An evolutionary rearrangement of the Xp11.3-11.23 region in 3p21.3, a region frequently deleted in a variety of cancers.

In searching for a tumor suppressor gene in the 3p21.3 region, we isolated two genes, RBM5 and RBM6. Sequence analysis indicated that these genes share similarity. RBM5 and-to a lesser extent-RBM6 also have similarity to DXS8237E at Xp11.3-11.23, which maps less than 20 kb upstream of UBE1. A homologue of UBE1, UBE1L, is located at 3p21. 3. FISH analysis showed that the distance between UBE1L and RBM5 in 3p21.3 is about 265 kb. DXS8237E and UBE1 on the X chromosome have the same orientation, whereas on chromosome 3 the orientation of RBM5 and that of RBM6 are opposite to the orientation of UBE1L. Presumably, part of the Xp11.3-11.23 region has duplicated to chromosome 3. Part of this region on chromosome 3 may subsequently have duplicated again within the same chromosomal region. Inversion at some stage of the evolution of the human genome would explain the change in orientation of the genes on chromosome 3 compared with that of the genes on the X chromosome.     

How to obtain good morphology and antigen detection in the same tissue section?

Most human and animal biopsy samples are routinely embedded in paraffin since this enables the pathologist or researcher to obtain excellent morphology and simplifies storage. Nevertheless, in many cases, the antigen of interest cannot be detected in paraffin section. The alternative available for good immunohistochemistry is preparation of cryosections, which usually provide decent antigen preservation and are frequently used for immunofluorescence. However, cryosections often do not provide efficient morphological details of tissues and cells for pathologic evaluation. In order to obtain good antigen preservation and improve tissue and cell morphology after freezing, we tested three different fixations and freezing methodologies and compared them to routine formaldehyde fixation and paraffin embedding. As a model system, we selected the epithelium of the rat urinary bladder and trachea. On all samples, haematoxylin and eosin staining was performed as well as immunofluorescence with antibodies against tight junction protein ZO-1 and against intermediate filament cytokeratin 7. The best compromise between morphology and immunofluorescence was obtained with “sucrose impregnation prior to freezing” method. Moreover, this procedure is also quicker in comparison to standard paraffin section preparation. To check the clinical relevance of our study, this method was used for human biopsy samples of neoplastic urothelial and bronchial mucosa lesions. Besides good immunofluorescence results, the morphology of these samples was well preserved. We therefore propose that cryosection preparation with sucrose impregnation prior to freezing should be further exploited in other clinical and veterinary applications, since it enables good morphology and antigen preservation.     

Microwave‐assisted solid‐phase peptide synthesis at 60 °C: alternative conditions with low enantiomerization

Several conditions have been used in the coupling reaction of stepwise SPPS at elevated temperature (SPPS‐ET), but we have elected the following as our first choice: 2.5‐fold molar excess of 0.04–0.08 M Boc or Fmoc‐amino acid derivative, equimolar amount of DIC/HOBt (1:1) or TBTU/DIPEA (1:3), 25% DMSO/toluene, 60 °C, conventional heating. In this study, aimed to further examine enantiomerization under such condition and study the applicability of our protocols to microwave‐SPPS, peptides containing L ‐Ser, L ‐His, L ‐Cys and/or L ‐Met were manually synthesized traditionally, at 60 °C using conventional heating and at 60 °C using microwave heating. Detailed assessment of all crude peptides (in their intact and/or fully hydrolyzed forms) revealed that, except for the microwave‐assisted coupling of L ‐Cys, all other reactions occurred with low levels of amino acid enantiomerization (<2%). Therefore, herein we (i) provide new evidences that our protocols for SPPS at 60 °C using conventional heating are suitable for routine use, (ii) demonstrate their appropriateness for microwave‐assisted SPPS by Boc and Fmoc chemistries, (iii) disclose advantages and limitations of the three synthetic approaches employed. Thus, this study complements our past research on SPPS‐ET and suggests alternative conditions for microwave‐assisted SPPS. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Neutrophils exhibit distinct phenotypes toward chitosans with different degrees of deacetylation: implications for cartilage repair

Introduction  

Osteoarthritis is characterized by the progressive destruction of cartilage in the articular joints. Novel therapies that promote resurfacing of exposed bone in focal areas are of interest in osteoarthritis because they may delay the progression of this disabling disease in patients who develop focal lesions. Recently, the addition of 80% deacetylated chitosan to cartilage microfractures was shown to promote the regeneration of hyaline cartilage. The molecular mechanisms by which chitosan promotes cartilage regeneration remain unknown. Because neutrophils are transiently recruited to the microfracture site, the effect of 80% deacetylated chitosan on the function of neutrophils was investigated. Most studies on neutrophils use preparations of chitosan with an uncertain degree of deacetylation. For therapeutic purposes, it is of interest to determine whether the degree of deacetylation influences the response of neutrophils to chitosan. The effect of 95% deacetylated chitosan on the function of neutrophils was therefore also investigated and compared with that of 80% deacetylated chitosan.     

Molecular sieves provoke multiple substitutions in the enzymatic synthesis of fructose oligosaccharide–lauryl esters

The cause of discrepancies in the literature regarding the specificity of immobilized Candida antarctica lipase B in the acylation of oligosaccharides was examined. Molecular sieves, generally used to control the water content during acylation reactions, turned out to have an important role in this. It was proven that molecular sieves alone can catalyze the acylation of fructose oligomers using vinyl laurate, leading to multiple substitution of the oligomers. This effect was the most profound at conditions unfavorable for the enzyme, because this resulted in a relatively high concentration of the chemically produced adducts. The enzyme alone catalyzed the formation of monosubstituted oligomers. It was proven that even solvent pre-drying by molecular sieves already causes the release of catalyzing compounds to the liquid, leading to subsequent catalysis. These findings should be taken into account when applying molecular sieves in this type of reactions in the future. Molecular sieves could, moreover, be used as a catalyst when multiple substitution is desired.     

Polyphasic characterization of “<Emphasis Type="Italic">Aspergillus nidulans</Emphasis> var. <Emphasis Type="Italic">roseus</Emphasis>” ATCC 58397

Polyphasic characterization of the echinocandin B producer Aspergillus nidulans var. roseus ATCC 58397 strain was carried out to elucidate its taxonomical status. According to its carbon source utilization and secondary metabolite spectrum as well as the partial β-tubulin, calmodulin, and γ-actin gene sequences, A. nidulans var. roseus belongs to the Emericella rugulosa species. Auxotroph mutants of A. nidulans var. roseus ATCC 58397 and E. rugulosa CBS 171.71 and CBS 133.60 formed stable heterokaryons on minimal medium with several A. nidulans strains, and in the case of A. nidulans var. roseus, even cleistothecia were developed.