In vitro replication of mouse hepatitis virus strain A59.

An in vitro replication system for mouse hepatitis virus (MHV) strain A59 was developed using lysolecithin to produce cell extracts. In extracts of MHV-infected cells, radiolabeled UMP was incorporated at a linear rate for up to 1 h into RNA, which hybridized to MHV-specific cDNA probes and migrated in denaturing formaldehyde-agarose gels to the same position as MHV genomic RNA. The incorporation of [32P]UMP into genome-sized RNA in vitro correlated with the observed increase of [3H]uridine incorporation in MHV-infected cells labeled in vivo. Incorporation of [32P]UMP into genome-sized RNA was inhibited when extracts were incubated with puromycin. The addition to the assay of antiserum to the MHV-A59 nucleocapsid protein N inhibited synthesis of genome-sized RNA by 90% compared with the addition of preimmune serum. In contrast, antiserum to the E1 or E2 glycoproteins did not significantly inhibit RNA replication. In vitro-synthesized RNA banded in cesium chloride gradients as a ribonucleoprotein complex with the characteristic density of MHV nucleocapsids isolated from virions. These experiments suggest that ongoing protein synthesis is necessary for replication of MHV genomic RNA and indicate that the N protein plays an important role in MHV replication.     

Primary structure analysis proves that protein phosphatases 2C1 and 2C2 are isozymes

The two recently discovered forms of protein phosphatase 2C, termed 2C1 and 2C2, were digested with CNBr or trypsin, and several peptides corresponding to two regions of the protein were sequenced. These studies revealed close homology between the two enzymes with 49 identities over the 62 residues that could be compared directly. The results establish that protein phosphatases 2C1 and 2C2 are the products of different genes. The C-terminus of protein phosphatase 2C2 has also been identified.     

The effect of dietary alteration of prostaglandin synthesis on blood pressure and the reversal of hypertension in the one-kidney, one-clip rat

This study examined the effect of diet-induced changes in prostaglandin synthesis on systolic blood pressure in one-kidney, one clip (1k, 1C) hypertensive rats and on the fall in blood pressure after unclipping. It tested the hypothesis that inhibition of prostaglandin synthesis exacerbates hypertension in this model and prevents complete reversal after unclipping. Rats with sustained hypertension within 8 weeks of renal artery clipping were fed synthetic diets supplemented to 20% of total energy with either safflower oil (linoleic acid) or a mixture of cod liver oil (90%) (containing eicosapentaenoic acid) and linseed oil (10%) (containing linolenic acid) for 4 weeks. The latter oil mixture resulted in a predictable reduction in kidney PGE2 and 6-keto PGF1 alpha (hydrolysis product of PGI2), aortic 6-keto PGF1 alpha and serum TXB2. However, at the end of 4 weeks dietary treatment there were no differences in systolic blood pressure between the two diet groups, and the blood pressure fall 24 hours after unclipping was similar. These findings therefore do not support a role for prostanoids in the maintenance or reversal of 1K, 1C hypertension.     

Relationships among negative life events, physiological reactivity, and health symptomatology

College undergraduates classified as high (n = 25) and low (n = 25) on recent life stress participated in an experiment involving a novel laboratory stressor. Heart rate and pulse arrival time (PAT) were measured during baseline, anticipation, testing, and recovery periods of the experiment. The results did not replicate those obtained by Pardine and Napoli in that high and low life stress subjects did not show differential physiological reactions. In addition, regression analyses failed to demonstrate that physiological reactivity moderated the relationship between life stress and subsequent self-reported psychiatric or physical health symptomatology. The present findings demonstrated neither the stress-buffering effects of physiological reactivity nor a relationship between life stress and reactivity when the latter was conceptualized as an outcome.     

Self referral to an accident and emergency department for another opinion.

OBJECTIVE--To determine whether patients referring themselves to an accident and emergency department for another opinion after consulting their general practitioner present with serious illness, show any risk factors for being admitted, or are more likely to be patients of particular practitioners. DESIGN--Six month prospective survey. SETTING--District general hospital''s accident and emergency department, receiving 42,000 new patients a year. PATIENTS--180 Patients identified as attending for another opinion having already consulted a general practitioner. INTERVENTIONS--Classified as admission, referral to specialist clinic, follow up in accident and emergency department, or referral back to general practitioner. END POINT--Admission, with an analysis of admitted patients. MEASUREMENTS AND MAIN RESULTS--General outcome, diagnostic category, age, time of attendance, time since seen by general practitioner, and name of general practitioner were recorded. Forty seven patients were admitted, 99 were discharged back to the general practitioner (62 without a letter), and two died. Patients were most likely to be admitted if they attended within 24 hours after seeing a general practitioner, were aged under 5, or presented with respiratory or gastrointestinal complaints. Some general practitioners were overrepresented. CONCLUSIONS--Important disorders present in this way, and therefore these patients should be seen by a doctor. Information about these attendances could be useful to general practitioners in reviewing their performance.     

Reproductive performance of Coopworth ewes following oral doses of zearalenone before and after mating

The reproductive performance of Coopworth ewes after administration of zearalenone was determined in two trials. In Trial I, zearalenone was administered to groups of 33 ewes at rates of 0, 1.5, 3.0, 6.0, 12.0 and 24.0 mg/ewe/day for 10 days, starting on Day 7 of the oestrous cycle before mating. There was a linear decline (P less than 0.001) in ovulation rate with dose of zearalenone; also cycle length decreased and duration of oestrous increased with increasing dose levels. Reductions in the incidence of ovulation and in fertilization were seen only at doses of 12 and 24 mg. In Trial 2, groups of 50 ewes were given the same range of doses of zearalenone for 10 days, starting 5 days after mating to entire rams. There was no effect of zearalenone treatment after mating on pregnancy rate or embryonic loss. These results indicate that the effects of zearalenone, administered orally, on ewe reproduction, at the dose levels examined, were restricted to ewes exposed before mating. Intakes of zearalenone of 3 mg/ewe/day or more during this period would be reflected as depressed ovulation rates and lower lambing percentages.     

Biochemical analysis of CRM 66. A nonfunctional Pseudomonas aeruginosa exotoxin A

Pseudomonas aeruginosa exotoxin A (ETA) is an ADP-ribosyltransferase which inactivates protein synthesis by covalently attaching the ADP-ribose portion of NAD+ onto eucaryotic elongation factor 2 (EF-2). A direct biochemical comparison has been made between ETA and a nonenzymatically active mutant toxin (CRM 66) using highly purified preparations of each protein. The loss of ADP-ribosyltransferase activity and subsequent cytotoxicity have been correlated with the presence of a tyrosine residue in place of a histidine at position 426 in CRM 66. In the native conformation, CRM 66 demonstrated a limited ability (by a factor or at least 100,000) to modify EF-2 covalently and lacked in vitro and in vivo cytotoxicity, yet CRM 66 appeared to be normal with respect to NAD+ binding. Upon activation with urea and dithiothreitol, CRM 66 lost ADP-ribosyltransferase activity entirely yet CRM 66 retained the ability to bind NAD+. Replacement of Tyr-426 with histidine in CRM 66 completely restored cytotoxicity and ADP-ribosyltransferase activity. These results support previous findings from this laboratory (Wozniak, D. J., Hsu, L.-Y., and Galloway, D. R. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8880-8884) which suggest that the His-426 residue of ETA is not involved in NAD+ binding but appears to be associated with the interaction between ETA and EF-2.