Gene conversion-like sequence transfers in a mouse antibody transgene: antigen selection allows sensitive detection of V region interactions based on homology.

Gene conversion is important for antibody diversification in chickens, rabbits and cows. In mice, however, conversion events appear to be infrequent among endogenous antibody genes. DNA sequence transfer events that resemble gene conversions have been reported for a mouse H chain transgene (VVC(mu)) that contains two closely spaced homologous VDJ segments. Surprisingly, these reported VVC(mu) sequence transfers were found frequently among mouse B cells responding to immunization. Transgene sequence transfers could be occurring at high frequency in responding VVC(mu) B cells or could be occurring at lower frequency with subsequent amplification by preferential antigen selection. To distinguish these possibilities, we have analyzed a second transgene (InVVC(mu)) that is identical to VVC(mu) except that the two VDJ regions have been exchanged in position. We find that transgene sequence transfers are much less frequent among responding B cells in InVVC(mu) mice, demonstrating the importance of selection in the frequent transgene conversions observed in VVC(mu) mice. These results suggest that mice, like other species, can use gene conversion to diversify antibodies. Such diversification events are apparently infrequent, however, and might only be detected among endogenous Ig genes with a favorable arrangement of V genes and an antigenic stimulation that selects cells with conversions. For both VVC(mu) and InVVC(mu) mice, conversion-like sequence transfers are strongly correlated with somatic hypermutation. Based on these results, we hypothesize that, in mice, gene conversions represent infrequent alternative reactions of a homology-based DNA repair process that is central in the somatic hypermutational mechanism.     

Origin of Fueguian‐Patagonians: An approach to population history and structure using R matrix and matrix permutation methods

A complicated history of isolation between Fueguian and Patagonian groups (originated by the appearance of the Straits of Magellan) as much as differences in population structure and life strategies constitute important factors in the clustering pattern of those groups. The aim of this work was to test several hypotheses about population structure and history of Fueguian‐Patagonians to propose a model that incorporates predictions for future studies. R matrix methods and matrix permutation analyses were performed upon a data matrix of craniofacial measurements of 441 skulls divided into nine samples pertaining to six Patagonian and three Fueguian populations. Association of biological distances with three matrices representing several settlement patterns was tested using matrix permutation tests. Results of R matrix study show that the minimum genetic distance obtained confirms separation between Fueguians and Patagonians. Moreover, an analysis of residual variances from the expected regression line confirms admixture between Andean and Pampean populations and Araucanian groups, consistent with ethnohistorical observations. A model representing a long history of isolation between Fueguian and Patagonians, rather than a model emphasizing differences in life‐strategies, presented the best correlation with the biological distance matrix. Because similar results were already obtained in archaeological, molecular, and morphological studies, a model for the settlement of Tierra del Fuego is proposed. It is summarized by four main hypotheses that can be tested independently by different disciplines in the future. Am. J. Hum. Biol. 14:308–320, 2002. © 2002 Wiley‐Liss, Inc.     

Structural and functional analysis of a new desmin variant causing desmin‐related myopathy

Desmin‐related myopathy is a familial or sporadic disease characterized by skeletal muscle weakness and cardiomyopathy as well as the presence of intracytoplasmic aggregates of desmin‐reactive material in the muscle cells. Previously, two kinds of deletions and eight missense mutations have been identified in the desmin gene and proven to be responsible for the disorder. The present study was conducted to determine structural and functional defects in a pathogenic desmin variant that caused a disabling disorder in an isolated case presenting with distal and proximal limb muscle weakness and cardiomyopathy. We identified a novel heterozygous Q389P desmin mutation located at the C‐terminal part of the rod domain as the causative mutation in this case. Transfection of desmin cDNA containing the patient’s mutation into C2.7, MCF7, and SW13 cells demonstrated that the Q389P mutant is incapable of constructing a functional intermediate filament network and has a dominant negative effect on filament formation. We conclude that Q389P mutation is the molecular event leading to the development of desmin‐related myopathy. Hum Mutat 18:388–396, 2001. © 2001 Wiley‐Liss, Inc.     

Combined use of positive and negative immunomagnetic isolation followed by real-time RT-PCR for detection of the circulating tumor cells in patients with colorectal cancers

To establish a novel molecular diagnostic method of detecting circulating tumor cells (CTCs) LS174T colon cancer cells were serially diluted with normal blood. Additional peripheral blood samples were collected from 25 patients with colorectal carcinoma. Mononuclear cells (MNCs) were collected, equally divided into four parts, and then cancer cells were enriched by four methods: method A, nonimmunobead method; method B, negative immunobead method: CD45 immunomagnetic beads were used to deplete the leukocytes; method C, positive immunobead method: Ber-EP4 immunomagnetic beads were used to enrich cancer cells; method D, negative-and-positive immunobead method: CD45 immunomagnetic beads were first used to deplete the leukocytes from MNC and then Ber-EP4 immunomagnetic beads were used to enrich cancer cells. Finally, real-time quantitative RT-PCR was used to monitor mRNA expression of ₂-mircoglobulin (2M) and carcinoembryonic antigen (CEA). The relative CEA mRNA values were corrected with reference to 2M mRNA, to CEA mRNA/2M mRNA ratios according to a CEA mRNA external standards prepared with tenfold serial dilutions (1–10⁴ IS174T cells) of cDNA and 2M mRNA external standards prepared with tenfold serial dilutions (10²–10⁷ leukocytes) of cDNA. In recovery experiments a significant correlation between the number of cancer cells and CEA mRNA expression was found when CD45 or Ber-EP4 immunomagnetic beads were used alone. A highly significant correlation was found when CD45 and Ber-EP4 immunomagnetic beads were used successively. The sensitivity of method D was one cancer cell per milliliter of blood. Circulating cancer cells were detected in 19 of 25 patients with colorectal cancers. The relative CEA mRNA value obtained by method D was the smallest. The positive detection rate of circulating cancer cells in patients at Dukes B, C, and D stages were 25.0% (1/4), 83.3% (10/12), and 88.9% (8/9). Combinative use of immunomagnetic isolation followed by real-time RT-PCR is a useful technique to detect circulating tumor cells in patients with colorectal carcinomas. Applying negative and positive immunomagnetic beads successively yields the highest correlation with amount of tumor cells.     

Diversity of rotavirus strains among children with acute diarrhea in China: 1998-2000 surveillance study

As part of a national rotavirus surveillance activity, we collected fecal specimens from 3,177 children with acute diarrhea in 10 regions of China between April 1998 and April 2000 and screened them for rotavirus. Rotavirus was detected in 41% (n = 1,305) of specimens, and in these, G1 was the predominant serotype (72.6%), followed by G3 (14.2%), G2 (12.1%), G4 (2.5%), G9 (0.9%), and G untypeable (0.7%). Among 327 G-typed strains tested for P genotype, 14 different P-G combinations were identified, with the globally common strains P[8]G1, P[4]G2, P[8]G3, and P[8]G4 representing 75.6% of all typed rotavirus strains. Among the uncommon strains, 11 were P[6]G9, and others included P[6]G1, P[6]G3, and five novel P-G combinations (P[9]G1, P[4]G1, P[4]G3, P[4]G4, and P[8]G2). Our results indicate that while the common rotavirus strains remain predominant, the diversity of strains is much greater than was previously recognized.     

Molecular cytogenetic characterization of nasopharyngeal carcinoma cell lines and xenografts by comparative genomic hybridization and spectral karyotyping

Nasopharyngeal carcinoma (NPC) cell lines and xenografts represent valuable models for functional and therapeutic studies on this common malignancy in Southeast Asia. The karyotypic information in most NPC cell lines and xenografts, however, remains largely unclear to date. We have characterized the chromosomal aberrations in six commonly used human NPC cell lines and xenografts using the molecular cytogenetic technique of comparative genomic hybridization (CGH). Genomic imbalances identified in cell lines were further correlated with structural abnormalities indicated from spectral karyotyping (SKY) analysis. CGH revealed consistent overrepresentations of 8q (six out of six cases) with a smallest overlapping region identified on 8q21.1q22. Other common gains included 7p (4/6 cases), 7q (4/6 cases), 12q (4/6), and 20q (4/6 cases), where minimal overlapping regions were suggested on 7p15p14, 7q11.2q21, and 12q22q24.1. Common losses were detected on 3p12p21 (4/6 cases) and 11q14qter (4/6 cases). Although SKY analysis on cell lines revealed predominantly unbalanced rearrangements, reciprocal translocations that involved chromosome 2 [i.e., t(1;2), t(2;3), and t(2;4)] were suggested. Furthermore, SKY examination illustrated additional breakpoints on a number of apparently balanced chromosomes. These breakpoints included 3p21, 3q26, 5q31, 6p21.1p25, 7p14p22, and 8q22. Our finding of regional gains and losses and breakpoints represents information that may contribute to NPC studies in vitro.     

异种脱细胞真皮基质在开放性乳突病变切除术中的应用

目的探讨开放性乳突病变切除术后加速乳突术腔上皮化及缩小乳突术腔的有效办法。方法63例行开放性乳突病变切除术的患者按照复查治疗方法分为两组，异种脱细胞真皮组36例，应用异种脱细胞真皮基质覆盖术腔，碘仿纱条组27例，以碘仿纱条填塞术腔。术后随访两组患者，对比观察术腔愈合情况和上皮化时间。结果异种脱细胞真皮组术腔上皮化时间为2～4周，平均2．2周；碘仿纱条组术腔上皮化时间为9～35周，平均13．7周，异种脱细胞真皮组的上皮化时间短于碘仿纱条组（P〈0．01）。结论异种脱细胞真皮基质可促进上皮组织再生，减少术后感染及肉芽发生，加速乳突腔上皮化，提高开放性乳突病变切除术的疗效。     

Nkx2.5‐negative myocardium of the posterior heart field and its correlation with podoplanin expression in cells from the developing cardiac pacemaking and conduction system

Recent advances in the study of cardiac development have shown the relevance of addition of myocardium to the primary myocardial heart tube. In wild‐type mouse embryos (E9.5–15.5), we have studied the myocardium at the venous pole of the heart using immunohistochemistry and 3D reconstructions of expression patterns of MLC‐2a, Nkx2.5, and podoplanin, a novel coelomic and myocardial marker. Podoplanin‐positive coelomic epithelium was continuous with adjacent podoplanin‐ and MLC‐2a‐positive myocardium that formed a conspicuous band along the left cardinal vein extending through the base of the atrial septum to the posterior myocardium of the atrioventricular canal, the atrioventricular nodal region, and the His‐Purkinje system. Later on, podoplanin expression was also found in the myocardium surrounding the pulmonary vein. On the right side, podoplanin‐positive cells were seen along the right cardinal vein, which during development persisted in the sinoatrial node and part of the venous valves. In the MLC‐2a‐ and podoplanin‐positive myocardium, Nkx2.5 expression was absent in the sinoatrial node and the wall of the cardinal veins. There was a mosaic positivity in the wall of the common pulmonary vein and the atrioventricular conduction system as opposed to the overall Nkx2.5 expression seen in the chamber myocardium. We conclude that we have found podoplanin as a marker that links a novel Nkx2.5‐negative sinus venosus myocardial area, which we refer to as the posterior heart field, with the cardiac conduction system. Anat Rec, 290:115–122, 2007. © 2006 Wiley‐Liss, Inc.     

Carboxymethyl-histaminocarbonylmethylamylose as a mimetic system of chymotrypsin in the catalytic hydrolysis of esters

A new type of polysaccharide host, carboxymethyl-histaminocarbonylmethylamylose ( 2b ), containing carboxylic, imidazolyl and hydroxyl groups in the backbone, was used as a mimetic system for chymotrypsin in the catalytic hydrolysis of 3-acetoxy-N-dodecylpyridinium iodide ( 1 ). The substrate is located in the hydrophobic cavity of the amylose helix. The apparent saturation, the entropy-favored kinetics and the pronounced catalytic efficiency (9 times higher than that of a system consisting of the same concentration of carboxymethylamylose and histamine) show that 2b is a good enzyme model in which the definite binding site, active center and self-organization characteristics are present. Most distinctly, the pH-rate constant profile of the hydrolysis of 1 and 2b is of bell-type and has an optimum at pH 7,98, which is very close to 7,90 for chymotrypsin. In conclusion, the charge relay mechanism is also involved in the catalytic effect of 2b .