Cytomegalovirus basic phosphoprotein (pUL32) binds to capsids in vitro through its amino one-third.

The cytomegalovirus (CMV) basic phosphoprotein (BPP) is a component of the tegument. It remains with the nucleocapsid fraction under conditions that remove most other tegument proteins from the virion, suggesting a direct and perhaps tight interaction with the capsid. As a step toward localizing this protein within the molecular structure of the virion and understanding its function during infection, we have investigated the BPP-capsid interaction. In this report we present evidence that the BPP interacts selectively, through its amino one-third, with CMV capsids. Radiolabeled simian CMV (SCMV) BPP, synthesized in vitro, bound to SCMV B-capsids, and C-capsids to a lesser extent, following incubation with either isolated capsids or lysates of infected cells. Human CMV (HCMV) BPP (pUL32) also bound to SCMV capsids, and SCMV BPP likewise bound to HCMV capsids, indicating that the sequence(s) involved is conserved between the two proteins. Analysis of SCMV BPP truncation mutants localized the capsid-binding region to the amino one-third of the molecule--the portion of BPP showing the greatest sequence conservation between the SCMV and HCMV homologs. This general approach may have utility in studying the interactions of other proteins with conformation-dependent binding sites.

A model of gastric emptying using paracetamol absorption in intensive care patients.

OBJECTIVES: To describe the range and factors which may affect gastric emptying in the ICU patient. DESIGN: Validation sample. SETTING: The adult Intensive Care Unit (ICU) of a teaching hospital. PATIENTS: Twenty-seven ICU patients, aged 18-65 years were studied within 3 days of their ICU admission. All patients had normal hepatic and renal chemistry and had no contraindications to enteral feeding. MEASUREMENTS AND MAIN RESULTS: The area under the concentration curve from 0-60 min (AUC60) of a paracetamol absorption test was used as the measure of gastric emptying. The variables of the presence or absence of bowel sounds, volume of gastric aspirate ( > 50 ml or < 50 ml), an estimated risk of death (ROD), an APACHE II score calculated 24 h before the study, a pHi measurement, the use of dopamine (2.5-5 microg/kg, yes or no) and of opioids were included in a multiple regression analysis. Using Pearson correlation, AUC60 was positively correlated with the estimated ROD (r = 0.50, p < 0.05). There was a statistically significant difference in the mean AUC60 between those patients who did, and those who did not, receive dopamine (t = 3.06, p < 0.005). On multiple regression analysis the only variable which was significantly associated with AUC60 was estimated ROD, which accounted for 25% of the variance in AUC60. CONCLUSION: The results suggest that there is a wide range in gastric emptying in critically ill patients. The results may be due to the case mix of the patients. The use of dopamine may adversely affect gastric emptying and requires further investigation in the ICU patient. Prediction of gastric emptying is difficult in these patients and further investigation is necessary in order to improve our understanding of this process.

Human cytomegalovirus capsid assembly protein precursor (pUL80.5) interacts with itself and with the major capsid protein (pUL86) through two different domains.

We have used the yeast GAL4 two-hybrid system to examine interactions between the human cytomegalovirus (HCMV) major capsid protein (MCP, encoded by UL86) and the precursor assembly protein (pAP, encoded by UL80.5 and cleaved at its carboxyl end to yield AP) and found that (i) the pAP interacts with the MCP through residues located within the carboxy-terminal 21 amino acids of the pAP, called the carboxyl conserved domain (CCD); (ii) the pAP interacts with itself through a separate region, called the amino conserved domain (ACD), located between amino acids His34 and Arg52 near the amino end of the molecule; (iii) the simian CMV (SCMV) pAP and AP can interact with or replace their HCMV counterparts in these interactions, whereas the herpes simplex virus pAP and AP homologs cannot; and (iv) the HCMV and SCMV maturational proteinase precursors (ACpra, encoded by UL80a and APNG1, respectively) can interact with the pAP and MCP. The ACD and CCD amino acid sequences are highly conserved among members of the betaherpesvirus group and appear to have counterparts in the alpha- and gammaherpesvirus pAP homologs. Deleting the ACD from the HCMV pAP, or substituting Ala for a conserved Leu in the ACD, eliminated detectable pAP self-interaction and also substantially reduced MCP binding in the two-hybrid assay. This finding indicates that the pAP self-interaction influences the pAP-MCP interaction. Immunofluorescence studies corroborated the pAP-MCP interaction detected in the GAL4 two-hybrid experiments and showed that nuclear transport of the MCP was mediated by pAP but not AP. We conclude that the pAP interacts with the MCP, that this interaction is mediated by the CCD and is influenced by pAP self-interaction, and that one function of the pAP-MCP interaction may be to provide a controlled mechanism for transporting the MCP into the nucleus.

Cytomegalovirus "missing" capsid protein identified as heat-aggregable product of human cytomegalovirus UL46.

Capsids of human and simian strains of cytomegalovirus (HCMV and SCMV, respectively) have identified counterparts for all but one of the protein components of herpes simplex virus (HSV) capsids. The open reading frames (ORFs) for the CMV and HSV counterpart proteins are positionally homologous in the two genomes. The HSV capsid protein without a recognized counterpart in CMV is VP19c, a 50-kDa element of the intercapsomeric "triplex." VP19c is encoded by HSV ORF UL38, whose positional homolog in the HCMV genome is UL46. The predicted protein product of HCMV UL4A6, however, has essentially no amino acid sequence similarity to HSV VP19c, is only two-thirds as long, and was not recognized as a component of CMV capsids. To identify and learn more about the protein encoded by HCMV UL46, we have expressed it in insect cells from a recombinant baculovirus and tested for its presence in CMV-infected human cells and virus particles with two UL4A6-specific antipeptide antisera. Results presented here show that this HCMV protein (i) has a size of approximately 30 kDa as expressed in both recombinant baculovirus-infected insect cells and HCMV-infected human cells; (ii) has a homolog in SCMV; (iii) is a capsid component and is present in a 1:2 molar ratio with the minor capsid protein (mCP), encoded by UL85; and (iv) interacts with the mCP, which is also shown to interact with itself as demonstrated by the GAL4 two-hybrid system; and (v) aggregates when heated and does not enter the resolving gel during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a characteristic that accounts for it eluding detection until now. We call this protein the mCP-binding protein, and on the basis of the characteristics that it shares with HSV VP19c, we conclude that the HCMV mCP-binding protein is the functional as well as genetic homolog of HSV VP19c.

Human cytomegalovirus (HCMV) smallest capsid protein identified as product of short open reading frame located between HCMV UL48 and UL49.

The capsid of cytomegalovirus contains an abundant, low-molecular-weight protein whose coding sequence within the viral genome had not been identified. We have used a combination of biochemical and immunological techniques to demonstrate that this protein, called the smallest capsid protein in human cytomegalovirus, is encoded by a previously unidentified 225-bp open reading frame (ORF) located between ORFs UL48 and UL49. This short ORF, called UL48/49, is the positional homolog of herpes simplex virus ORF UL35 (encoding capsid protein VP26) and shows partial amino acid sequence identity to positional homologs in human herpes viruses 6 and 7.

Effect of education on oxygen mask placement.

We have investigated the effect of nurse and patient education on oxygen mask placement after surgery. In 15 patients, each studied for 8 h, masks remained positioned correctly in two and were removed a total of 37 times in the others. Median total removal time per patient (4 min 3 s) was less than in previous studies and maximum total time of mask removal was 1 h 33 min 57 s. Masks were removed for longer than 10 min on four occasions in two patients. None of these removals was for nursing care. This is an improvement compared with previous studies.

Molecular properties of pyruvate formate-lyase activating enzyme.

Pyruvate formate-lyase is a radical-containing enzyme that catalyzes the nonoxidative cleavage of pyruvate via a postulated homolytic mechanism. The formation of this enzymic radical in vitro requires an activating system composed of PFL-activating enzyme, S-adenosylmethionine, ferrous ion, a reduced flavin, DTT, and pyruvate as an allosteric effector. The need for large quantities of PFL-activating enzyme for biochemical and biophysical studies on the mechanism of protein radical formation has prompted us to clone the act gene and overexpress the gene product in Escherichia coli. Using PCR technology, the act gene was isolated and subcloned into various expression vectors. The overexpression of the protein was as high as 30-50% of the total cellular protein. However, the majority of the protein resided in the form of insoluble inclusion bodies. A procedure was developed to denature and isolate the inclusion bodies followed by refolding under anaerobic conditions. This purification method affords 5 mg of purified protein from 1 g of cells. Biochemical characterization demonstrated that the enzyme can bind one Fe(II) per protein monomer, and the protein did not exhibit any visible chromophore as previously observed. Co(II) and Cu(II) can be reconstituted into the protein with similar stoichiometries. Kinetic studies showed that the rate of radical formation was independent of ionic strength and the Km's for SAM and inactive PFL were determined to be 2.8 and 1.2 microM, respectively. Fluorescent binding data revealed that the Kd for SAM binding to the activating enzyme alone was comparable to the Km for SAM in the PFL activation indicating that the binding site for SAM resides on AE.(ABSTRACT TRUNCATED AT 250 WORDS)

Video surveillance of oxygen administration by mask in postoperative patients.

Patients may not receive prescribed oxygen because the oxygen face mask becomes displaced. Using video, we have observed the position of the face mask in 20 postoperative patients and recorded the timing and the events associated with mask displacement. Correct placement of the mask was confirmed at the start of the 8-h study period from 22:00 on the first night after operation. The mask remained on continuously and positioned correctly in only one patient. In the other 19 patients, it was removed 64 times (range 1-10 times per patient). The mask was removed 45 times for nursing tasks such as mouth care and temperature measurement and these represented 70% of the total number of times that the mask was not in position. Other reasons for removal were vomiting, retching and removal by the patient. The mask remained off a median time of 6 min 55 s per episode (range 46 s to 7 h 46 min 57 s) and per patient a median of 1 h 6 min 48 s (range 1 min to 7 h 46 min 57 s). Mask removal resulted in an average decrease in oxygen saturation of 4%. Oxygen by mask at 4 litre min-1 maintained an average saturation > or = 95% in most, but not all, of the patients.

The potency of pancuronium at the adductor pollicis and diaphragm in infants and children.

To measure the potency of pancuronium at the diaphragm and adductor pollicis in infants and children, train-of-four stimulation was applied to the ulnar and phrenic nerves under N2O-halothane anesthesia. The force of contraction of the adductor pollicis was measured and compared with the diaphragmatic electromyogram (EMG). Cumulative dose response curves were determined for pancuronium in 18 patients divided equally into three age groups: 0-1 yr, 1-3 yr, and 3-10 yr. The potency of pancuronium at both muscles decreased with increasing age (P less than 0.05), while the adductor pollicis:diaphragm potency ratio remained constant. The mean doses (+/- SEM) required to depress adductor pollicis first twitch responses by 90% (ED90) were 42 +/- 3.3 micrograms/kg in the 0-1-yr group, 47 +/- 4.2 micrograms/kg in the 1-3-yr group, and 62 +/- 4.1 micrograms/kg in the 3-10-yr group. Corresponding figures for the diaphragm were 70 +/- 4.3 micrograms/kg, 81 +/- 5.1 micrograms/kg, and 101 +/- 4.4 micrograms/kg, respectively. The ED90 ratios (diaphragm ED90/adductor pollicis ED90) in the three age groups were 1.69 +/- .07, 1.75 +/- .14, and 1.64 +/- .09, respectively. These results are consistent with similar rates of maturation of the diaphragm and the adductor pollicis muscles in infancy and childhood. Thus, train-of-four monitoring of the adductor pollicis is likely to overestimate the degree of neuromuscular blockade of the diaphragm in pediatric patients.