Detection of polyomaviral DNA in clinical samples from immunocompromised patients: correlation with clinical disease

Characterization of the region between HLA-B and the TNF loci in the human MHC revealed the presence of duplicated loci, named CL1 and CL2, that included repeat sequences. Development and use of a PCR typing methodology that amplified both CL microsatellites simultaneously indicated that PCR product patterns analysed on native agarose gels were allelic (Abraham et al., 1992). The purpose of the current study was to determine the molecular explanation for the unique patterns achieved. Sequence analysis of the CL1 locus from 32 chromosomes representing 10 ancestral haplotypes indicated that six alleles were present. The CL microsatellites also provided an opportunity to study the evolutionary relationships between MHC haplotypes from different racial groups. Sequence comparison of closely related ancestral haplotypes from different racial groups suggested that the CL1 microsatellite has not changed in the period since divergence.     

Recombinant scrapie-like prion protein of 106 amino acids is soluble

The N terminus of the scrapie isoform of prion protein (PrPSc) can be truncated without loss of scrapie infectivity and, correspondingly, the truncation of the N terminus of the cellular isoform, PrPC, still permits conversion into PrPSc. To assess whether additional segments of the PrP molecule can be deleted, we previously removed regions of putative secondary structure in PrPC; in the present study we found that deletion of each of the four predicted helices prevented PrPSc formation, as did deletion of the stop transfer effector region and the C178A mutation. Removal of a 36-residue loop between helices 2 and 3 did not prevent formation of protease-resistant PrP; the resulting scrapie-like protein, designated PrPSc106, contained 106 residues after cleavage of an N-terminal signal peptide and a C-terminal sequence for glycolipid anchor addition. Addition of the detergent Sarkosyl to cell lysates solubilized PrPSc106, which retained resistance to digestion by proteinase K. These results suggest that all the regions of proposed secondary structure in PrP are required for PrPSc formation, as is the disulfide bond stabilizing helices 3 and 4. The discovery of PrPSc106 should facilitate structural studies of PrPSc, investigations of the mechanism of PrPSc formation, and the production of PrPSc-specific antibodies.     

Use of a gonadotropin-releasing hormone agonist in the evaluation of postmenopausal virilization due to ovarian hyperthecosis. A case report

BACKGROUND: Hyperthecosis in a postmenopausal woman is a very rare cause of virilization, and only five cases have been reported previously. CASE: A woman presented with a nine-year history of increasing hirsutism and a mild virilization beginning in the perimenopausal period. Initial androgen metabolite concentrations suggested attenuated late-onset adrenal hyperplasia, but a trial of dexamethasone treatment was ineffective. Subsequent use of leuprolide acetate resulted in a biochemical and clinical improvement in the signs and symptoms. CONCLUSION: This case is unique because gonadotropin-releasing hormone agonist administration was utilized as both a diagnostic and therapeutic modality.     

Instrumental treatment of recurrent lithiasis of biliary tract with stenosis of hepatico-jejunal anastomosis

The optimal treatment for recurrent stones of the biliary tract in patients previously treated by hepatic-jejunal anastomosis with Roux en Y loop is debated and the criteria are not fully defined. Age, cardiopulmonary diseases, diabetes, multiple previous surgery of the biliary tract, size, number, and type of stones are some of the factors to evaluate before choosing the most convenient approach. We present two high surgical risk patients with recurrent biliary tract stones previously treated with hepatic-jejunal anastomosis and Roux en Y loop, and treated successfully. Percutaneous lithotripsy was used and in one extracorporeal lithotripsy was associated. These techniques offered a good treatment for these patients, but we must consider the high cost of these procedures. The criteria to choose the best option should consider the risk factors and the cost effectiveness of the various possibilities.     

Evaluation of a breast biopsy phantom for learning freehand ultrasound-guided biopsy of the liver

RATIONALE AND OBJECTIVES: We evaluated whether a breast biopsy phantom device would aid in the development of skills in freehand ultrasound liver biopsy. METHODS: Three radiologists who were inexperienced in freehand biopsy of the liver were observed. Each radiologist was timed and scored during attempts to biopsy lesions created in a beef liver. The time required for biopsy and the success of each pass was recorded. A commercially available breast biopsy phantom was then used by each of these radiologists during two 20-min practice sessions. Posttraining testing on the beef liver was performed in the same manner as pretraining testing. RESULTS: Freehand biopsy practice using the breast biopsy phantom resulted in a reduction in the mean time required for biopsy from 32 to 17 sec. Each of the three subjects reduced the mean time required for successful biopsy after training using the breast biopsy phantom. The total number of lesions missed was reduced from 14 of 43 to 0 of 45. CONCLUSION: Practice using the ultrasound breast biopsy phantom improves performance in freehand ultrasound biopsy of the liver.     

Mapping protein-protein interactions by affinity-directed mass spectrometry

A precise and rapid method for identifying sites of interaction between proteins was demonstrated; the basis of the method is direct mass spectrometric readout from the complex to determine the specific components of the proteins that interact--a method termed affinity-directed mass spectrometry. The strategy was used to define the region of interaction of a protein growth factor with a monoclonal antibody. A combination of proteolytic digestion and affinity-directed mass spectrometry was used to rapidly determine the approximate location of a continuous binding epitope within the growth factor. The precise boundaries of the binding epitope were determined by affinity-directed mass spectrometric analysis of sets of synthetic peptide ladders that span the approximate binding region. In addition to the mapping of such linear epitopes, affinity-directed mass spectrometry can be applied to the mapping of other types of molecule-molecule contacts, including ligand-receptor and protein-oligonucleotide interactions.     

Quantitative cardiac perfusion: a noninvasive spin-labeling method that exploits coronary vessel geometry

A patient referred for preoperative investigation of prolonged bleeding and easy bruising was found to have increased thrombin and reptilase times; however, the thrombin catalysed release of fibrinopeptides A and B was normal. Analysis of five other family members, spanning three generations, indicated that three had a similar defect and suggested autosomal dominant inheritance. Non-reducing SDS-PAGE of purified fibrinogen from affected individuals showed that the 340 kD form of their fibrinogen ran as a doublet. SSCP (single-stranded conformational polymorphism) analysis of exon 5 of the A alpha gene, which encodes the C-terminal half of the chain, confirmed the presence of a mutation. Cycle sequencing of PCR amplified DNA revealed a 13 base pair deletion (nt 4758-4770), resulting in a frame-shift at Ala 475, which translates as four new amino acids before terminating at a new stop codon (-476His-Cys-Leu-Ala-Stop). The presence of a circulating truncated A alpha chain was confirmed when SDS-PAGE gels were probed with an alpha chain specific antisera; which showed that the variant A alpha chain comigrated with gamma chains. The truncation results in a variant A alpha chain with a deletion of 131 amino acids (480-610), and four new amino acids at the C-terminal.     

FAD-mediated enzymatic conversion of NAD+ to NADH: application to chiral synthesis of L-lactate

Electroenzymatic reduction of NAD+ to NADH for subsequent use in enzymatic synthesis has been carried out at carbon electrodes bearing lipoamide dehydrogenase (LiDH) immobilized under a Nafion film. The self-mediated electron transfer was made possible by an excess of flavin adenine dinucleotide (FAD) entrapped together with LiDH. Results were compared to those obtained with a similar electrode containing both LiDH and a polymeric form of FAD (pFAD) prepared by anodic polymerization of FAD.     

Modified sperm stress test: a simple assay that predicts sperm-related abnormal in-vitro fertilization

Loss of sperm motility is associated with the process of sperm senescence and occurs at different rates within a given normal or abnormal sperm population. Reactive oxygen species attack cell membrane phospholipids, generating fatty acid peroxides and other degradation products, that also have deleterious effects on sperm motility and fertilizing ability. The objective of this investigation was to study a modification of the original sperm stress test (MOST), changing the culture medium to one offering transitional metals and shortening the total test time, to ascertain whether it can predict fertilization under these laboratory conditions. A total of 41 semen samples was obtained from patients undergoing in-vitro fertilization (IVF) at our institution. Semen samples were grouped into those producing total fertilization rates (FR) within normal limits (>50%) and those showing low total FR (<50%). The normal FR group had a significantly greater MOST mean value than the low FR group (0.71 versus 0.44). Furthermore, there was a statistically significant correlation between the MOST score and ungrouped fertilization rates (r = 0.53, P = 0.0004). Diagnostic statistics for MOST ratio values predicting <50% FR showed an optimal threshold of 0.39. Collectively, sensitivity, specificity, positive predictive value and negative predictive value have their largest values at this threshold. Taking into account the above mentioned threshold figures, there is a significant association between MOST and FR categories (P = 0.0009). In conclusion, MOST is a simple assay that has significant predictive value for sperm related IVF abnormalities.