Human immunodeficiency virus 1 (HIV-1)-specific reverse transcriptase (RT) inhibitors may suppress the replication of specific drug-resistant (E138K)RT HIV-1 mutants or select for highly resistant (Y181C-->C181I)RT HIV-1 mutants

Mutant HIV-1 that expresses a Glu138-->Lys substitution in its RT [(E138K)RT] is resistant to the HIV-1-specific RT inhibitor 2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5"-(4"-amino-1",2"- oxathiole-2",2"-dioxide)pyrimidine (TSAO). However, cell cultures infected with this mutant were completely protected against virus-mediated destruction by micromolar concentrations of the HIV-1-specific RT inhibitors tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and -thione (TIBO), nevirapine, and bis(heteroaryl)piperazine (BHAP). In contrast, cells infected with a virus mutant that expresses a Tyr181-->Cys substitution in its RT [(Y181C)RT] were not protected by nevirapine and TIBO and were only temporarily protected by BHAP. HIV-1 mutant that emerged under the latter conditions contained a Cys181-->Ile substitution in their RT [(LC181I)RT]. This mutant proved highly resistant to all HIV-1-specific RT inhibitors tested, except for several 1-(2-hydroxyethoxymethyl)-6-(phenylthio)thymine (HEPT) derivatives. When recombinant (C181I)RT was evaluated for susceptibility to the HIV-1-specific RT inhibitors, it was resistant to all inhibitors except the HEPT compounds. Since a (Y181F)RT HIV mutant strain was isolated from cells infected with (Y181C)RT HIV-1 and treated with BHAP, we postulate that the Ile codon was derived from a Cys-->Phe transversion mutation (TGT-->TTT), followed by a Phe-->Ile transversion mutation (TTT-->ATT).     

Development and characterization of a purified diet to identify obesity-susceptible and resistant rat populations

A purified moderately high fat diet has been developed to examine diet-induced obesity in rats. Male Sprague-Dawley rats were fed this or an AIN-76A diet for 15 wk and energy metabolism indices were monitored. Food intake, body weight and water balance indices were recorded on a weekly or daily basis. Over the 15-wk period, rats fed the experimental diet diverged into two groups differing in the rate of body weight gain. Animals were labeled as "gainers" or "resisters" depending on their susceptibility to obesity. Following the dietary period, rats were decapitated and trunk blood was collected for glucose and insulin measurements. Gainers consumed slightly more energy than resisters over the experimental period (P < 0.05), but due to greater fecal energy loss, absorbed energy did not differ. Hence gainers became obese without significantly altered energy retention. Urinary creatinine, urea nitrogen and water balance were not different between the groups and consequently could not explain body weight differences. Further, gainers had significantly greater plasma glucose concentration than controls, indicating a potential for these animals to become diabetic. Results suggest metabolic differences must account for the divergence in weight gain observed in the two groups. The dietary model characterized in this study should provide a useful tool to study diet-induced obesity and to determine its underlying mechanism.     

Mammary gland expression of transgenes and the potential for altering the properties of milk

Transgenic animals are a useful in vivo experimental model for assessing the ability and impact of foreign gene expression in a biological system. Transgenic mice are most commonly used, while transgenic sheep, goats, pigs and cows have also been developed for specific, "applied" purposes. Most of the work directed at targeting expression of transgenes to the mammary gland of an animal, by using a milk gene promoter, has been with the intent of either studying promoter function or recovering the desired protein from the milk. Transgenic technology can also be used to alter the functional and physical properties of milk resulting in novel manufacturing properties. The properties of milk have been altered by adding a new protein with the aim of improving the milk, not of recovering the protein for other uses.     

US-guided core breast biopsy: use and cost-effectiveness

OBJECTIVES: We sought to determine, using serial echocardiography, the hydrodynamic mechanisms involved in the occurrence of hemolysis after mitral valve repair. BACKGROUND: Recently, fluid dynamic simulation models have identified distinct patterns of mitral regurgitant flow disturbances in patients with mitral prosthetic hemolysis that were associated with high shear stress and may therefore produce clinical hemolysis. Rapid acceleration, fragmentation, and collision jets were associated with high shear stress and hemolysis whereas slow deceleration and free jets were not. METHODS: We reviewed serial echocardiographic studies of 13 consecutive patients with hemolytic anemia after mitral valve repair who were referred for mitral reoperation between January 1985 and December 1996 (group 1). Thirteen patients undergoing reoperation for mitral regurgitation after mitral valve repair but without hemolysis served as controls (group 2). RESULTS: The mitral regurgitant jet was central in origin in 12 group 1 patients and 9 group 2 patients (Fisher exact test, p= 0.3). The other patients had para-ring regurgitation. Group 1 patients had collision (n=11), rapid acceleration (n=2) or fragmentation (n=1) jets whereas group 2 patients had slow deceleration (n=11) or free jets (n=2) (Fisher exact test, p < 0.0001). One patient with hemolysis had both collision and rapid acceleration jets. The "culprit" jet could be identified on the postbypass transesophageal echocardiography (TEE) study in only 1 patient at the time of initial mitral repair. Twelve group 1 patients underwent reoperation, with subsequent resolution of hemolysis in all patients. At reoperation, the initial repair was found to be intact in 8 (67%) patients. CONCLUSION: Distinct patterns of flow disturbance associated with high shear stress were identified by color Doppler imaging in patients with hemolysis after mitral valve repair. The majority (92%) of these color flow disturbances were not present during intraoperative postbypass TEE study after initial mitral repair and subsequently developed in the early postoperative period.     

Measurement of amide hydrogen exchange by MALDI-TOF mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) was used to determine amide proton/deuteron (H/D) exchange rates. The method has broad application to the study of protein conformation and folding and to the study of protein-ligand interactions and requires no modifications of the instrument. Amide protons were allowed to exchange with deuterons in buffered D2O at room temperature, pD 7.25. Exchanged deuterons were "frozen" in the exchanged state by quenching at pH 2.5, 0 degree C and analyzed by MALDI-TOF MS. The matrix mixture consisted of 5 mg/mL alpha-cyano-4-hydroxycinnamic acid, acetonitrile, ethanol, and 0.1% TFA. The matrix was adjusted to pH 2.5, and the chilled MALDI target was rapidly dried. Deuteration of amide protons on cyclic AMP-dependent protein kinase was measured after short times of incubation in deuterium by pepsin protein digestion and MALDI-TOF MS analysis. The unseparated peptic digest was analyzed in a single spectrum of the mixture. From five spectra, H/D exchange rates were determined for some 40 peptides covering 65% of the protein sequence.     

Isoseparation curves: a mechanism for optimizing off-axis dose homogeneity of intact breast irradiation

The purpose of this paper is to determine whether using off-axis isoseparation curves to optimize the collimator rotation angle improves dose homogeneity. Eleven intact breast irradiation patients underwent computerized tomography (CT) treatment planning with 1 cm abutting slices. Central plane treatment planning, using 6 MV photons, tissue inhomogeneity corrections, and isocentric opposed tangent treatment fields, was performed. Collimators were rotated to match chest wall slope through the use of a beam's-eye-view setting. Patient separations were measured from the apex of the breast to the posterior field border on each axial CT slice. Sagittal-plane isoseparation curves were constructed from these measurements. Using these curves, the collimator rotation that minimized off-axis separation differences was determined. A comparison of off-axis dose inhomogeneity was performed for patients with a > or =10 degrees difference between this optimized collimator angle and the angle determined by chest wall slope. These comparative treatment plans differed only with respect to collimator angle rotation. The mean optimal collimator rotation angle differed significantly from the mean rotation angle which matched the chest wall slope (5.4 degrees vs. 11 degrees, respectively, P < 0.001). Four of the 11 patients had rotation angle differences of 10 degrees. In these patients, the optimization of collimator angle reduced the percentage of breast volume to "that" received > or =110% of the prescribed dose. For the patient with the largest breast size to the patient with the smallest breast size the decreases were, respectively, 5% (15% to 10%), 3% (24% to 21%), 1% (4% to 3%), and 1% (0.9% to 0%). The mean reduction in dose inhomogeneity was greatest in the inferior breast quadrants. At 6 cm and 4 cm off axis, the mean reductions in the percentages of the breast tissue to "that" received 110% of the prescribed dose were respectively 15.1% and 5.3 %. Optimizing the collimator angle through the use of isoseparation curves decreases dose inhomogeneity. The greatest improvements are in the inferior quadrants of the intact breast. The improved dose homogeneity may have clinical relevance in the treatment of patients with large breast sizes.     

The flavin-containing monooxygenase 2 gene (FMO2) of humans, but not of other primates, encodes a truncated, nonfunctional protein

Flavin-containing monooxygenases (FMOs) are NADPH-dependent flavoenzymes that catalyze the oxidation of heteroatom centers in numerous drugs and xenobiotics. FMO2, or "pulmonary" FMO, one of five forms of the enzyme identified in mammals, is expressed predominantly in lung and differs from other FMOs in that it can catalyze the N-oxidation of certain primary alkylamines. We describe here the isolation and characterization of cDNAs for human FMO2. Analysis of the sequence of the cDNAs and of a section of the corresponding gene revealed that the major FMO2 allele of humans encodes a polypeptide that, compared with the orthologous protein of other mammals, lacks 64 amino acid residues from its C terminus. Heterologous expression of the cDNA revealed that the truncated polypeptide was catalytically inactive. The nonsense mutation that gave rise to the truncated polypeptide, a C --> T transition in codon 472, is not present in the FMO2 gene of closely related primates, including gorilla and chimpanzee, and must therefore have arisen in the human lineage after the divergence of the Homo and Pan clades. Possible mechanisms for the fixation of the mutation in the human population and the potential significance of the loss of functional FMO2 in humans are discussed.     

Analogues of capsaicin with agonist activity as novel analgesic agents: structure-activity studies. 4. Potent, orally active analgesics

Structural features of three regions of the capsaicin molecule necessary for agonist properties were delineated by a previously reported modular approach. These in vitro agonist effects were shown to correlate with analgesic potency in rodent models. Combination of optimal structural features from each of these regions of the capsaicin molecule have led to highly potent agonists (eg., 1b). Evaluation in vivo established that 1b had analgesic properties but poor oral activity, short duration of action, and excitatory side effects which precluded further development of this compound. Preliminary metabolism studies had shown that the phenol moiety of 1b was rapidly glucuronidated in vivo, providing a possible explanation for the poor pharmacokinetic profile. Subsequent specific modification of the phenol group led to compounds 2a-j, which retained in vitro potency. The in vivo profiles of two representatives of this series, 2a,h, were much improved over the "parent" phenol series, and they are candidates for development as analgesic agents.     

Supramalleolar osteotomy for ankle valgus in myelomeningocele

Oral fibroblasts stimulated invasion of oral-carcinoma cells into the collagen matrix. The mechanisms of the fibroblast-induced stimulation of invasiveness was further investigated by examining cell motility and proteolytic activity of tumor cells, using mainly an adenoid-cystic-carcinoma cell line (ACCS) and normal fibroblasts from gingival tissues. Conditioned medium from the fibroblasts grown in serum-free medium was fractionated on a Superdex 200 pg column, and Peak 1 eluted at 200 to 300 kDa and Peak 2 eluted at 50 to 100 kDa were found to contain different specific activity. Treatment of ACCS cells with Peak 1 resulted in an increase in the production of proteolytic enzymes. Peak 2 stimulated both chemotaxis and chemokinesis of ACCS cells. A chemotactic factor was purified from the heparin-unbound fraction of Peak 2 by anion exchange and hydrophobic chromatography, and was named "fibroblast-derived motility factor (FDMF)". At 1 microg/ml, FDMF stimulated chemotaxis of ACCS cells by 4-fold compared with unstimulated controls. Characterization of the physicochemical properties of FDMF suggested that it might be different from any known motility factors. Exposure of ACCS cells to FDMF resulted in reduced amounts of actin stress fiber in the cytoplasm and induction of tyrosine phosphorylation of several cellular proteins detectable 30 to 60 min after treatment. These FDMF-induced changes were blocked by pre-treatment either with genistein or with pertussis toxin. These findings suggest that FDMF may be a novel protein which stimulates cell motility via a signaling pathway mediated by a pertussis-toxin-sensitive G protein and tyrosine phosphorylation.     

Perinuclear antineutrophil cytoplasmic antibodies in patients with Crohn's disease define a clinical subgroup

BACKGROUND & AIMS: Antineutrophil cytoplasmic antibodies (ANCA) have been consistently detected in a subgroup of patients with Crohn's disease (CD). This study was designed to determine whether serum ANCA expression in patients with CD characterizes an identifiable clinical subgroup. METHODS: The study population consisted of 69 consecutive patients with an established diagnosis of CD as determined by a combination of characteristic clinical, radiographic, endoscopic, and histopathologic criteria. Sera from the patients were analyzed for the presence of ANCAs using the fixed neutrophil enzyme-linked immunosorbent assay (ELISA) assay. Perinuclear ANCA (pANCA)-positive and cytoplasmic ANCA (cANCA)-positive results by ELISA were confirmed by indirect immunofluorescence staining. Clinical profiles of the ANCA-positive patients with CD were compared with those of patients with CD not expressing ANCA (ANCA-negative). RESULTS: pANCA-positive patients with CD have endoscopically and/or histopathologically documented left-sided colitis and symptoms of left-sided colonic inflammation, clinically reflected by rectal bleeding and mucus discharge, urgency, and treatment with topical agents. One hundred percent of patients with CD expressing pANCA had "UC-like" features. CONCLUSIONS: In patients with CD, serum pANCA expression characterizes a UC-like clinical phenotype. Stratification of CD by serum pANCA provides evidence of heterogeneity within CD and suggests a common intestinal mucosal inflammatory process among a definable subgroup of patients with CD and UC expressing this marker.