Glucocorticoid-regulated stability of a constitutively expressed mouse mammary tumor virus glycoprotein.

Glucocorticoids regulate the transport and processing of mouse mammary tumor virus (MMTV) glycoproteins in viral-infected HTC rat hepatoma cells. To begin to determine the role of cellular components involved in this steroid-mediated response, a constitutively expressed MMTV glycoprotein gene containing a mutation in the endoproteolytic cleavage site was used to simplify the viral maturation products. Expression of the uncleavable MMTV glycoprotein gene in transfected HTC rat hepatoma cells demonstrated that treatment with the synthetic glucocorticoid dexamethasone resulted in a 5-fold increase in the steady state level of the intracellular and cell surface MMTV glycoproteins. Under these conditions, dexamethasone did not alter the level of MMTV glycoprotein transcripts. Pulse-chase radiolabeling with [35S]methionine demonstrated that dexamethasone did not affect the apparent rate of MMTV glycoprotein translation, and an analysis of oligosaccharide side-chain structure by endoglycosidase-H digestion revealed that glucocorticoids did not alter the 45-min endoplasmic reticulum to Golgi transit time. Pulse-chase kinetic analysis of 4-h pulse-labeled cells revealed that the half-life of the mature glycosylated MMTV polyprotein, gp78, was 105 min in glucocorticoid-treated cells and 45 min in untreated cells. Taken together, our results suggest that glucocorticoids increase the stability of MMTV glycoproteins by a posttranslational mechanism and that this effect may be occurring relatively early in the exocytic pathway.

Evidence for a protein-trafficking gene that rescues the defective glucocorticoid-regulated transport and Golgi retention of mouse mammary tumor virus glycoproteins in a rat hepatoma cell-sorting variant.

Glucocorticoids regulate the trafficking of cell surface mouse mammary tumor virus (MMTV) glycoproteins in the virus-infected rat hepatoma cell line M1.54. The CR4 rat hepatoma sorting variant, which is derived from M1.54 cells by immunoselection, is uniquely defective in the glucocorticoid-regulated transport of MMTV glycoproteins. Indirect immunofluorescence of fixed permeabilized cells and subcellular fractionation of isolated microsomes revealed that variant CR4 cells retain the MMTV glycoproteins in Golgi-like membranes after glucocorticoid treatment. The variant CR4 phenotype can be complemented by interspecies cell fusions with human HepG2 hepatoma cells and by DNA rescue with genomic fragments isolated from either human or rat hepatoma cells. Transfected wild-type genomic fragments rescue the sorting defect in CR4 at a frequency consistant with a single genetic locus, whereas homologous transfection with CR4 genomic DNA has no effect. Thus, complementation of a rat hepatoma cell-sorting variant supports the existence of a novel protein-trafficking activity encoded by the human or rat genomes that acts in trans in the Golgi to selectively mediate the sorting of cell surface MMTV glycoproteins in glucocorticoid-treated cells.

Altered effects of glucocorticoids on the trafficking and processing of mouse mammary tumor virus glycoproteins constitutively expressed in rat hepatoma cells in the absence of nonglycosylated viral components.

We have documented previously that glucocorticoid hormones modulate the posttranslational localization of cell surface mouse mammary tumor virus (MMTV) glycoproteins in the viral-infected M1.54 rat HTC hepatoma cell line. To determine whether glucocorticoids affect the trafficking of individually synthesized MMTV glycoproteins, HTC cells were transfected with a constitutively expressed MMTV glycoprotein gene lacking the viral phosphoprotein and polymerase genes. This construct also allows equivalent levels of MMTV glycoproteins to be compared in the presence or absence of glucocorticoids. Indirect immunofluorescence and immunoprecipitation of radiolabeled cells revealed that in transfected cells the transmembrane MMTV glycoproteins are efficiently expressed, transported to the cell surface, and proteolytically cleaved in the presence or in the absence of the synthetic glucocorticoid dexamethasone. Cell surface immunoprecipitation of [35S]methionine-labeled cells showed that the level of plasma membrane gp78 appeared to be stimulated 2-fold after dexamethasone treatment, even though fluorescence-activated cell sorting revealed no discernible change in the total concentration of cell surface MMTV glycoproteins. Analysis of oligosaccharide side chain maturation through a pulse-chase radiolabeling revealed that the rate of rough endoplasmic reticulum-Golgi transport was essentially identical in dexamethasone-treated and untreated transfected cells and was similar to that observed in dexamethasone-treated M1.54 cells. Thus, in contrast to viral-infected hepatoma cells, mostly constitutive cellular machinery mediates the trafficking and maturation of cell surface MMTV glycoproteins expressed outside of the proviral context. Taken together, our results suggest that the glucocorticoid-stimulated synthesis of nonglycosylated viral components may contribute to or be responsible for the regulated trafficking of MMTV glycoproteins observed in viral-infected rat hepatoma cells.

Glucocorticoid-regulated and constitutive trafficking of proteolytically processed cell surface-associated glycoproteins in wild type and variant rat hepatoma cells.

Glucocorticoids regulate the trafficking of mouse mammary tumor virus (MMTV) glycoproteins to the cell surface in the rat hepatoma cell line M1.54, but not in the immunoselected sorting variant CR4. To compare the localization of MMTV glycoproteins to another proteolytically processed glycoprotein, both wild type M1.54 cells and variant CR4 cells were transfected with a human insulin receptor (hIR) expression vector, pRSVhIR. The production of cell surface hIR was monitored in dexamethasone-treated and -untreated wild type M1.54 and variant CR4 cells by indirect immunofluorescence, direct plasma membrane immunoprecipitation, and by [125I] insulin binding. In both wild type and variant rat hepatoma cells, hIR were localized at the cell surface in the presence or in the absence of 1 microM dexamethasone. In contrast, the glucocorticoid-regulated trafficking of cell surface MMTV glycoproteins occurred only in wild type M1.54 cells. We conclude that the hIR, which undergoes posttranslational processing reactions similar to MMTV glycoproteins, does not require glucocorticoids to be transported to the plasma membrane and is representative of a subset of cell surface glycoproteins whose trafficking is constitutive in rat hepatoma cells. Thus, MMTV glycoproteins and hIR provide specific cell surface markers to characterize the glucocorticoid-regulated and constitutive sorting pathways.

Empiric antimicrobial therapy of domestically acquired acute diarrhea in urban adults.

From June 1985 to September 1987, 202 adults were enrolled in a randomized, double-blinded study comparing ciprofloxacin (500 mg) with sulfamethoxazole and trimethoprim (160 mg/800 mg) or placebo for adults with acute diarrhea. All patients were treated on the day of presentation and received medication on a twice-daily schedule (every 12 hours) for 5 days. Bacterial isolates from these patients included 35 Campylobacter, 18 Shigella, and 15 Salmonella. Treatment at the time of presentation with ciprofloxacin compared with placebo shortened the duration of diarrhea (2.4 vs 3.4 days), and increased the percentage of patients cured or improved by treatment days 1, 3, 4, and 5. Similar significant differences for sulfamethoxazole and trimethoprim compared with placebo were not seen.

A survey of furin substrate specificity using substrate phage display.

The substrate specificity of furin, a mammalian enzyme involved in the cleavage of many constitutively expressed protein precursors, was studied using substrate phage display. In this method, a multitude of substrate sequences are displayed as fusion proteins on filamentous phage particles and ones that are cleaved can be purified by affinity chromatography. The cleaved phage are propagated and submitted to additional rounds of protease selection to further enrich for good substrates. DNA sequencing of the cleaved phage is used to identify the substrate sequence. After 6 rounds of sorting a substrate phage library comprising 5 randomized amino acids (xxxxx), virtually all clones had an RxxR motif and many had Lys, Arg, or Pro before the second Arg. Nine of the selected sequences were assayed using a substrate-alkaline phosphatase fusion protein system. All were cleaved after the RxxR, and some substrates with Pro or Thr in P2 were also found to be cleaved as efficiently as RxKR or RxRR. To further elaborate surrounding determinants, we constructed 2 secondary libraries (xxRx(K/R)Rx and xxRxPRx). Although no consensus developed for the latter library, many of the sequences in the the former library had the 7-residue motif (L/P)RRF(K/R)RP, suggesting that the furin recognition sequence may extend over more than 4 residues. These studies further clarify the substrate specificity of furin and suggest the substrate phage method may be useful for identifying consensus substrate motifs in other protein processing enzymes.

Selected overview of nongynecologic surgical intra-abdominal infections. Prophylaxis and therapy.

True prophylaxis of intra-abdominal nongynecologic infections is limited to elective, nonemergency surgery and is best shown in three clean-contaminated surgical procedures. All of these have an infection rate of approximately 10 to 20 percent and include all colon resection surgery, most gastric surgery, and about one third of the cholecystectomies for chronic calculous cholecystitis. Each of these three surgical procedures has a somewhat different pattern of bacterial pathogens. The most useful comparative studies of early preoperative therapy have been performed in cases of suspected appendicitis (50 percent of which usually show perforation or gangrene at the time of surgery) and penetrating abdominal wounds (80 percent of which usually enter some part of the bowel and theoretically soil the peritoneum). These procedures are usually classified as contaminated, with a 20 to 30 percent infection rate, or dirty, with a more than 30 percent infection rate, depending upon several factors. Comparative investigations of intraoperative and postoperative antibiotic therapy of established intra-abdominal infections are more difficult to obtain because of the heterogeneity of the sites, organisms, and medical and surgical therapy. The initial pathogens causing secondary peritonitis and hepatic, perirectal, diverticular, and most other types of intraperitoneal abscesses are mixed coliforms and anaerobes, with emphasis on the anaerobes. Retroperitoneal abscesses, pancreatic abscesses, and biliary tract infections are predominantly caused by coliforms. The organisms responsible for these early infections are usually community-acquired rather than more antibiotic-resistant hospital-acquired bacteria. Considering the availability of a large number of effective broad-spectrum antibacterial agents and therapeutic combinations, it has become increasingly difficult to assess the rightful place of any new prospective antimicrobial regimen unless it has quite unique characteristics. Most empiric therapy in established intra-abdominal infection studies have compared gentamicin and clindamycin, the most popular regimen in the United States over the past 15 years, with a cephalosporin, broad-spectrum penicillin, or aminoglycoside, either alone or together with clindamycin or metronidazole. Results have usually been considered similar in most studies, although in some studies, agents with limited Bacteroides fragilis activity, such as cefamandole or cefaperazone, have been considered inferior. Most new prophylactic regimens have been compared with the first-generation cephalosporins and, again, similar results have been obtained between the groups with two exceptions. Cepha

The G-protein inhibitor, pertussis toxin, inhibits the secretion of brain-derived neurotrophic factor.

Secretion of neurotrophins is critical for the delivery of neurotrophic support. Brain-derived neurotrophic factor is targeted to a regulated secretory pathway in neurons as well as the neurosecretory AtT-20 cells. Here, we show that pertussis toxin, which inactivates Gi and Go G proteins, inhibits up to 50% of the regulated release of brain derived neurotrophic factor by AtT-20 cells. To determine whether pertussis toxin-sensitive G proteins may regulate brain-derived neurotrophic factor release in vivo, the effect of intraocular pertussis toxin was assessed on the isthmo-optic nucleus in the developing chick visual system. The isthmo-optic nucleus projects axons from the midbrain to innervate retinal amacrine cells and depends on target-derived brain-derived neurotrophic factor between embryonic days 13 and 17 (E13-17). During this period approximately 50% of isthmo-optic neurons are eliminated by programmed cell death. Intraocular pertussis toxin administered at E13 increased cell death of isthmo-optic neurons by 42%, whereas injections at E19 had no effect. Co-injection of brain-derived neurotrophic factor with pertussis toxin rescued approximately 50% of isthmo-optic neurons from enhanced cell death, although overall retinal brain derived neurotrophic factor protein levels were unaffected by pertussis toxin. Retrograde transport of exogenous 125I-labeled brain derived neurotrophic factor from the retina to the midbrain was increased by co-administration of pertussis toxin, possibly owing to diminished competition from endogenously released brain-derived neurotrophic factors for the receptors that mediate retrograde axonal transport. These data suggest that the release of a major fraction of brain-derived neurotrophic factor in the secretory pathway in vitro and in vivo is regulated by the activity of pertussis toxin-sensitive G proteins.

Heat stability of two candidate international reference preparations for recombinant human tissue factor.

Adequate heat stability of international reference preparations (IRP) for thromboplastin (tissue factor) is an essential requirement. Accelerated degradation testing was performed by three laboratories on two candidate IRP for recombinant human tissue factor. Heat treatment of these candidates resulted in slight shortening of the PT, contrasting with heat-induced prolongation of the PT observed with a conventional human brain derived IRP. Heat stability of these candidates was improved when compared with the stability of previous recombinant human tissue factor preparations. The PT-ratio did not change significantly when the candidates were stored for 28 days at 44 degrees C. It can therefore be concluded that both candidates are acceptable with regard to stability.

In vitro activity of new quinoxaline compounds against Campylobacter species and Clostridium difficile.

SC-44914 and SC-44942-A are two new quinoxaline compounds with a spectrum of activity similar to that of metronidazole. We studied the activity of SC-44914 and SC-44942-A against 35 Campylobacter jejuni, 30 C. coli, and 20 Clostridium (Cl.) difficile and compared it with that of metronidazole by utilizing an agar dilution method. The quinoxalines had little activity against the C. jejuni and C. coli [minimum inhibitory concentration (MIC)90 > or = 64 micrograms/ml]. SC-44914 and SC-44942-A had excellent activity against Cl. difficile (MIC90 < or = 0.06 micrograms/ml for SC 44914, and 0.5 micrograms/ml for SC-44942A).

In vitro activity of tosufloxacin against bacterial enteric pathogens.

The in vitro activity of tosufloxacin (A-61827), a new quinolone antibiotic, was compared with that of four other quinolones against 162 bacterial enteric pathogens. Susceptibility testing was performed by using an agar dilution method. The minimal inhibitory concentration (MIC) was defined as the lowest concentration of antibiotic without visible growth. Tosufloxacin was the most active agent against Campylobacter jejuni and C. coli, and ciprofloxacin was the most active agent against Salmonella, Shigella, and Vibrio. The frequency of spontaneous point mutational resistance to tosufloxacin for three C. jejuni ranged from 1.2 x 10(-9) to 5.1 x 10(-13). No significant differences in mutational frequency were seen among the quinolones.

A pseudoepidemic due to Salmonella typhimurium.

A pseudoepidemic due to Salmonella typhimurium occurred in the clinical microbiology laboratory of a university hospital and involved 10 patients. One patient received "unnecessary" antibiotics. Investigation of the events implicated a contaminated rubber pipette bulb. Such bulbs should be considered as a possible source of false-positive cultures.

Fulminant community-acquired infectious diseases: diagnostic problems.

The processes presented here do not represent an all-inclusive list of fulminant infectious diseases. Some of the more common acute, overwhelming infections of the central nervous system and lungs are covered elsewhere in this issue. We have selected less common, potentially catastrophic syndromes that might be recognized earlier if certain historical clues, physical findings, or laboratory abnormalities are appreciated. Specific and effective therapy is available for most of the diseases we have chosen. Meningitis due to Naegleria fowleri, a free-living ameba that may invade the central nervous system through the cribriform plate in persons swimming in brackish water, and hemorrhagic mediastinitis due to inhalation of Bacillus anthracis, which is acquired in occupational exposure to goat's hair, wool, or an animal with anthrax, are other examples but are lacking in proven effective therapy. Although most physicians quickly consider exotic and overwhelming infections in the severely compromised patient, fewer recognize this risk in the diabetic, cirrhotic, or healthy person with a unique occupational or travel history. During the present epidemic of AIDS, previous exposure to the HTLV-3 virus must be considered in all severely ill patients. The proper use of new diagnostic tests may permit the physician to intercede effectively if these life-threatening diseases are suspected.

Composition of A-150 tissue-equivalent plastic.

In recent years, the use of tissue-equivalent materials has become quite common in fast-neutron dosimetry, with the A-150 plastic developed by Shonka et al. probably the most popular. Information on this specific plastic is scantily reported in the literature and as a consequence a preponderance of authors unknowingly reference an article by Shonka describing an early version of a tissue substitute plastic but having a different elemental composition than the present A-150 formulation. We have reviewed the results of 21 chemical analyses which have occurred over a time span of four years on a total of 14 samples of A-150 plastic and based on these data and the formulation of the plastic, have arrived at a suggested composition for A-150 tissue-equivalent plastic. The ambiguities of water absorption by nylon, one of the components of the plastic, and the uncertainty this reflects in the composition of the plastic were evaluated.

Using a database to analyze core basic science content in a problem-based curriculum.

As medical schools critically reevaluate their methods of instruction and as the number of innovative programs increases, the content delivered across disciplines must be carefully reviewed. However, few methods of content analysis have been applied to problem-based programs. In 1989-90 and 1990-91, the authors analyzed the distribution of basic science content in the 53 cases in the problem-based curriculum of Rush Medical College of Rush University. They developed a content vocabulary and created a database using a widely available computer software program. The content areas specific to each case were identified by faculty using the content vocabulary. To determine whether these content areas were actually identified by the students participating in the problem-solving sessions, the authors surveyed the 36 student participants in the classes of 1993 and 1994 and also interviewed the 15 faculty facilitators of the sessions. The surveys and interviews demonstrated that over 90% of the content areas identified by the faculty were actually covered by the students. The authors conclude that the database assists in their review of the curriculum for omission and redundancy. Other uses and limitations of this method are also discussed.

Campylobacter fetus subsp. jejuni: experience in a large Chicago medical center.

From January 1979 through December 1979, 2069 stool specimens received by our laboratory for enteric culturing were additionally examined for the presence of Campylobacter fetus subsp. jejuni. The study population included inpatients, outpatients and hospital food handlers. Patients were included regardless of symptoms. Enteric pathogens were identified as follows: Salmonella, 27 isolates; Campylobacter fetus subsp. jejuni, 26 isolates; and Shigella, 11 isolates. Twenty-five of 26 patients with stool cultures positive for Campylobacter fetus subsp. jejuni had an acute diarrheal illness. Diarrhea, fever and chills were the most common symptoms. In most patients the disease was self-limited, requiring only supportive therapy. A seasonal variation was noted, with 14 of the 26 patients presenting between July and September.

The office approach to the sexually transmitted diseases: Part I.

Sexually transmitted diseases (STDs) are diagnosed in 10 million patients per year in the United States. The infected individuals come from all walks of life and all age groups. They may present with either genital or nongenital signs and symptoms. Most individuals are treated as outpatients, but more than a quarter of a million hospital admissions for STDs are necessary each year. These numbers will rise precipitously as the number of AIDS cases increases. More than 10,000 deaths per year are caused by STDs, primarily because of AIDS, cervical carcinoma, and hepatitis B induced cirrhosis and hepatoma. Physicians must become highly skilled in the diagnosis and treatment of the common STDs caused by herpes simplex virus, Neisseria gonorrhoeae, and Chlamydia trachomatis. Simple office microscopic skills are needed for the diagnosis of vaginitis, cervicitis, and urethritis, and all physicians should be encouraged to develop these skills. Physicians will need to keep abreast of the rapidly evolving changes in the diagnosis and treatment of STDs.

The office approach to the sexually transmitted diseases: Part II.

All physicians share a responsibility for educating the public in the expanding risks of sexually transmitted diseases, and in the vital importance of safe sexual practices. Both the public health implications of undiagnosed STDs and the adverse social consequences of an incorrect diagnosis emphasize the need to comprehensively and precisely diagnose these diseases, despite the problems encountered in obtaining material for culture. Current interest in AIDS should not obscure the facts that genital herpes continues to increase in prevalence, and that syphilis no longer is declining. The many extragenital syndromes associated with STDs, such as perihepatitis, polyarthritis, and acute septic states make it necessary to evaluate all sexual contacts.

Multiplane fracture of the distal humerus.

A unique but complex intraarticular fracture of the distal humerus is described. The term multiplane fracture has been applied to highlight a fracture pattern that features not only the well recognized T fracture lines in the sagittal and horizontal planes but also a coronal fracture of the trochlea. The Herbert screw was used in five cases to achieve stable fixation of the purely articular coronal fracture. At a follow-up of 20 months, on average, all fractures healed with an acceptable functional result. No radiographic evidence of avascular necrosis was seen in the follow-up radiographs.

A urine preservative system to maintain bacterial counts. A laboratory and clinical evaluation.

The urinary tract is a common site of infection in the hospitalized, institutionalized, or ambulatory patient population. Ideally, urine should be cultured immediately or refrigerated up to 24 hours for quantitative examination for microorganisms. In the evaluation of patients at their homes or in long-term care facilities, rapid plating or refrigeration may not be practical. This is also true when evaluating small children in whom external collection devices are required to obtain a specimen. Because of these limitations, we evaluated a urine preservative and transport system, the Sage Products Urine Culture Tube, in a study of 1469 clinical specimens. This tube utilizes boric acid (1.1% final concentration) as a preservative. The Urine Culture Tube was easy to use and was as effective as refrigeration in maintaining bacterial counts. This system may be particularly useful where rapid transport or refrigeration is limited.