First cleavage of enucleated rat eggs following transplantation of karyoplast removed from pronuclear eggs stored at 2 to 6 degrees C for various durations.

Pronuclear rat eggs were cultured for 24 to 48 hours at 37 degrees C after storage at 2 to 6 degrees C for 0 to 216 hours in medium. Very high proportions (89 to 97%) of eggs cleaved to the two-cell stage after storage for 0 to 48 hours. The proportion, however, decreased rapidly in eggs stored for 72 hours (60%), and eggs stored for more than 120 hours cleaved poorly. However, when male and female pronuclei from stored eggs were transplanted into enucleated fresh eggs, 92 to 100% of the fused eggs with karyoplast stored for 0 to 144 hours cleaved. Although the cleavage rate was reduced to 50% when karyoplast from eggs stored for 168 hours was transplanted, this reduction was not significant. Complete loss of cleaving ability was observed in fused eggs with the karyoplast stored for 216 hours. These results clearly indicate that the pronuclei of rat eggs can be stored for a longer period than the cytoplasm at low temperatures (2 to 6 degrees C).

Expression of hemagglutinin of Haemophilus paragallinarum serotype A in Escherichia coli.

The genomic DNA of Haemophilus paragallinarum (Hpg) serotype A strain 221 was cloned into vector plasmid pBR322. The recombinant plasmids were introduced into Escherichia coli (E. coli) strain C600. Subsequently, a total of 277 transformants were obtained. One, designated strain no. 6, expressed hemagglutination activity against chicken erythrocytes. Strain no. 6 contained the recombinant plasmid pNV102, and DNA of about 2.57 kb was inserted into pNV102. When strain no. 6 was cured of pNV102, the strain lost hemagglutination activity. When the cured strain was retransformed with pNV102, hemagglutination activity was restored. E. coli strain no. 6 reacted with monoclonal antibody specific to the hemagglutinin of Hpg serotype A in a dot-blotting analysis. Chickens immunized with the inactivated strain no. 6 produced the hemagglutination inhibition (HI) antibody, and chickens possessing the HI antibody showed protection against challenge exposure by Hpg strain 221.

Detection of plasmid DNA in Erysipelothrix rhusiopathiae isolated from pigs with chronic swine erysipelas.

Forty-three strains of Erysipelothrix rhusiopathiae, isolated from pigs with chronic swine erysipelas, were examined for the presence of plasmid DNA by agarose gel electrophoresis and electron microscopy. Seven of these strains were found to contain plasmids of which number were varied from 1 to 6. The plasmids ranged from 1.4 to 86 kb in size. This is the first reported evidence for plasmid DNA in E. rhusiopathiae. The functions of the plasmids are unknown at present.

The boundaries of business: commentaries from the experts.

The World Leadership Survey, which began a worldwide dialogue on a set of important issues facing managers in the 1990s, continues with commentaries from four recognized experts, each of whom addresses the survey results from a different perspective. Kenichi Ohmae, chairman of McKinsey and Company in Tokyo, addresses "The Perils of Protectionism." Ohmae argues that the old definitions of national boundaries and corporate interests reflect obsolete economic theories. The real test of national well-being, Ohmae suggests, should be the economic welfare of a nation's citizens. Sylvia Ann Hewlett, economist and former director of the Economic Policy Council in New York, analyzes the survey in terms of "The Human Resource Deficit." According to Hewlett, four principles should guide corporate strategies in the 1990s: human resource development should move up the scale of corporate priorities; a family-friendly workplace will attract and keep talented workers; companies will take limited direct responsibility for training and education; the private sector will promote public investment in social issues. James E. Austin, the Richard P. Chapman Professor of Business at the Harvard Business School, writes about "The Developing-Country Difference." In developing countries, Austin observes, managers display attitudes and follow practices that diverge from those in developed nations. In particular, the role of government, investments in education and technology, and environmental concerns set these nations apart. Michel Crozier, president of the Centre de Sociologie des Organisations in Paris, writes about "The Changing Organization." In the 1990s, Crozier argues, managers need to break from old management theories and practice, questioning hierarchy, control, distance, access to information-the whole managerial system.

Decrease in antimicrobial susceptibility of Bordetella bronchiseptica caused by antigenic modulation and phase variation.

The susceptibilities to 20 antimicrobial agents of Bordetella bronchiseptica variants in C mode and degraded phases were compared with those of their parents in X mode. Increases in MICs of 4- to 32-fold were observed for ampicillin, carbenicillin, erythromycin, novobiocin, rifampin, chloramphenicol, nalidixic acid, and oxolinic acid.

R plasmid with carbadox resistance from Escherichia coli of porcine origin.

Escherichia coli isolates of porcine fecal origin from a farm where the antibacterial agent carbadox was used were examined for resistance to carbadox (Cdxr). Of 72 strains examined, 24 showed resistance to this drug. All 24 Cdxr strains, except one, were also resistant to tetracycline (Tcr), streptomycin (Smr), spectinomycin (Spcr), sulfadimethoxine (Sur), kanamycin (Kmr), ampicillin (Apcr), or a combination of tetracycline, streptomycin, spectinomycin, sulfadimethoxine, and ampicillin. The Cdxr character was invariably transmissible by conjugation to E. coli K-12 jointly with other drug resistance, with the resistance patterns present in transconjugants being Cdxr Smr Spcr Apcr or Cdxr Smr Spcr Sur Apcr. About 25% of these transconjugants simultaneously lost the resistance to carbadox, streptomycin, spectinomycin, and ampicillin or carbadox, streptomycin, spectinomycin, sulfadimethoxine, and ampicillin or carbadox, streptomycin, spectinomycin, sulfadimethoxine, and ampicillin in the presence of acriflavine. Agarose gel electrophoretic analysis of deoxyribonucleic acid from a transconjugant showed a single plasmid deoxyribonucleic acid molecule with a molecular weight of about 28 x 10(6), which was capable of transforming E. coli C to Cdxr Smr Spcr Apcr. This resistance was transmissible by conjugation as a unit to other K-12 strains. These results confirmed the presence of an R plasmid specifying the Cdxr character in the host strain.

Antibiotic resistance of Erysipelothrix rhusiopathiae isolated from pigs with chronic swine erysipelas.

The susceptibility of 258 isolates of Erysipelothrix rhusiopathiae from slaughtered pigs affected with chronic erysipelas in Japan to antimicrobial agents was determined. A total of 111 (43.0%) strains showed resistance to erythromycin, oleandomycin, oxytetracycline, or dihydrostreptomycin. Plasmids were not detected. This is the first report of resistance of E. rhusiopathiae to these antibiotics.

Phosphorylation sites of protein kinase C delta in H2O2-treated cells and its activation by tyrosine kinase in vitro.

Protein kinase C delta (PKC delta) is normally activated by diacylglycerol produced from receptor-mediated hydrolysis of inositol phospholipids. On stimulation of cells with H(2)O(2), the enzyme is tyrosine phosphorylated, with a concomitant increase in enzymatic activity. This activation does not appear to accompany its translocation to membranes. In the present study, the tyrosine phosphorylation sites of PKC delta in the H(2)O(2)-treated cells were identified as Tyr-311, Tyr-332, and Tyr-512 by mass spectrometric analysis with the use of the precursor-scan method and by immunoblot analysis with the use of phosphorylation site-specific antibodies. Tyr-311 was the predominant modification site among them. In an in vitro study, phosphorylation at this site by Lck, a non-receptor-type tyrosine kinase, enhanced the basal enzymatic activity and elevated its maximal velocity in the presence of diacylglycerol. The mutation of Tyr-311 to phenylalanine prevented the increase in this maximal activity, but replacement of the other two tyrosine residues did not block such an effect. The results indicate that phosphorylation at Tyr-311 between the regulatory and catalytic domains is a critical step for generation of the active PKC delta in response to H(2)O(2).

Highly sensitive detection of viral RNA genomes in blood specimens by an optimized reverse transcription-polymerase chain reaction.

A protocol was developed for a highly sensitive detection of viral RNA in blood specimens by reverse transcription coupled with a nested polymerase chain reaction. Using Japanese encephalitis virus (JEV) as a model, the optimized reverse transcription-polymerase chain reaction (ORTPCR) detects as few as 3-5 virions in 0.1 ml of whole blood specimens. The effectiveness of this assay system is confirmed by diagnosis of human hepatitis C viral (HCV) infection.

A maxi Cl- channel coupled to endothelin B receptors in the basolateral membrane of guinea-pig parietal cells.

1. To study endothelin (ET) receptors in guinea-pig stomach, ET-binding assays and in vitro autoradiography were performed on fundic cell suspensions and on sections of the fundus, respectively. ETA and ETB receptor subtypes were found to coexist in the parietal cells. 2. Endothelin 1 (ET-1) added to the (basolateral) bathing solution was found to activate noisy whole-cell Cl- currents within about 1 min in both single, isolated parietal cells and those within gastric glands obtained from the fundus. 3. ET-1-induced Cl- currents were rapidly blocked by a Cl- channel blocker (NPPB) added to the (basolateral) bathing solution in a concentration-dependent manner with a half-maximum inhibition concentration of 33 microM. 4. The anion selectivity sequence of the ET-1-induced conductance was I- > Br- > Cl- > F-, corresponding to Eisenman's sequence I. 5. Changes in extracellular pH between 5 and 8 did not affect the ET-1-induced activation of Cl- currents. 6. Similar activating effects were also observed with ET-3 and a specific ETB receptor agonist (IRL1620). An ETB receptor antagonist (IRL1720) prevented the ET-1 effect, whereas an ETA-selective antagonist (FR139317 or BQ123) failed to antagonize the ET-1 effect. 7. In the whole-cell mode, unitary Cl- channel events could be observed in association with ET-1-activated macroscopic currents. The single-channel conductances were around 200 and 350 pS at negative and positive membrane potentials, respectively. 8. It is concluded that gastric parietal cells of guinea-pig possess pH-insensitive 'maxi' Cl- channels coupled to ETB receptors in the basolateral membrane.