Discovery of potent, selective, orally active benzoxazepine-based Orexin-2 receptor antagonists

During our efforts to identify a series of potent, selective, orally active human Orexin-2 Receptor (OX2R) antagonists, we elucidated structure-activity relationship (SAR) on the 7-position of a benzoxazepine scaffold by utilizing Hammett σ(p) and Hansch-Fujita π value as aromatic substituent constants. The attempts led to the discovery of compound 1m, possessing good in vitro potency with over 100-fold selectivity against OX1R, good metabolic stability in human and rat liver microsome, good oral bioavailability in rats, and in vivo antagonistic activity in rats by oral administration.     

Urea sensitization caused by separation of Helicobacter pylori RNA polymerase beta and beta' subunits

BACKGROUND: The beta and beta' subunits of RNA polymerase are fused in all Helicobacters, but separate in most other taxa. Prior studies had shown that this fusion is not essential for viability in culture or in vivo, but had not tested it for potentially important quantitative effects on phenotype. METHODS: The effect of separating rpoB and rpoC sequences on Helicobacter pylori growth was tested in culture and during mouse infection. RESULTS: Derivatives of strains X47 and SS1 carrying this "rpoBCsplit" allele colonized mice less vigorously than their wild-type parents in competition tests. With X47 rpoBCsplit, this reduced vigor was evident in wild-type mice, whereas with SS1 rpoBCsplit it was seen only in cytokine IL-10- and IL-12beta-deficient mice. In culture, the rpoBCsplit allele sensitized each of four strains tested (X47, SS1, 88-3887, and AM1) to urea, a metabolite that is secreted into the gastric mucosa; urea sensitization was more severe in X47 than in SS1 genetic backgrounds. The rpoBCsplit allele also caused poorer growth on Ham's F12 agar, a nutritionally limiting medium, but had little effect on sensitivity to mild acidity. CONCLUSIONS: H. pylori's normal RNA polymerase beta-beta' subunit fusion contributes quantitatively to fitness. We propose that urea, although important to H. pylori in vivo, also be considered inhibitory; and that H. pylori's natural beta-beta' subunit fusion helps it cope with urea exposure.     

Function of Pax1 and Pax9 in the sclerotome of medaka fish

We examined the expression and functions of Pax1 and Pax9 in a teleost fish, the medaka Oryzias latipes. While Pax1 and Pax9 show distinct expression in the sclerotome in amniotes, we could not detect the differential expression of Pax1 and Pax9 in the developing sclerotome of the medaka. Furthermore, unlike the mouse, in which Pax1 is essential for development of the vertebral body, and where the neural arch is formed independent of either Pax1 or Pax9, our morpholino knockdown experiments revealed that both Pax1 and Pax9 are indispensable for the development of the vertebral body and neural arch. Therefore, we conclude that after gene duplication, Pax1 and Pax9 subfunctionalize their roles in the sclerotome independently in teleosts and amniotes. In Stage-30 embryo, Pax9 was strongly expressed in the posterior mesoderm, as was also observed for mouse Pax9. Since this expression was not detected for Pax1 in the mouse or fish, this new expression in the posterior mesoderm likely evolved in Pax9 of ancestral vertebrates after gene duplication. Two-month-old fish injected with Pax9 morpholino oligonucleotide showed abnormal morphology in the tail hypural skeletal element, which may have been related to this expression.     

Some common properties of lectins from marine algae

Twelve kinds of lectins isolated from four species of marine algae, Boodlea coacta (Chlorophyta) and Hypnea japonica, Carpopeltis flabellata and Solieria robusta (Rhodophyta), were compared for their chemical and biological properties. These lectins were proteins or glycoproteins, similar to terrestrial plant lectins. However, unlike most terrestrial plant lectins, they had a small molecular size (4,200 to 25,000 daltons), were mostly monomeric, and had no affinity for monosaccharides. They strongly agglutinated trypsin-treated rabbit erythrocytes, and their activities commonly were inhibited by glycoproteins bearing N-glycans. From hemagglutination-inhibition tests with various glycoproteins and related compounds, it was found that B. coacta lectins recognize high-mannose N-glycans; H. japonica lectins complex N-glycans, and C. flabellata and S. robusta lectins recognize both types of N-glycans.     

Preliminary crystallographic study of phospholipase A2 from the venom of Trimeresurus flavoviridis (habu snake)

Crystallization and a preliminary crystallographic study of Trimeresurus flavoviridis (habu snake) phospholipase A2 (PLA2) were carried out. Although crystals were obtained from various solutions, crystals suitable for X-ray analysis could be obtained from polyethylene glycol solutions only when a repeated seeding technique was applied starting from twinned crystals. The crystal is monoclinic with space group P21, with a = 44.1, b = 55.7, c = 48.8 A, and beta = 92.4 degrees. An asymmetric unit contains a dimer consisting of two identical subunits made of 122 amino acids. The crystal reflects X-rays beyond 2.5 A. A Pt derivative gave a good isomorphous crystal.     

Functional assembly of the Na+/H+ antiporter of Helicobacter pylori from partial fragments in vivo

Functional assembly of the Helicobacter pylori Na+/H+ antiporter (HPNhaA) from partial fragments was studied. Expression plasmids encoding a series of complementary N- and C-terminal fragment pairs containing the transmembrane domains (TMs) were constructed by inserting a stop or a start codon into each of the loop regions of NhaA. HPNhaA fragments alone or complementary fragment pairs were expressed in DeltanhaA Escherichia coli, and fragment integration into the membrane and antiporter activity were measured. TM1-10, TM1-11, TM2-12, TM6-12, and TM10-12 were found in the membrane fraction, while the other fragments were not. While no single fragment displayed antiporter activity, simultaneous expression of fragments in certain pairs, such as TM1-2 + TM3-12, TM1-8 + TM9-12, or TM1-11 + TM12, reconstituted antiporter activity. With the exception of TM12, all of the fragments in the pairs were detected in the membrane. No single fragments expressed alone for these pairs were found in the membrane, except for TM1-11, suggesting that the interaction between the fragments in these pairs stabilized the fragments and enabled reconstitution of HPNhaA. We also found that the simultaneous expression of three complementary fragments (TM1-2 + TM3-8 + TM9-12) reconstituted HPNhaA activity. Other pairs that were found in the membrane (TM1-5 + TM6-12, TM1-10 + TM11-12, and TM1 + TM2-12) did not reconstitute antiporter activity, suggesting that they may not have the proper conformation. These results revealed that the ability to reconstitute antiporter activity depends on the split position in the loop regions and the interaction between complementary fragment pairs. We propose that formation of the active HPNhaA molecule is initiated by the interaction of short-lived intermediates and maintained by the increased stability of the intermediates within the resulting complex.     

Phosphopeptide enrichment by aliphatic hydroxy acid-modified metal oxide chromatography for nano-LC-MS/MS in proteomics applications

We developed novel methods for phosphopeptide enrichment using aliphatic hydroxy acid-modified metal oxide chromatography (MOC). Titania and zirconia were successfully applied to enrich phosphopeptides with the aid of aliphatic hydroxy acids, such as lactic acid and beta-hydroxypropanoic acid, to reduce the interaction between acidic non-phosphopeptides and the metal oxides. These methods removed the vast majority of non-phosphopeptides from phosphoprotein standard digests, and large numbers of phosphopeptides could be readily identified. The methods were coupled with nano-LC-MS/MS systems without difficulty. Recovery of phosphopeptides in MOC varied greatly from peptide to peptide, ranging from a few percent to 100%, and the average was almost 50%. Repeatability and linearity were satisfactory. In an examination of the cytoplasmic fraction of HeLa cells, more than 1000 phosphopeptides were identified using lactic acid-modified titania MOC and beta-hydroxypropanoic acid-modified zirconia MOC, respectively. The overlap between phosphopeptides enriched by these two methods was 40%, and the combined results provided 1646 unique phosphopeptides. To our knowledge, this is the first successful application of a single MOC-based approach to phosphopeptide enrichment from complex biological samples such as cell lysates.     

Modulation of control of muscle sympathetic nerve activity during orthostatic stress in humans

We tested the hypothesis that orthostatic stress would modulate the arterial baroreflex (ABR)-mediated beat-by-beat control of muscle sympathetic nerve activity (MSNA) in humans. In 12 healthy subjects, ABR control of MSNA (burst incidence, burst strength, and total activity) was evaluated by analysis of the relation between beat-by-beat spontaneous variations in diastolic blood pressure (DAP) and MSNA during supine rest (CON) and at two levels of lower body negative pressure (LBNP: -15 and -35 mmHg). At -15 mmHg LBNP, the relation between burst incidence (bursts per 100 heartbeats) and DAP showed an upward shift from that observed during CON, but the further shift seen at -35 mmHg LBNP was only marginal. The relation between burst strength and DAP was shifted upward at -15 mmHg LBNP (vs. CON) and further shifted upward at -35 mmHg LBNP. At -15 mmHg LBNP, the relation between total activity and DAP was shifted upward from that obtained during CON and further shifted upward at -35 mmHg LBNP. These results suggest that ABR control of MSNA is modulated during orthostatic stress and that the modulation is different between a mild (nonhypotensive) and a moderate (hypotensive) level of orthostatic stress.     

Preoperative detection of pleural adhesions by respiratory dynamic computed tomography

Background

Video-assisted thoracic surgery (VATS) plays an important role in thoracic surgery because it is less invasive. However, the existence of severe pleural adhesions may make VATS difficult and complicated. The aim of this study was to assess the utility of inspiration and expiration computed tomography (respiratory dynamic CT (RD-CT)) in evaluation of pleural adhesions preoperatively.

Methods

RD-CT was performed on 107 patients undergoing thoracotomies (both VATS and open). We assessed synchronous motion during respiration on RD-CT. Comparing the results of RD-CT and intraoperative findings, we assessed the utility of preoperative evaluation.

Results

A negative correlation between sliding score and adhesion grade was revealed. Sliding score in adhesion negative patients was significantly higher than that in adhesion positive patients (P?<?0.0001). The sensitivity of RD-CT was 63.6%, specificity was 74.1%, and accuracy was 72%. Among 62 patients with a CT-Respiration Ratio of less than 0.65, the sensitivity of RD-CT was 77.8%, specificity was 86.8%, and accuracy was 85.5%.

Conclusions

RD-CT may be clinically useful for detecting the presence of pleural adhesions. It can be adopted as one of the criteria for deciding the surgical approach.

     

Prosaposin Facilitates Sciatic Nerve Regeneration In Vivo

Abstract: Prosaposin, a multifunctional protein, is the precursor of saposins, which activate sphingolipid hydrolases. In addition to acting as a precursor for saposins, prosaposin has been shown to rescue hippocampal CA1 neurons from lethal ischemic damage in vivo and to promote neurite extension of neuroblastoma cells in vitro. Here we show that prosaposin, when added to a collagen-filled nerve guide after sciatic nerve transection in guinea pigs, increased dramatically the number of regenerating nerve fibers within the guide. To identify the target neurons of prosaposin during peripheral nerve regeneration, we determined the degree of atrophy and chromatolysis of neurons in the spinal anterior horn and dorsal root ganglia on the prosaposin-treated and untreated side. The effect of prosaposin on large spinal neurons and small neurons of the dorsal root ganglion was more conspicuous. Subsequent immunohistochemistry demonstrated that the atrophy of cholinergic large neurons in the anterior horn is prevented to significant extent by prosaposin treatment. These findings suggest that prosaposin promotes peripheral nerve regeneration by acting on α-motor neurons in the anterior horn and on small sensory neurons in the dorsal root ganglion. The present study raises the possibility of using prosaposin as a tool for the treatment of peripheral nerve injuries.