Progressive Effect of α-Phenyl-N-Tert-Butyl Nitrone (PBN) on Rat Embryo Development in Vitro

In the present study we demonstrated the effects of the spin-trapping agent α-phenyl-N-tert-butylnitrone (PBN) on the in vitro development of rat embryos at the early stage. In rat embryos, PBN increased the speed of the first cleavage and had no toxicity during pregnancy after embryo culture. These results showed that reactive oxygen species (ROIs) that were formed by activating molecular oxygens through redox reactions regulated the speed of development for early-stage embryos. Thus, PBN caused a decrease in the level of ROIs and toxicity and an in increase in the level of the development of rat embryos. On the other hand, PBN could not decrease the 2-cell block in vitro nor increase the blastulation rate, in contrast to the fact that a scavenger of superoxide anions, SOD, is effective in doing so for mouse embryos. From these results it was concluded that free radicals play an important role in the in vitro development of rat embryos at the early stage, but play no role in the decrease of the 2-cell block or their blastulation rate. It should be noted that PBN had no toxicity for embryonic development at the 2-cell stage.     

Localized axis determinant in the early cleavage embryo of the goldfish, Carassius auratus

 The teleost dorsoventral axis cannot be distinguished morphologically before gastrulation. In order to examine whether the yolk cell affects axis determination, we bisect early cleavage embryos of the goldfish, Carassius auratus. When the vegetal yolk hemisphere is removed by bisection along the equatorial plane at the 2-cell stage, the embryos develop abnormally and exhibit a symmetrical morphology. No dorsal structures, such as notochord, somites and neural tube, differentiate and no embryonic shield is formed during gastrulation. In addition, no goosecoid mRNA is expressed before gastrulation. The frequency of abnormality decreases as the age at which the vegetal yolk hemisphere is removed increases. Most embryos removed at the 32-cell stage develop normally. Their morphological phenotype is similar to that of a Xenopus ventralized embryo generated by ultraviolet irradiation on the vegetal hemisphere soon after fertilization. We also observed that, when the embryos were bisected along the first cleavage plane at the 2-cell stage, the proportion of pairs of embryos of which one embryo developed normally was 44.8%. These results indicate that the vegetal yolk hemisphere of the early cleavage embryo of the goldfish contains axis determination factor(s), which are necessary for generation of dorsal structures. Furthermore, it is suggested that these determinant(s) are distributed asymmetrically within the vegetal yolk hemisphere. Received: 25 May 1996 / Accepted: 19 September 1996     

Isolation and Characterization of Mumps Virus Strains in a Mumps Outbreak with a High Incidence of Aseptic Meningitis

In 1993, mumps with a high incidence of aseptic meningitis became prevalent in Akita prefecture, Japan. Three mumps virus isolates obtained from the nonvaccine-associated cases lacked the BamHI restriction cleavage site of the P gene, like the Urabe strain (Yamada, A. et al, Vaccine 8: 553-557). However, four additional nucleotide substitutions were found in the determined region of 157 bp. Fourteen of 19 cases from which mumps virus showing the Urabe-like RFLP profile was detected were complicated with symptomatic meningitis, whereas there were only four cases of meningitis among 23 individuals infected with the wild type showing no Urabe-like RFLP profile (non-“Urabe-like” wild-type). The incidence of meningitis was over 70% among patients infected with the “Urabe-like” wild-type virus. The “Urabe-like” wild-type disappeared after February 1994 in the epidemic area and was replaced by the non-“Urabe-like” wild-type. Patients infected with the “Urabe-like” wild-type lived in a closed colony, in which there were two instances of transmission between siblings. Thus this outbreak was transient and narrowly localized.     

Effects of acute hyperthermia on the carotid baroreflex control of heart rate in humans

 The purpose of this study was to examine the effect of hyperthermia on the carotid baroreceptor-cardiac reflexes in humans. Nine healthy males underwent acute hyperthermia (esophageal temperature ∼38.0° C) produced by hot water-perfused suits. Beat-to-beat heart rate (HR) responses were determined during positive and negative R-wave-triggered neck pressure steps from +40 to −65 mm Hg during normothermia and hyperthermia. The carotid baroreceptor-cardiac reflex sensitivity was evaluated from the maximum slope of the HR response to changes in carotid distending pressure. Buffering capacity of the HR response to carotid distending pressure was evaluated in % from a reference point calculated as (HR at 0 mm Hg neck pressure−minimum HR)/HR range ×100. An upward shift of the curve was evident in hyperthermia because HR increased from 57.7±2.4 beats/min in normothermia to 88.7±4.1 beats/min in hyperthermia (P<0.05) without changes in mean arterial pressure. The maximum slope of the curve in hyperthermia was similar to that in normothermia. The reference point was increased (P<0.05) during hyperthermia. These results suggest that the sensitivity of the carotid baroreflex of HR remains unchanged in hyperthermia. However, the capacity for tachycardia response to rapid onset of hypotension is reduced and the capacity for bradycardia response to sudden hypertension is increased during acute hyperthermia. Received: 14 October 1996 /Revised: 16 January 1997 / Accepted: 21 January 1997     

An integrated method for the enrichment and selective isolation of Actinokineospora spp. in soil and plant litter

AIMS: To devise and evaluate a method for isolating the rare, zoosporic actinomycetes, Actinokineospora spp. in soil and plant litter. METHODS AND RESULTS: The newly developed method consists of two enrichment stages followed by plating on a selective medium. The source material is initially incubated with calcium carbonate to multiply the population of Actinokineospora spp., and is then air-dried. The second stage consists of rehydration-centrifugation, in which the amended substrate is immersed in phosphate buffer-soil extract to liberate actinomycete zoospores, and nonmotile microbial associates are then eliminated by centrifugation. Portions of the supernatant enriched with zoospores are plated on humic-acid vitamin agar supplemented with fradiomycin, kanamycin, nalidixic acid and trimethoprim. We examined 39 soil and plant-litter samples taken from fields, forests and stream banks. The proposed method consistently enriched and selectively isolated Actinokineospora spp. in 17 samples. Evidence for antimicrobial activity was found in most of the isolates. CONCLUSION: A combination of enrichment and a medium containing selective antibiotics can be used successfully for efficient isolation of certain rare actinomycete taxa. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of new methodologies with which to isolate rare actinomycetes is of great importance to extend our understanding of their ecology, taxonomy and bioactivity.     

Intermembrane distance in multilamellar vesicles of phosphatidylcholine depends on the interaction free energy between solvents and the hydrophilic segments of the membrane surface

To investigate the interaction of the surface of biomembranes with solvents systematically, we have studied the structure and phase behavior of multilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) in dimethylformamide (DMF)-water mixture by X-ray diffraction and differential scanning calorimetry. The solubility of phosphorylcholine, which is the same molecular structure as the head-group of phosphatidylcholine (PC), decreased with an increase in DMF concentration. This result indicates that DMF is a poor solvent for the hydrophilic segments of the surface of the PC membrane, and interaction free energy of the hydrophilic segments of the membrane surface with solvents increases with an increase in DMF concentration. X-ray diffraction data indicated that DPPC-MLVs were in the bilayer gel phase from 0 to 80% (v/v) DMF, and that the spacing (lamellar repeat period) and intermembrane distance of DPPC-MLV decreased with an increase in DMF concentration. Main transition temperature and pre-transition temperature of DPPC-MLV increased with an increase in DMF concentration, and above 50% (v/v) DMF there was no pre-transition. In the interaction of POPC-MLV with DMF, X-ray diffraction data indicated that POPC-MLVs were in L(alpha) phase (liquid-crystalline phase) from 0 to 80% (v/v) DMF, and that the spacing and intermembrane distance of POPC-MLV decreased with an increase in DMF concentration. These results are discussed by the change of the interaction free energy between the hydrophilic segments of the membrane surface and solvents. As DMF concentration increases, this interaction free energy may increase, resulting in the decrease of the intermembrane distance of PC-MLVs.     

Antibacterial activity of bovine lactoferrin and its peptides against enterohaemorrhagic Escherichia coli O157:H7

The antimicrobial activities of bovine lactoferrin (bLF), its pepsin hydrolysate (bLFH) and the active peptide lactoferricin^® B (LFcinB) against four clinical isolates of enterohaemorrhagic Escherichia coli O157:H7 were studied. The MICs against these isolates were 3 mg ml⁻¹ for bLF, 0·1–0·2 mg ml⁻¹ for bLFH and 8–10 μg ml⁻¹ for LFcinB in 1% Bactopeptone broth. LFcinB killed these bacteria within 3 h at concentrations above 10 μg ml⁻¹. Transmission electron microscopy findings suggested that LFcinB acts on the bacterial surface and affects cytoplasmic contents. LFcinB was shown to influence the levels of verotoxins in the culture supernatant fluid of an E. coli 0157:H7 strain. These results demonstrate that E. coli O157:H7 strains are susceptible to the antimicrobial effects of bLF and its peptides.     

Molecular characterization of thirteen gyrA mutations conferring nalidixic acid resistance in Bacillus subtilis

We isolated 607 independent nalidixic acid-resistant mutants from Bacillus subtilis. A 163 by DNA segment from a 5 portion of the gyrA gene was amplified from the DNA of each mutant strain. After heat denaturation, the product was subjected to gel electrophoresis to detect conformational polymorphism of single-strand DNA (PCR-SSCP analysis). Mobility patterns of the two DNA strands from all the mutant strains examined differed from those of the parental wild-type strains. The patterns were classified into 13 types, and the DNA sequence of each type was determined. A unique sequence alteration was found in mutants belonging to each of the 13 types, defining 13 gyrA alleles. Eight were single base pair substitutions, four were substitutions of two consecutive base pairs, and one was a substitution of three consecutive base pairs. Only three amino acid residues (Ser-84, Ala-85, and Glu-88) were altered in the deduced amino acid sequences of the mutated genes. We conclude that molecular typing based on the PCR-SSCP method is a powerful technique for the exhaustive identification of allelic variants among mutants selected for a phenotypic trait.     

Oncogene overexpression and de novo drug-resistance in human prostate cancer cells

We have isolated a variant [PC3(R)] of the human prostate PC3 tumor cell line which showed resistance to several anticancer drugs. Studies to evaluate the mechanisms of resistance to anticancer drugs in the PC3(R) cell line indicated that mdrl was not overexpressed. Studies also indicated that activities of topo I and topo II were not different in these cell lines, nor was there any difference in the formation of drug-induced KCl-SDS precipitable complexes, including that topoisomerases were not involved in the development of resistance in PC3(R) cells. While the activity of glutathione S-transferase and total glutathione levels were also similar in these cell lines, the glutathione peroxidase activity in PC3(R) cells was 5-fold lower than in PC3 cells. ] Furthermore, proto-oncogene expression for c-myc, and H-ras was significantly higher in resistant cell than in sensitive cells, indicating that the amplication of early response genes may play a role in the emergence of de novo resistance in PC3(R) cells.