Efficient autoproteolytic processing of the MHV-A59 3C-like proteinase from the flanking hydrophobic domains requires membranes

The replicase gene of the coronavirus MHV-A59 encodes a serine-like proteinase similar to the 3C proteinases of picornaviruses. This proteinase domain is flanked on both sides by hydrophobic, potentially membrane-spanning, regions. Cell-free expression of a plasmid encoding only the 3C-like proteinase (3CLpro) resulted in the synthesis of a 29-kDa protein that was specifically recognized by an antibody directed against the carboxy-terminal region of the proteinase. A protein of identical mobility was detected in MHV-A59-infected cell lysates. In vitro expression of a plasmid encoding the 3CLpro and portions of the two flanking hydrophobic regions resulted in inefficient processing of the 29-kDa protein. However, the efficiency of this processing event was enhanced by the addition of canine pancreatic microsomes to the translation reaction, or removal of one of the flanking hydrophobic domains. Proteolysis was inhibited in the presence of N-ethylmaleimide (NEM) or by mutagenesis of the catalytic cysteine residue of the proteinase, indicating that the 3CLpro is responsible for its autoproteolytic cleavage from the flanking domains. Microsomal membranes were unable to enhance the trans processing of a precursor containing the inactive proteinase domain and both hydrophobic regions by a recombinant 3CLpro expressed from Escherichia coli. Membrane association assays demonstrated that the 29-kDa 3CLpro was present in the soluble fraction of the reticulocyte lysates, while polypeptides containing the hydrophobic domains associated with the membrane pelletes. With the help of a viral epitope tag, we identified a 22-kDa membrane-associated polypeptide as the proteolytic product containing the amino-terminal hydrophobic domain.     

Metabolic efficiency during arm and leg exercise at the same relative intensities

This study was conducted to compare gross efficiency (GE), net efficiency (NE), work efficiency (WE), and delta efficiency (DE) between arm crank and cycle exercise at the same relative intensities. Eight college-aged males underwent two experimental trials presented in a randomized counterbalanced order. During each trial subjects performed three intermittent 7-min exercise bouts separated by 10-min rest intervals on an arm or semirecumbent leg ergometer. The power outputs for the three bouts of arm crank or cycle exercise corresponded to 50, 60, and 70% of the mode-specific VO2peak. GE, NE, and WE were determined as the ratio of Kcal.min-1 equivalent of power output to Kcal.min-1 of total energy expended, energy expended above rest and energy expended above unloaded exercise, respectively. DE was determined as the ratio of the increment of Kcal.min-1 of power output above the previous lower intensity to the increment of kcal.min-1 of total energy expended above the previous lower intensity. GE and NE did not differ between arm crank and cycle exercises. However, WE was lower (P < 0.05) during arm crank than cycle exercise at 50, 60, and 70% VO2peak. DE was also lower (P < 0.05) during arm crank than cycle exercise at delta 50-60 and at delta 60-70% VO2peak. It is concluded metabolic efficiency as determined by work and delta efficiency indices was lower during arm crank compared with cycle exercise at the same relative intensities. These findings add to the understanding of the difference in metabolic efficiency between upper and lower body exercise.     

The ''no suicide contract'': helpful or harmful?

1. Contracting for Safety (CFS) has become an integral part of nursing practice but never has been subjected to scientific scrutiny. 2. Clinical reasons for avoiding the use of contracting with some patient populations exist, including that some patients may construe CFS as a failure of empathy by the caregiver. 3. CFS can, in some instances, be helpful in establishing a therapeutic relationship or aiding in assessment, but never should be the sole basis for determining a patient's lethality.     

Response of mouse plantaris muscle to functional overload: comparison with rat and cat

Functional overload (FO) of a muscle by removing its synergists results in a compensatory hypertrophy of the muscle. However, the extent of the response appears to be dependent, at least in part, on the activity and/or loading levels of the muscle following surgery. Thus, differences in the inherent physical activity levels across species may be an important factor to consider. In the present study, the effects of 8 weeks of FO on the isometric mechanical properties of the plantaris of mice (highly active) were determined and the findings compared with the results from previous studies performed on the plantaris of rats (highly active) and cats (less active). FO resulted in approximately a doubling of the mass, the physiological cross-sectional area and the maximum tetanic tension per unit cross-sectional area, was similar in the plantaris of control and FO mice. Isometric twitch speed properties were unaffected, but the tension enhancement in response to an increase in the rate of stimulation showed the pattern of a "faster" muscle following FO. The fatigue resistance of the plantaris in FO mice was significantly higher than in control mice. Although the degree of hypertrophy that occurred in the mouse plantaris was similar to that observed after FO in rats and in cats that are exercised intermittently at high intensities, there were differences in the mechanical properties that may be related to the adaptability of species and/or the behavioral responses to the overload.     

Beneficial effect of vitamin E administration on nitric oxide function in subjects with hypercholesterolaemia

Study of the solubilization of commercial grades of soya phosphatidylcholine (SPC) with different purities by bile salts (BS) indicated that only highly pure grades of SPC are suitable for the preparation of clear solutions of BS/SPC-mixed micelles (BS/SPC-MM). The solubilizing capacity of different BS towards SPC increased in the following order; Sodium cholate (SC) < sodium deoxycholate (SDC) < sodium glycocholate (SGC). Moreover, egg phosphatidylcholine (EPC) was solubilized to a higher extent than SPC. Furthermore, the solubility study of different drugs in the prepared MM showed substantial enhancement of solubility, the extent of which is essentially affected by the chemical nature of the drug and the composition of MM. Benzodiazepine drugs such as clonazepam, tetrazepam, diazepam, and lorazepam displayed higher affinity for MM compared with BS alone, whereas steroidal drugs, such as estradiol, prednisolone and progesterone, compared with benzodiazepines, displayed relatively higher affinity for BS alone. The solubilizing capacity of MM for the different drugs was increased to different degrees by the addition of benzyl alcohol which was comparable to the solubility of the drug in pure benzyl alcohol. The interaction between benzyl alcohol and the drug in MM could be proved by NMR.     

Posttreatment with eicosatetraynoic acid decreases lung edema in guinea pigs exposed to phosgene: the role of leukotrienes

Acetylenic acids such as 5,8,11,14-eicosatetraynoic acid (ETYA), have been shown to be effective in preventing pulmonary edema formation (PEF). In phosgene-exposed guinea pigs, we examined the effects of ETYA on PEF, measured as real time lung weight gain (lwg). Pulmonary artery pressure (Ppa), airway pressure (Paw), perfusate leukotrienes (LT) C4/D4/E4/B4, and lung tissue lipid peroxidation (TBARS) were measured using the isolated, buffer-perfused lung model. Guinea pigs were challenged to 175 mg/m3 (44 ppm) phosgene for 10 minutes giving a concentration x time product of 1750 mg.min/m3 (437 ppm.min). Five minutes after removal from the exposure chamber, guinea pigs were treated, i.p., with 200 microL of 100 microM ETYA. 200 microL of 50 microM ETYA was added to the perfusate every 40 minutes, beginning at 60 minutes after start of exposure (t = 0). There were four groups in this study: air-treated, phosgene-exposed, ETYA-posttreated + phosgene, and ETYA-posttreated + air ETYA-posttreated + phosgene guinea pigs had significantly lower Ppa (P = .006), Paw (P = .009), and lwg (P = .016) compared with phosgene-exposed animals. Phosgene exposure reduced LTB4 compared with air-treated controls (P = .09). ETYA-posttreatment + phosgene had significantly increased perfusate LTB4 (P = .0006) compared with phosgene exposure only group. Total perfusate, LTC4 + LTD4 + LTE4, was not different between phosgene-exposed, air-treated or ETYA-posttreatment + phosgene over time. Posttreatment with ETYA significantly lowered TBARS formation, 206 +/- 13 versus 285 +/- 23 nmol/mg protein (P = .016), compared with phosgene-exposed lungs. Paradoxically, ETYA posttreatment decreased PEF and lipid peroxidation, but increased sulfidopeptide LT release from the lung during perfusion. We conclude that LTC4/D4/E4, and B4, may play different roles than previously thought for PEF in the isolated perfused lung model.     

Insulin-like growth factors cross the blood-brain barrier

Although evidence exists that insulin may cross the blood-brain barrier, little is known about the ability of insulin-like growth factors (IGF-I and -II) to cross this barrier. In the present studies, equimolar concentrations of equal specific activity 125I-labeled IGF-I, IGF-II, or insulin were infused into the carotid artery of anesthetized adult rats. The perfusions were carried out for 3 min in the presence or absence of excess unlabeled ligand or insulin, with three or more animals in each group. Immediately after the perfusion, brains were frozen and sectioned for autoradiography. All ligands were detected in choroid plexus, median eminence, and blood vessels, but [125I]IGF-I and -II were also prominently localized in brain parenchyma. Densitometric analysis of film autoradiographs (28-day exposure for all ligands) revealed that radiolabeled IGFs, especially IGF-I, were significantly more abundant throughout the forebrain than [125I]insulin, especially in the paraventricular nucleus, where [125I]IGF-I was 10-fold and [125I]IGF-II was 5-fold more abundant than [125I]insulin. The difference in [125I]IGF-I vs. [125I]insulin accumulation was confirmed by parallel measurements of radioactivity in anatomically matched brain sections using a gamma-spectrometer. The uptake of radiolabeled IGF-I, IGF-II, and insulin by brain parenchyma and vasculature was completely inhibited by excess (1,000-fold) unlabeled ligand; however, insulin (10,000-fold excess) did not completely abolish [125I]IGF-I and -II accumulation. Microscopic evaluation of nuclear emulsion-coated brain sections revealed that radioactivity associated with [125I]IGF-I and -II perfusions was selectively concentrated in capillaries and medium-sized parenchymal cells in the paraventricular nucleus and, to a lesser extent, the supraoptic nucleus and anterior nucleus of the thalamus, whereas in other brain regions the radioligands were mostly bound to capillaries. These results suggest that radiolabeled IGF-I and -II bind to brain capillaries and cross the blood-brain barrier into brain parenchyma more readily than radiolabeled insulin.