Immunohistochemical localization to pyloric antral G cells of peptides derived from porcine preprogastrin

Antibodies to the peptides (designated cryptic A and B) that flank the G34 region of pig progastrin were used in immunohistochemical studies of the gastrointestinal tract. In elution and restaining experiments, the same cells were revealed by the cryptic peptide antibodies, and by antibodies specific for C-terminus of G17 and N-terminus of G34. The cells reacting with the cryptic peptide antibodies were localized predominantly to antral mucosa. They were found in pig, ferret, dog and cat but not in man, guinea pig, rat or mouse; presumably in the latter species there are amino acid substitutions in the cryptic peptides that influence immunoreactivity with the present antibodies. The results indicate that progastrin production occurs only in G cells in the gut, and that a single population of cells produces all the predicted regions of progastrin.     

Determination of Leb-active oligosaccharides in urine of pregnant and lactating women by radioimmunoassay.

Antiserum obtained by immunizing a goat with lacto-N-difucohexaose I, a Leb blood-group hapten, coupled to poly-L-lysine was used in a radioimmunoassay to detect Leb-active oligosaccharides (chiefly lacto-N-difucohexaose I) in urine of 138 pregnant and lactating women of different ABO and Lewis blood groups. Specificity of the method was determined by comparing inhibitory activities of 18 oligosaccharides. Only women who belonged to the Leb blood group excreted Leb-active oligosaccharides in urine. Leb-active oligosaccharides increase during pregnancy, reaching levels up to approximately 70 mumol lacto-N-difucohexaose I equivalents per pmol creatinine in the third trimester and early post-partum period. Excretion varies considerably but tends to be highest in those individuals who strongly express the Leb antigen on their red blood cells or who belong to blood group O. Leb-active oligosaccharides were detected in plasma of a few individuals but at concentrations 1000-fold lower than in urine.     

Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system.     

Quantified proarrhythmic potential of selected human embryonic stem cell-derived cardiomyocytes

To improve proarrhythmic predictability of preclinical models, we assessed whether human ventricular-like embryonic stem cell-derived cardiomyocytes (hESC-CMs) can be selected following a standardized protocol. Also, we quantified their arrhythmogenic response and compared this to a contemporary used rabbit Purkinje fiber (PF) model. Multiple transmembrane action potentials (AP) were recorded from 164 hESC-CM clusters (9 different batches), and 12 isolated PFs from New Zealand White rabbits. AP duration (APD), early afterdepolarizations (EADs), triangulation (T), and short-term variability of repolarization (STV) were determined on application of the I_Kr blocker E-4031 (0.03/0.1/0.3/1 μM). Isoproterenol (0.1 μM) was used to assess adrenergic response. To validate the phenotype, RNA isolated from atrial- and ventricular-like clusters (n = 8) was analyzed using low-density Taqman arrays. Based on initial experiments, slow beating rate (< 50 bpm) and long APD (> 200 ms) were used to select 31 ventricular-like clusters. E-4031 (1 μM) prolonged APD (31/31) and induced EADs only in clusters with APD90 > 300 ms (11/16). EADs were associated with increased T (1.6 ± 0.2 vs 2.0 ± 0.3?) and STV (2.7 ± 1.5 vs 6.9 ± 1.9?). Rabbit PF reacted in a similar way with regards to EADs (5/12), increased T (1.3 ± 0.1 vs 1.9 ± 0.4?), and STV (1.2 ± 0.9 vs 7.1 ± 5.6?). According to ROC values, hESC-CMs (STV 0.91) could predict EADs at least equivalent to PF (STV 0.69). Isoproterenol shortened APD and completely suppressed EADs. Gene expression analysis revealed that HCN1/2, KCNA5, and GJA5 were higher? in atrial/nodal-like cells, whereas KCNJ2 and SCN1B were higher? in ventricular-like cells (?P < 0.05). Selection of hESC-CM clusters with a ventricular-like phenotype can be standardized. The proarrhythmic results are qualitatively and quantitatively comparable between hESC-CMs and rabbit PF. Our results indicate that additional validation of this new safety pharmacology model is warranted.     

Determination of small amounts of noradrenaline using a fluorescence histochemical method

Summary Using the Hayashi method, the distribution of glucosaminidase was studied throughout sequential molar development. Conspicuous activity was observed in certain cellular components of the stellate reticulum and dental papilla. The outer enamel epithelium, stratum intermedium and ameloblasts displayed less intense reactions.Supported by PHS Grant No. 2800-02 —Tooth Germ Development, National Institute of Dental Research, National Institutes of Health.Lieutenant Commander, Dental Corps, United States Navy.     

Analysis of binding properties and specificity through identification of the interface forming residues (IFR) for serine proteases <Emphasis Type="Italic">in silico</Emphasis> docked to different inhibitors

Background  

Enzymes belonging to the same super family of proteins in general operate on variety of substrates and are inhibited by wide selection of inhibitors. In this work our main objective was to expand the scope of studies that consider only the catalytic and binding pocket amino acids while analyzing enzyme specificity and instead, include a wider category which we have named the Interface Forming Residues (IFR). We were motivated to identify those amino acids with decreased accessibility to solvent after docking of different types of inhibitors to sub classes of serine proteases and then create a table (matrix) of all amino acid positions at the interface as well as their respective occupancies. Our goal is to establish a platform for analysis of the relationship between IFR characteristics and binding properties/specificity for bi-molecular complexes.     

Effects of the parkinsonism-inducing neurotoxin MPTP and its metabolite MPP+ on sympathetic adrenergic nerves in mouse iris and atrium

The effect of systemic administration of the parkinsonism-inducing neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and its metabolite MPP+ (1-methyl-4-phenylpyridine) on sympathetic adrenergic nerves in mouse iris and atrium has been investigated employing histo- and neurochemical techniques. The results indicate that MPTP does not have any potent neurotoxic effects on sympathetic adrenergic nerves. The effects of MPTP noted appear mainly to be restricted to a noradrenaline (NA) -depleting action and an acutely transient impairment of the NA uptake mechanism. This latter effect could be counteracted by monoamine oxidase inhibition. MPP+ was found to have more potent neurotoxic actions than MPTP as reflected i.e. by a patchy loss of histochemically demonstrable adrenergic nerves in iris which persisted for at least 7 days. Pretreatment with the NA uptake blocker desipramine antagonised the effects of MPP+, indicating that neurotoxicity is mediated via the NA uptake mechanism. The difference in neurotoxic potency of MPTP between sympathetic adrenergic nerves and central catecholamine neurons might be related to differences in metabolism of MPTP in the CNS and the periphery and/or due to the sympathetic adrenergic nerves being more resistant towards the cytotoxic actions following MPTP administration.