway activity were 51.9% (95% CI = 45.0–59.9) and 73.6% (95% CI = 67.8–79.9), respectively. In conclusion, the Hippo pathway plays an important role in the progression of ccRCC. Low Hippo pathway activity is associated with poor outcomes in ccRCC, indicating the tumour suppressor function of this pathway.     

Effects of subclass change on the structural stability of chimeric,humanized, and human antibodies under thermal stress

To address how changes in the subclass of antibody molecules affect their thermodynamic stability, we prepared three types of four monoclonal antibody molecules (chimeric, humanized, and human) and analyzed their structural stability under thermal stress by using size‐exclusion chromatography, differential scanning calorimetry (DSC), circular dichroism (CD), and differential scanning fluoroscopy (DSF) with SYPRO Orange as a dye probe. All four molecules showed the same trend in change of structural stability; the order of the total amount of aggregates was IgG1 < IgG2 < IgG4. We thus successfully cross‐validated the effects of subclass change on the structural stability of antibodies under thermal stress by using four methods. The T_h values obtained with DSF were well correlated with the onset temperatures obtained with DSC and CD, suggesting that structural perturbation of the CH2 region could be monitored by using DSF. Our results suggested that variable domains dominated changes in structural stability and that the physicochemical properties of the constant regions of IgG were not altered, regardless of the variable regions fused.     

New diagnostic techniques for the detection of organic acidemias

We describe an improved procedure for the extraction of organic acids which is equally suitable for urine, plasma and amniotic fluid and gives similar high recoveries for a wide variety of organic acids, without interferences from phosphate, sulfate, or urea. Internal standards are added, the oxoacids are converted to the pentafluorobenzyloximes; the sample is concentrated, acidified, chromatographed on a small column of silicic acid, and the effluent neutralized and dried. After forming the trimethylsilyl derivatives, gas chromatography is done on a 0.53 mm X 30 m low-polarity bonded-phase fused silica capillary column. For gas chromatographic-mass spectrometric quantification, the peak areas at the m/z of a characteristic fragment ion for each acid relative to the peak areas of ions of the internal standards are related to standard curves.