Genotyping the hemophilia inversion hotspot by use of inverse PCR

BACKGROUND: Factor VIII intron 22 inversions (Inv22) cause 40%-45% of severe cases of hemophilia A in all human populations. Currently, Inv22 can be analyzed either by Southern blotting or by rapid long-distance-PCR-based approaches. We describe an alternative method using inverse-PCR (I-PCR). METHODS: I-PCR involved 3 steps: (a) BclI restriction; (b) self-ligation of restriction fragments, providing BclI rings; and (c) standard multiplex-PCR analysis. PCR was achieved by use of a set of 3 primers that yielded a 487-bp amplicon for the nonrearranged intragenic allele and a 559-bp amplicon for the Inv22 allele. Specific primer sites were targeted by masking relevant regions for human repeats and low-complexity DNA. Inv22 I-PCR was applied to samples from 16 individuals (8 women and 8 men) representing 24 X chromosomes previously genotyped by Southern blotting. Additionally, we evaluated the sensitivity and the ability to assess eventual Inv22 carrier mosaicisms by experiments using artificial DNA mixtures (Inv22 + no-Inv22 male samples). RESULTS: Results for previously genotyped samples agreed with results of Southern blot analyses. As expected, cell composition of the artificial mosaic was linearly reflected by the relative intensities of Inv22 signals. I-PCR was estimated to detect Inv22-positive cells at concentrations as low as approximately 5%. CONCLUSION: The proposed technique provides a rapid tool for Inv22 genotyping.     

Primary sellar esthesioneuroblastoma

Summary We describe an extremely rare case of a primary intrasellar esthesioneuroblastoma.     

Diverse roles for the third complementarity determining region of the heavy chain (H3) in the binding of immunoglobulin Fv fragments to DNA, nucleosomes and cardiolipin

Autoantibodies to DNA and chromatin employ junctional diversity and somatic mutations to generate or enhance antigen recognition. To define the role of diversity generating mechanisms in the etiology of autoantibodies to nuclear antigens, the heavy (H) chain of a murine autoantibody, 3H9, was used in its somatically mutated or germ-line form in conjunction with its own or with heterologous CDR3 (H3) domains. The resulting H chains were expressed together with the 3H9 light (L) chain as single-chain Fv (scFv) in Escherichia coli and assayed for binding to DNA, nucleosomes, or cardiolipin by enzyme-linked immunosorbent assay. All recombinant scFv exhibited nearly identical binding to cardiolipin. In contrast, the binding to nuclear antigens was drastically reduced by the reversion of mutations in 3H9 or the exchange of H3, such that only 3H9 itself bound strongly to single-stranded DNA, double-stranded DNA and nucleosomes. The results illustrate diverse interactions between a single combining site and different autoantigens. The analysis of these interactions suggests that the 3H9 VH domain, as encoded by the germ line, directs binding to cardiolipin, whereas structural determinants of H3, in concert with the remainder of the combining site, guide the maturation of antibody binding toward nuclear autoantigens.     

Complexation of poly(monobenzyl itaconate) and poly(N-vinyl-2-pyrrolidone) in dilute solution

Upon mixing dilute solutions (10^?4 – 10^?2 mol/l) of poly(monobenzyl itaconate) (PMBI) and poly(N-vinyl-2-pyrrolidone) (PVP) in methanol, soluble polycomplex particles are formed. Here we report on the spectrophotometric determination of the turbidity of different solutions of PVP and PMBI as a function of polymer concentration and PVP molecular weight. Viscosity measurements were also performed. The polycomplex is weak. Polycomplex particles are more compact and much larger than separated chains of polyacid and polybase. No definite stoichiometry is observed and a microgel-like structure is proposed.     

The effect of cannabinoid receptor agonist WIN 55,212–2 on anxiety‐like behavior and locomotion in a genetic model of absence seizures in the elevated plus‐maze

GAERS and NEC rats were treated with cannabinoid 1/2 receptor agonist WIN 55,212‐2 2 mg/kg and tested on the Elevated Plus‐Maze.     

Pentadecapeptide BPC 157 (PL 14736) improves ligament healing in the rat

We improved medial collateral ligament (MCL) healing throughout 90 days after surgical transection. We introduced intraperitoneal, per‐oral (in drinking water) and topical (thin cream layer) peptide therapy always given alone, without a carrier. Previously, as an effective peptide therapy, stable gastric pentadecapeptide BPC 157 (GEPPPGKPADDAGLV, an anti‐ulcer peptide effective in inflammatory bowel disease therapy (PL 14736)) particularly improved healing of transected tendon and muscle and wound healing effect including the expression of the early growth response 1 (egr‐1) gene. After MCL transection BPC 157 was effective in rats when given once daily intraperitoneally (10 µg or 10 ng/kg) or locally as a thin layer (1.0 µg dissolved in distilled water/g commercial neutral cream) at the site of injury, first application 30 min after surgery and the final application 24 h before sacrifice. Likewise, BPC 157 was effective given per‐orally (0.16 µg/ml in the drinking water (12 ml/day/rat)) until sacrifice. Commonly, BPC 157 µg‐ng‐rats exhibited consistent functional, biomechanical, macroscopic and histological healing improvements. Thus, we suggest BPC 157 improved healing of acute ligament injuries in further ligament therapy. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1155–1161, 2010     

Stable gastric pentadecapeptide BPC 157 in trials for inflammatory bowel disease (PL-10, PLD-116, PL 14736, Pliva, Croatia). Full and distended stomach, and vascular response.

Gastric pentadecapeptide BPC 157 (GEPPPGKPADDAGLV, M.W. 1419, safe in clinical trials for inflammatory bowel disease (PL 10, PLD 116, PLD 14736, Pliva, Croatia)) has a particular cytoprotective/adaptive cytoprotective activity. The cytoprotective/adaptive cytoprotection researches largely neglect that stomach distension could per se jeopardize the mucosal integrity, with constantly stretched mucosa and blood vessels, and sphincters more prone for reflux induction. After absolute alcohol instillation in fully distended rat stomach, gastric, esophageal and duodenal lesions occur. Throughout next 3 min, left gastric artery blood vessels clearly disappear at the serosal site, indicative for loss of vessels both integrity and function. Contrary, constant vessels presentation could predict the benefi cial effect of applied agent. After pentadecapeptide BPC 157 instillation into the stomach the vessels presentation remains constant, and lesions of stomach, esophagus, and duodenum are inhibited. Standards (atropine, ranitidine, omeprazole) could only slightly improve the vessels presentation compared to control values, and they have only a partial effect on the lesions. In this review we emphasize BPC 157 unusual stability, and some of its important effects: effectiveness against various lesions in gastrointestinal tract, on nitric oxide (NO)-system, and NO-agents effects, on somatosensory neurons, salivary glands function, recovery of AMP-ADP-ATP system, endothelium protection, effect on endothelin, and on angiogenesis promotion. It also antagonizes other alcohol effects, including acute and chronic intoxication. Given peripherally, it counteracts the consequence of central dopamine system disturbances (receptor blockade), and induces serotonin release in substantia nigra. Therapeutic potential of BPC 157 as a cytoprotective agent is also seen in its capability to heal various wounds. Given directly into the stomach, BPC 157 instantly recovers disturbed lower esophageal and pyloric sphincter pressure in rats after 12–20 months of untreated esophagitis. All these could be suggestive for its role as a natural protectant in gastric juice with particular function throughout stomach distension.