Characterization of K+ Channels in the Basolateral Membrane of Rat Tracheal Epithelia

To study K⁺ channels in the basolateral membrane of chloride-secreting epithelia, rat tracheal epithelial monolayers were cultured on permeable filters and mounted into an Ussing chamber system. The mucosal membrane was permeabilized with nystatin (180 μg/ml) in the symmetrical high K⁺ (145 mm) Ringer solution. During measurement of the macroscopic K⁺ conductance properties of the basolateral membrane under a transepithelial voltage clamp, we detected at least two types of K⁺ currents: one is an inwardly rectifying K⁺ current and the other is a slowly activating outwardly rectifying K⁺ current. The inwardly rectifying K⁺ current is inhibited by Ba²⁺. The slowly activating K⁺ current was potentiated by cAMP and inhibited by clofilium, phorbol 12-myristae 13-acetate (PMA) and lowering temperature. This is consistent with the biophysical characteristics of I _SK channel. RT-PCR analysis revealed the presence of I _SK cDNA in the rat trachea epithelia. Although 0.1 mm Ba²⁺ only had minimal affect on short-circuit current (I _sc) induced by cAMP in intact epithelia, 0.1 mm clofilium strongly inhibited it. These results indicate that I _SK might be important for maintaining cAMP-induced chloride secretion in the rat trachea epithelia. Received: 1 March 1996/Revised: 5 August 1996     

Isolation of a root-specific cDNA encoding a ns-LTP-like protein from the roots of bean (Phaseolus vulgaris L.) seedlings

A root-specific cDNA clone, PVR3, was isolated from a bean (Phaseolus vulgaris L.) root cDNA library by a differential screening procedure. The nucleotide sequence of PVR3 contains an open reading frame coding for an 11.14 kDa polypeptide of 102 amino acid residues; the first 25 amino acids correspond to the sequence characteristic of a signal peptide. Comparison of the deduced PVR3 polypeptide sequence with the polypeptide sequences of previously cloned genes indicates that PVR3 may encode a ns-LTP-like protein. Molecular modelling of the PVR3 protein predicts that it has a three-dimensional structure that is similar to the three-dimensional model determined from the maize ns-LTP. The PVR3 mRNA accumulated mainly in the roots of young seedlings. It can be detected at low levels in flowers, but it is not detected in other organs. Genomic Southern blot analysis indicates that the genomic DNA corresponding to PVR3 cDNA is encoded by a single gene or small gene family in the bean genome.     

Development of in vitro matured and fertilized bovine oocytes co-cultured with Buffalo Rat Liver cells

This study was designed to evaluate the efficacy of Buffalo Rat Liver cells (BRLC) monolayers in supporting the development of in vitro matured and fertilized (IVM/IVF) bovine oocytes through to the hatched blastocyst stage compared to the commonly used co-culture system of bovine oviduct epithelial cells (BOEC). Cumulus oocyte complexes (COCs) obtained from 2- to 6-mm ovarian follicles at slaughter were matured for 24 h in TCM-199 supplemented with FBS and hormones (FSH, LH and estradiol 17-beta). In vitro fertilization (IVF) was performed using 1 x 10(6) percoll separated frozen-thawed spermatozoa in 1 ml of IVF-TL medium containing 18 to 20 matured oocytes. After 20 to 22 h of sperm exposure, 584 presumptive zygotes in 2 separate trials were randomly assigned to 3 treatment groups (BRLC co-culture, BOEC co-culture and control, consisting of medium alone). Zygotes were cultured in CZB media, a simple semi-defined medium, without glucose for the first 2 d, transferred to M199/FBS (TCM-199-HEPES supplemented with 20% HTFBS, 1 mM Sodium pyruvate), and cultured for an additional 8 days. Cleavage and development to morula and various blastocyst stages were recorded between d 3 and 11 after the start of IVF. Overall average cleavage rate was 75% (440 584 ) and did not vary across the treatments or trials. The proportion of embryos that reached the morula stage in both co-culture systems did not differ (P > 0.05) and was significantly higher (P > 0.05) compared to the control group. However, the percentage of the number of blastocysts, expanded blastocysts and hatched blastocysts varied across the treatment groups (P < 0.05), with the highest results obtained in the BRLC co-culture system. The production of blastocysts in BOEC co-culture was inconsistent between the 2 trials where a significant difference (40.6 vs 53.0%; P > 0.05) was observed. Rate of development to the blastocyst stage was similar between the 2 co-culture systems, with most of the embryos reaching the blastocyst stage by d 8 post insemination. The results of this study show that BRLC from a commercially available established cell line offer a more reliable alternative to a BOEC co-culture system for in vitro maturation, fertilization and culture of bovine embryos.     

Development of IVM-IVF produced 8-cell bovine embryos in simple,serum-free media after conditioning or Co-culture with buffalo rat liver cells

Development of 8-cell bovine embryos derived from in vitro matured/in vitro fertilized (IVM/IVF) oocytes was evaluated in two simple, serum-free media (CZB and SOM) with buffalo rat liver cells co-culture (BRLC) or after conditioning compared to a commonly used, serum-supplemented complex medium TCM-199. In a 3 x 4 factorial design, 578 eight-cell embryos were randomly assigned to 12 treatment groups. The factors were: first, type of culture medium (M199/FBS, CZBg and SOM), and second, the use of BRLC (as co-culture or to condition media for 24 hr and 48 hr) and unconditioned media. Development to morula was not affected by the type of medium, but co-culture and 48 hr conditioning within media type resulted in better development when compared to the 24-hr conditioned or unconditioned groups. Blastocyst development in SOM (38.9%) was different (P < 0.05) than in CZBg (46.6%) and M199/FBS (48.7%) and was lowest in the unconditioned group (27.8%) followed by 24 hr conditioned (33.3%), 48 hr (56.3%), and co-culture (59.6%). No blastocyst expansion was observed with unconditioned media and 24 hr conditioned SOM. Significant differences (P < 0.05) were found among all treatment groups except the co-culture and 48-hr conditioned groups. Hatching occurred only with co-culture and 48-hr conditioned groups of M199/FBS and CZBg media. These data show that CZB with glucose conditioned by BRLC monolayers for 48 hr can support the development of IVM/IVF produced bovine embryos to blastocyst compared to culture in TCM-199 with serum. © 1994 Wiley-Liss, Inc.     

Self-cleavage activity of the genomic HDV ribozyme in the presence of various divalent metal ions.

To identify the divalent metal ions that can support the self-cleavage activity of the genomic ribozyme of human hepatitis delta virus (HDV), we tested the activity of various divalent metal ions in the ribozyme reactions catalyzed by HDV88 (683-770 nt) and 88DI3 (HDV88 with the sequence from 740-752 nt deleted). Among various metal ions tested, Mg2+, Mn2+, Ca2+ and Sr2+ efficiently supported the self-cleavage reactions of the HDV88 and 88DI3 ribozymes. In the case of the 88DI3 ribozyme, other divalent metal ions, such as Cd2+, Ba2+, Co2+, Pb2+ and Zn2+, were also able to support the self-cleavage reaction to some extent (< 10%). In the presence of spermidine (0.5 mM), the cleavage reaction was promoted at lower concentrations of effective divalent metal ions. The HDV ribozyme represents the only example of ribozyme to date of a ribozyme that catalyzes the self-cleavage reaction in the presence of Ca2+ ions as efficiently as it does in the presence of Mg2+ ions.     

Secretion of nuclease across the outer membrane of Serratia marcescens and its energy requirements.

Extracellular secretion of Serratia marcescens nuclease occurs as a two-step process via a periplasmic intermediate. Unlike other extracellular proteins secreted by gram-negative bacteria by the general secretory pathway, nuclease accumulates in the periplasm in its active form for an unusually long time before its export into the growth medium. The energy requirements for extracellular secretion of nuclease from the periplasm were investigated. Our results suggest that the second step of secretion across the outer membrane is dependent upon the external pH; acidic pH effectively but reversibly blocks extracellular secretion. However, electrochemical proton gradient, and possibly ATP hydrolysis, are not required for this step. We suggest that nuclease uses a novel mechanism for the second step of secretion in S. marcescens.     

Crystallization,molecular replacement solution,and refinement of tetrameric β-amylase from sweet potato

Sweet potato β-amylase is a tetramer of identical subunits, which are arranged to exhibit 222 molecular symmetry. Its subunit consists of 498 amino acid residues (Mr 55,880). It has been crystallized at room temperature using polyethylene glycol 1500 as precipitant. The crystals, growing to dimensions of 0.4 mm × 0.4 mm × 1.0 mm within 2 weeks, belong to the tetragonal space group P4₂2₁2 with unit cell dimensions of a = b = 129.63 Å and c = 68.42 Å. The asymmetric unit contains 1 subunit of β-amylase, with a crystal volume per protein mass (V_M) of 2.57 ų/Da and a solvent content of 52% by volume. The three-dimensional structure of the tetrameric β-amylase from sweet potato has been determined by molecular replacement methods using the monomeric structure of soybean enzyme as the starting model. The refined subunit model contains 3,863 nonhydrogen protein atoms (488 amino acid residues) and 319 water oxygen atoms. The current R-value is 20.3% for data in the resolution range of 8–2.3 Å (with 2 σ cut-off) with good stereochemistry. The subunit structure of sweet potato β-amylase (crystallized in the absence of α-cyclodextrin) is very similar to that of soybean β-amylase (complexed with α-cyclodextrin). The root-mean-square (RMS) difference for 487 equivalent Cα atoms of the two β-amylases is 0.96 Å. Each subunit of sweet potato β-amylase is composed of a large (α/β)₈ core domain, a small one made up of three long loops [L3 (residues 91–150), LA (residues 183–258), and L5 (residues 300–327)], and a long C-terminal loop formed by residues 445–493. Conserved Glu 187, believed to play an important role in catalysis, is located at the cleft between the (α/β)₈ barrel core and a small domain made up of three long loops (L3, L4, and L5). Conserved Cys 96, important in the inactivation of enzyme activity by sulfhydryl reagents, is located at the entrance of the (α/β)₈ barrel. © 1995 Wiley-Liss, Inc.     

Angiotensin-converting enzyme inhibitory and antihypertensive activities of pivalopril (RHC 3659-(S))

Pivalopril (RHC 3659-(S); (S)-N-cyclopentyl-N-(2-methyl-3-pivaloylthiopropionyl) glycine) is a new compound with a hindered sulfur group that has been compared to captopril for oral angiotensin-converting enzyme (ACE) inhibition in rats and dogs and antihypertensive activity in rats. In separate groups of conscious normotensive rats, pivalopril (0.03-1.0 mg/kg, orally [p.o.]) produced a dose-related antagonism of angiotensin I (AngI)-induced pressor effects. The ED50 for pivalopril and captopril was 0.1 mg/kg. In conscious normotensive dogs, pivalopril (incremental doses of 0.01-1.0 mg/kg, p.o.) produced a dose-related antagonism of AngI pressor effects. The ED50 was 0.17 mg/kg for pivalopril and 0.06 mg/kg for captopril. At equieffective doses the two compounds had similar durations of action. In sodium-deficient, conscious spontaneously hypertensive rats (SHR), pivalopril (1-100 mg/kg, p.o.) produced a dose-related reduction in mean arterial pressure. The potency and duration were similar to those of captopril. In the sodium-replete SHR, 5 days of oral dosing with pivalopril, 100 mg/(kg . day), decreased mean arterial pressure more effectively than captopril, 100 mg/(kg . day). No tolerance developed to the antihypertensive effect of either drug. It is concluded that pivalopril is a potent, orally effective ACE inhibitor and antihypertensive agent.