Protocol to enrich and analyze plasma membrane proteins.

This chapter describes a procedure for isolation and analysis of fractions enriched in plasma membranes from minute amounts of tissue. It consists of a method for extraction and fractionation of membranes and a method for enzymatic digestion of membrane proteins without use of detergents. The method for isolation of membranes comprises of a stepwise depletion of nonintegral membrane molecules from entire tissue homogenate by high-salt, carbonate, and urea washes followed by a treatment of the membranes with sublytic concentrations of a detergent and enrichment of the plasma membranes by a density gradient fractionation. Fluorometric assays for protein content and plasma membrane marker activity allow calculation of the yield and extent of plasma membrane enrichment. Reduction, carboxymethylation, and digestion with endoproteinase Lys-C are carried out on nonsolubilized membranes. The entire procedure allows processing and preparation of samples from 10-20 mg tissue, and therefore, can be extremely helpful for proteomic profiling of biopsy-size clinical samples.

Influence of low power cm-/mm-microwaves on cardiovascular function.

The present study has been designed to investigate physiological effects of short (15 min) exposure to low level energy microwaves (< 10 microW cm(-2)). To this end heart rate, PQ, QS and ST (electrocardiography), respiration (conductive stretch band around the thorax), skin temperature (thermocouple), skin conductance (Ag/AgCl electrodes), systolic and diastolic blood pressure (automatic cuff) were continuously recorded in a group of 50 test persons before, during and after a 15 min exposure to 3 microW cm(-2) high frequency (77 GHz) microwaves. After an equilibration period of 30 min the first group of test persons and after additional 30 min the second group of test persons were exposed. The study has been performed in a strict double blind design. While significant effects on the measured parameters were observed depending on time ('calming' effect), no significant difference was observed between exposure and sham exposure to microwaves. In view of the small scatter of the data the present study rules out physiologically relevant effects of low level energy on the autonomic nervous system and cardiovascular function.

Benefits of lifestyle modification in the pharmacologic treatment of obesity: a randomized trial.

BACKGROUND: Weight loss medications are recommended as an adjunct to diet and exercise modification but seem to be prescribed as a monotherapy by many physicians. This practice is likely to be associated with suboptimal weight loss. METHODS: This 1-year, randomized trial compared the effects of sibutramine hydrochloride used alone (ie, the drug-alone group) to sibutramine plus group lifestyle modification, prescribed with either a 5021- to 6276-kJ/d diet (1200-1500-kcal/d diet) (ie, the drug-plus-lifestyle group) or, for the first 4 months, a 4184-kJ/d diet (1000-kcal/d diet (ie, drug-plus-lifestyle with a portion-controlled diet [the combined treatment] group). Participants were 53 women with a mean (+/-SD) age of 47.2 +/- 9.8 years and weight of 101.3 +/- 9.7 kg. At baseline, they reported the number of pounds they expected to lose at the end of treatment. RESULTS: At month 12, patients treated with the drug alone lost (mean +/- SD) 4.1% +/- 6.3% of their initial body weight compared with significantly (P<.05) larger losses in the drug-plus-lifestyle group of 10.8% +/- 10.3% and the combined treatment group of 16.5% +/- 8.0%. Women in the 2 lifestyle groups achieved a significantly (P<.05) greater percentage of their expected weight loss than those in the drug-alone group and were significantly more satisfied with the medication and with changes in weight, health, appearance, and self-esteem (P<.05 for all). Significant reductions were observed at 12 months in triglyceride and low-density lipoprotein cholesterol levels but systolic and diastolic blood pressure both increased significantly (P<.05 for all). CONCLUSION: The addition of group lifestyle modification to the pharmacologic management of obesity significantly improved weight loss and patients' satisfaction with treatment outcome.

Sustained esophageal contraction: a marker of esophageal chest pain identified by intraluminal ultrasonography.

BACKGROUND & AIMS: Intraluminal pressure recording systems have not demonstrated predictable esophageal motor correlates of unexplained chest pain. This study used continuous high-frequency intraluminal ultrasonography to characterize esophageal contraction at the time of spontaneous and provoked chest pain. METHODS: Intraluminal pressure, pH, and ultrasound images of the esophagus were recorded for a maximum of 24 hours in 10 subjects with unexplained chest pain. Changes in esophageal muscle thickness were measured as a marker of muscle contraction. Ten additional subjects with suspected esophageal chest pain were studied after edrophonium chloride injection to provoke symptoms. Ten healthy subjects were studied as controls. RESULTS: Eighteen of 24 spontaneous chest pain episodes were preceded by a sustained esophageal contraction (SEC) detected on ultrasonography (mean duration, 68.0 seconds). This motor pattern was not accompanied by changes in intraluminal pressure. Four of 24 asymptomatic control periods were accompanied by SEC, although these contractions were of shorter mean duration (29.0 seconds; P < 0.001). SEC was observed in 5 subjects with a positive chest pain response to edrophonium and in none of the 5 subjects with a negative response. SEC was not detected in normal subjects. CONCLUSIONS: There is a strong temporal correlation between a previously unrecognized esophageal motor event, SEC, and both spontaneous and provoked esophageal chest pain.

The bovine leukemia virus encapsidation signal is composed of RNA secondary structures.

The encapsidation signal of bovine leukemia virus (BLV) was previously shown by deletion analysis to be discontinuous and to extend into the 5' end of the gag gene (L. Mansky et al., J. Virol. 69:3282-3289, 1995). The global minimum-energy optimal folding for the entire BLV RNA, including the previously mapped primary and secondary encapsidation signal regions, was analyzed. Two stable stem-loop structures (located just downstream of the gag start codon) were predicted within the primary signal region, and one stable stem-loop structure (in the gag gene) was predicted in the secondary signal region. Based on these predicted structures, we introduced a series of mutations into the primary and secondary encapsidation signals in order to explore the sequence and structural information contained within these regions. The replication efficiency and levels of cytoplasmic and virion RNA were analyzed for these mutants. Mutations that disrupted either or both of the predicted stem-loop structures of the primary signal reduced the replication efficiency by factors of 7 and 40, respectively; similar reductions in RNA encapsidation efficiency were observed. The mutant with both stem-loop structures disrupted had a phenotype similar to that of a mutant containing a deletion of the entire primary signal region. Mutations that disrupted the predicted stem-loop structure of the secondary signal led to similar reductions (factors of 4 to 6) in both the replication and RNA encapsidation efficiencies. The introduction of compensatory mutations into mutants from both the primary and secondary signal regions, which restored the predicted stem-loop structures, led to levels of replication and RNA encapsidation comparable to those of virus containing the wild-type encapsidation signal. Replacement of the BLV RNA region containing the primary and secondary encapsidation signals with a similar region from human T-cell leukemia virus (HTLV) type 1 or type 2 led to virus replication at three-quarters or one-fifth of the level of the parental virus, respectively. The results from both the compensatory mutants and BLV-HTLV chimeras indicate that the encapsidation sequences are recognized largely by their secondary or tertiary structures.

Helicobacter pylori: beyond peptic ulcer disease.

Beyond peptic ulcer disease, Helicobacter pylori infection is associated with intestinal-type gastric cancer and low-grade gastric mucosa-associated lymphoid tissue (MALT) lymphoma. It is also currently implicated as a possible cause of dyspepsia and extraintestinal disorders such as coronary artery disease, rosacea, chronic urticaria, and delayed growth in children. There are strong epidemiological data from large cohort studies linking H. pylori to gastric adenocarcinoma. Several cofactors, including early childhood acquisition of infection, strain-specific differences, genetic predisposition of the host, and the environment, appear to play a role in the progression of chronic gastritis to gastric cancer. H. pylori infection is seen in over 90% of MALT lymphomas, and about 70% of localized nonbulky tumors will undergo complete histological regression after eradication of the bacterium. Because follow-up data are limited to less than 2 years, those undergoing H. pylori eradication as primary therapy for MALT lymphoma require frequent histological surveillance for tumor recurrence. There are conflicting data from short-term studies regarding the effect of H. pylori eradication on dyspeptic symptoms. The decision to test or not for H. pylori in the dyspeptic patient may become easier when well-controlled studies with longer periods of follow-up become available. Because H. pylori induces a systemic inflammatory response, investigators are beginning to explore possible extraintestinal disease associations with the infection. The global prevalence of both peptic ulcer disease and gastric cancer has led to studies focusing on noninvasive screening for H. pylori in high-risk populations and prevention of primary infection by means of vaccination.

Beta-lactams versus glycopeptides in treatment of subcutaneous abscesses infected with Staphylococcus aureus.

The antibacterial efficacies of the beta-lactam antibiotics nafcillin and cefazolin were compared with those of the glycopeptide antibiotics vancomycin and teicoplanin in rats with subcutaneous abscesses infected with methicillin-susceptible Staphylococcus aureus. Animals were treated with antibiotics or diluent for 3 or 7 days. Rats receiving any antibiotic treatment, with the exception of teicoplanin-treated animals at day 7, had lower bacterial counts in their abscesses than did controls at days 3 and 7. Rats in the nafcillin and cefazolin treatment groups had lower bacterial counts in their abscesses than did rats in the vancomycin and teicoplanin treatment groups at days 3 and 7. The beta-lactam antibiotics were more effective therapy than the glycopeptide antibiotics in rats with subcutaneous abscesses infected with methicillin-susceptible S. aureus. In vitro, animal, and clinical data comparing beta-lactam and glycopeptide treatment of S. aureus infections were reviewed along with the resulting treatment recommendations.

[Integration of DNA oscillatory vector pSV neo to genome of NIH3T3: importance in investigations of transformation with c-Ha-ras oncogene]. / Integracja DNA wektora wahadlowego pSVneo do genomu komórek NIH3T3: znaczenie w badaniach transformacji onkogenem c-Ha-ras.

Main aim of this study was evaluation of application of stable clones of transforming cells, containing DNA of plasmid vector for the investigation of oncogenes. Plasmid vector was constructed basing on pSV2neo vector, containing activated oncogene c-Ha-ras-1, derived from pT 24-C3 which was followed by evaluation of phenotypic and genetic changes in standard line of NIH3T3 mouse fibroblast line after transfection with constructed plasmid. After two weeks of culture in selective conditions, transformants resistant to geneticin were obtained and analysis of clones was performed after transfection with constructed vector containing ras oncogene and after transfection pSV2neo. Analysis of efficiency of cloning and transformation basing on growth independent from placement and morphology and investigation of karyotypes demonstrated similar irregularities in both investigated groups and subchromosomal aberrations of NIH3T3 cells were even more frequent than in initial lines of NIH3T3 cells. Southern hybridization with pSV2neo-ras probe demonstrated that only restrictive DNA fragments, obtained by Pst1 enzyme contain copies of neo in cell genomes. Integration of gene cf geneticin-resistance increases thus normally unstable genetically NIH3T3 cells.

MR imaging of the brachial plexus.

The brachial plexus is difficult to evaluate with conventional radiologic techniques, including CT. However, it is well shown by MR imaging, which has direct multiplanar imaging capability and superior soft-tissue resolution [1-4]. We present our technique for evaluating the brachial plexus, discuss the anatomy, and illustrate normal and abnormal findings.

Principles of the design and operational considerations of large scale high performance liquid chromatography (HPLC) systems for proteins and peptides purification.

This review introduces concepts of design of large scale HPLC systems for purification of proteins and peptides. It is addressed to users of large scale HPLC systems to aid in system selection and help in customizing the design. Major techniques used for large scale HPLC purification of proteins and peptides are briefly reviewed. Engineering aspects of system design are discussed in detail. The review of selected relevant literature is provided as well as author's experience with the existing designs and his own systems. Commercial publications have been used in preparation of this review but only the most significant are listed as examples. The design process for any new system should be related to the performance of existing systems, if possible of a large scale. Laboratory data are also very useful in aiding the design process since they provide a lead, as to which chromatography techniques may succeed in providing required separation levels. The expertise needed for system design and operation comes from many areas: protein and peptide chemistry, chromatographic theory, mass transfer and hydrodynamics, machine design and material science. All these factors have to be blended together during the system design process. The controls must ensure flexibility in adapting to changing system configuration, depending on the operational requirements for various products. Extensive interfacing with the operator during the process run is essential. This work is focused mostly on system design and operation for reversed-phase chromatography and hydrophobic interaction chromatography, but it also covers aspects associated with other chromatographic techniques. The reviewed design principles would also apply to design other than HPLC large scale chromatography systems for biotechnology and pharmaceutical operations.

MR imaging of uterine carcinoma: correlation with clinical and pathologic findings.

Thirteen patients with clinical stages I and II endometrial carcinoma were examined with magnetic resonance (MR) imaging before surgery. Depth of invasion and stage of disease were assessed, and the results were compared with those from MR images of the surgical specimens and pathologic findings. Staging with MR imaging was accurate in 11 of 13 patients (85%). Our results agree with previous reports that MR imaging is an accurate, noninvasive method of assessing depth of myometrial invasion and cervical involvement. We anticipate that MR imaging will have an increasing role in treatment of patients with endometrial carcinoma.

Magnetic resonance imaging (MRI) effects on rat pineal neuroendocrine function.

This study investigated the effects of MRI on receptor-mediated activation of pineal gland indole biosynthesis. Exposure of rats to MRI reduced the effects of isoproterenol on pineal serotonin and N-acetylserotonin levels suggesting that strong magnetic fields and/or radio-frequency pulsing used in MRI inhibited beta-adrenergic activation of the gland. There was no effect of MRI on saline controls.

New retrovirus helper cells with almost no nucleotide sequence homology to retrovirus vectors.

We prepared retrovirus packaging cell lines containing gag-pol genes from spleen necrosis virus (expressed from a cytomegalovirus promoter and the simian virus 40 (SV40) polyadenylation sequences) and, on a separate vector, either the env gene from spleen necrosis virus (expressed from the Rous sarcoma virus promoter and the SV40 polyadenylation sequences) or the env gene from amphotropic murine leukemia virus (expressed from a cytomegalovirus promoter and the SV40 polyadenylation sequences). The nucleotide sequences in these packaging cell lines have almost no homology to the retrovirus vectors we used. Retrovirus vectors were produced from these new helper cell lines without any genetic interactions between the vectors and sequences in the helper cells and without transfer of the packaging sequences.

Computed tomography and magnetic resonance imaging of the pericardium.

Computed tomography is an established modality for the evaluation of the pericardium. It is used to evaluate complicated pericardial effusions, pericardial thickening, calcific pericarditis, pericardial cysts, postoperative changes and primary and metastatic neoplasms of the pericardium. Magnetic resonance imaging is being used with increasing frequency in the evaluation of pericardial disease. It offers advantages over computed tomography, including a potential for tissue characterization, absence of ionizing radiation or need for intravenous contrast medium, and the ability to scan in any plane. Disadvantages include greater cost, longer examination times and the inability to identify calcification positively.