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.     

Minimal genetic influences on plasma fibrinogen level in adult males in the NHLBI twin study

Plasma fibrinogen was determined in 189 twins participating at the Indiana center during the third examination of the NHLBI twin study with a mean age of 63 years. Moderate heritability estimates were obtained from 44 complete MZ pairs and 39 complete DZ pairs. After adjustment of fibrinogen levels for age and other confounding variables related to cardiovascular disease risk, the maximum likelihood heritability estimate was only 30% (p = 0.03). Plasma fibrinogen was most strongly associated with smoking and the presence of diabetes. Omitting all subjects with diabetes or cardiovascular disease further reduced the heritability estimates slightly, and most path models including genetic parameters provided no significant improvement in fit over a model determined solely by random environmental effects. Our results are consistent with the environment rather than genetic influences having a greater influence on the level of plasma fibrinogen.     

Evaluation of trainees' stay in general practice by the young physicians and their tutors

In 1991 a new reform for postgraduate trainees was introduced in Denmark. Since the reform all young doctors-as a compulsory part of their vocational training-have had to work for six months as general practice trainees, no matter their wishes for future specialization. The reform furthermore led to the general practice trainees being considerably younger than previously-the average length of postgraduate experience before working as a trainee declined from six to seven years to one to two years. In this light an investigation was conducted from August 1992 to July 1993. Seven hundred and fifty-eight questionnaires were sent to general practice trainees and their tutors in Denmark. Ninety-five percent of the questionnaires were returned, thus it was possible to evaluate the new compulsory training in general practice. In conclusion, the main part of the trainees had a positive assessment of the work conditions in general practice. However, a large part of the trainees indicated that their tutor doctors had set aside too short a time for supervision.     

Malignant transformation of NIH3T3 cells by overexpression of mot-2 protein

The murine mortalin genes, mot-1 and mot-2, are members of the hsp70 family of proteins and differ from each other by only two amino acid residues. Mot-1 is expressed in normal cells and has pancytosolic cellular distribution whereas mot-2 is found in the perinuclear region of immortal cells. We report here that a high level of expression of mot-2 protein resulted in malignant transformation of cells as analysed by anchorage independent growth and nude mice assays. A high level of protein expression is attributed to the 900 bp 3' untranslated region of the cDNA which does not have any transforming activity per se. Mortalin cDNA clones isolated from human transformed cells were also found to have transforming activity in similar assays and a high level of expression was apparent in some of the human immortalized cells that showed non-pancytosolic mortalin immunofluorescence. Taken together, the data suggest that nonpancytosolic mortalin may have a role in tumorigenesis.     

Distribution of myosin heavy chain isoforms in non-weight-bearing rat soleus muscle fibers

The effects of 14 days of spaceflight (SF) or hindlimb suspension (HS) (Cosmos 2044) on myosin heavy chain (MHC) isoform content of the rat soleus muscle and single muscle fibers were determined. On the basis of electrophoretic analyses, there was a de novo synthesis of type IIx MHC but no change in either type I or IIa MHC isoform proportions after either SF or HS compared with controls. The percentage of fibers containing only type I MHC decreased by 26 and 23%, and the percentage of fibers with multiple MHCs increased from 6% in controls to 32% in HS and 34% in SF rats. Type IIx MHC was always found in combination with another MHC or combination of MHCs; i.e., no fibers contained type IIx MHC exclusively. These data suggest that the expression of the normal complement of MHC isoforms in the adult rat soleus muscle is dependent, in part, on normal weight bearing and that the absence of weight bearing induces a shift toward type IIx MHC protein expression in the preexisting type I and IIa fibers of the soleus.     

Pulmonary and systemic fat embolization after medullary canal pressurization: a hemodynamic and histologic investigation in the dog

BACKGROUND: The potential to produce fat embolism may be important in determining the ideal method and timing of fracture treatment in patients with preexisting lung injury. METHODS: Four dogs underwent femoral and tibial canal reaming and pressurization. Blood gas samples were analyzed, and pulmonary arterial pressure was monitored at 1 and 72 hours. Animals were killed 72 hours postoperatively, and the lungs, kidneys, and brain were examined histologically and compared with equivalent specimens from four control dogs that had not undergone femoral and tibial canal reaming and pressurization. RESULTS: Postmortem, intravascular fat persisted for 72 hours after induction of pulmonary fat embolism. Mean PaO2 was unchanged from baseline at 72 hours after canal pressurization. Canal pressurization caused a sustained increase in pulmonary arterial pressure (p=0.02) for 1 hour after canal pressurization. The mean pulmonary edema score at 72 hours was 29+/-3. Only a scant polymorph infiltrate (zero to two polymorphs per high-power field) was present at any time. No hyaline membranes were seen at any time. The percentage area occupied by intravascular fat in the lungs was 0.0214+/-0.0058 at 72 hours. No signs of ischemia or inflammation were seen in either the cerebral or the renal specimens. CONCLUSION: This study is the first to show that intravascular fat persists in the lungs, kidneys, and brain for 72 hours after canal pressurization and, by itself, does not cause pathologic evidence of acute inflammation.     

Endothelial injury following experimental subarachnoid hemorrhage in rats: effects on brain blood flow

BACKGROUND: The leading cause of death and disability in patients suffering from aneurysmal subarachnoid hemorrhage (SAH) is cerebral vasospasm, a persistent, progressive, and often irreversible constriction of cerebral arteries. A wide array of pathological changes occur in cerebral arteries following SAH, with endothelial injury being the earliest and most consistent one. Since intact endothelium modulates many reflexes that influence vascular tone, damage to them may represent a significant contributor to cerebral vasospasm. METHODS: Changes in local cerebellar blood flow (LCBF) and pathological alterations in major cerebral arteries were studied and compared in rats at various time intervals following SAH. SAH induced by the subarachnoid injection of 0.3 ml of whole blood. Sham rats received a subarachnoid injection of 0.3 ml of isotonic saline. RESULTS: Except for an immediate but transient decrease, LCBF remained unchanged over a 3 day period following saline injection. Likewise, there were no pathological alterations in cerebral arteries of saline-injected rats. In contrast, the subarachnoid injection of whole blood produced significant changes in both LCBF and cerebral arteries. Within 30 minutes post-blood injection, LCBF became significantly decreased and remained so for 4 hours. However, within 24 hours, LCBF had returned to control levels where it remained for 3 days. Endothelial injury was observed in the basilar and middle cerebral arteries from 30 minutes through 4 hours, the same periods in which LCBF was significantly reduced. Within 24 hours, the time period in which LCBF had rebounded to control ranges, cerebral arteries showed no evidence of endothelial damage and resembled control cells. CONCLUSION: The results indicate a direct correlation between changes in LCBF and the structural integrity of endothelial cells in the early stages following SAH. The lack of chronically depressed LCBF (after 1 day) may be related to the quick structural repair of endothelium.     

Imaging features and clinical significance of perineural spread or extension of head and neck tumors

Perineural spread of head and neck tumors is a form of metastatic disease in which tumor disseminates to noncontiguous regions along the endoneurium or perineurium. Both computed tomography (CT) and magnetic resonance (MR) imaging can help detect perineural spread, although MR imaging is the modality of choice because of its multiplanar capability, its superior soft-tissue contrast, and the decreased amount of artifact from dental hardware. Perineural spread most commonly occurs in adenoid cystic carcinoma and squamous cell carcinoma. Nerve enlargement may lead to foraminal enlargement and, ultimately, to foraminal destruction, findings that are best seen at CT. Extension through the foramen ovale and involvement of the Meckel cave is best seen on coronal T1-weighted MR images, and nerve enhancement is best seen on fat-suppressed T1-weighted MR images. Other radiologic findings include obliteration of fat planes at foraminal openings, neuropathic atrophy, cavernous sinus enlargement, and replacement of the trigeminal subarachnoid cistern with soft tissue. The pathway of perineural tumor spread is predictable with knowledge of the pertinent cranial nerve anatomy; however, patients with radiologically or pathologically proved perineural spread may have normal nerve function at clinical examination. Therefore, it is imperative that the radiologist be familiar with both normal cranial nerve anatomy and the radiologic appearance and assessment of perineural tumor extension.     

Pharmacokinetics of tablet huperzine A in six volunteers

AIM: To study pharmacokinetics of tablet huperzine A (Hup-A) in Chinese volunteers to help establishing its drug administration schedule. METHODS: For 6 volunteers after a single oral dose of 0.99 mg, drug concentrations in plasma were assayed by reverse phase high pressure liquid chromatography (HPLC) at 0.5, 0.75, 1.0, 1.25, 1.5, 2, 4, 6, 8, and 10 h. The pharmacokinetic parameters were calculated with a 3P87 program by computer. RESULTS: The time course of plasma concentrations conformed to a one-compartment open model with a first order absorption. The pharmacokinetic parameters were as follows: T 1/2ka = 12.6 min, T 1/2ke = 288.5 min, Tmax = 79.6 min, Cmax = 8.4 micrograms L-1, AUC = 4.1 mg L-1 min. CONCLUSION: Hup-A was absorbed rapidly, distributed widely in the body, and eliminated at a moderate rate.