Characterization of a monoclonal antibody and its single-chain antibody fragment recognizing the nucleoside Triphosphatase/Helicase domain of the hepatitis C virus nonstructural 3 protein

We have produced a murine monoclonal antibody (MAb), ZX10, recognizing the NTPase/helicase domain of the hepatitis C virus (HCV) nonstructural 3 protein (NS3), from which we designed a single-chain variable fragment (ScFv). The ZX10 MAb recognized a discontinuous epitope of the NTPase/helicase domain, of which the linear sequence GEIPFYGKAIPL at residues 1371 to 1382 constitutes one part. cDNAs from variable regions coding for the heavy and light chains were cloned, sequenced, and assembled into the NS3-ScFv, which was inserted into procaryotic and eucaryotic expression vectors. Escherichia coli-expressed NS3-ScFv inhibited the binding of the ZX10 MAb to NS3, confirming a retained specificity. However, the ability to bind the peptide 1371-1382 had been lost. In vitro-translated NS3-ScFv and HCV NS3/NS4A were coprecipitated by antibodies to HCV NS4A, confirming the in vitro activity of the NS3 ScFv. Thus, we have designed a functional NS3 NTPase/helicase domain-specific ScFv which should be evaluated further with respect to disturbing enzymatic functions of the NS3 protein.     

Characterization of the translocated chromosome using fluorescencein situ hybridization and random amplified polymorphic DNA on twoTriticum aestivum—Thinopyrum intermedium translocation lines resistant to wheat streak mosaic or barley yellow dwarf virus

Fluorescencein situ hybridization (FISH) was used to determine the breakpoint of the translocation chromosome in two bread wheat (Triticum aestivum) germplasm lines withThinopyrum intermedium chromatin carrying resistance to either wheat streak mosaic virus (WSMV) or barley yellow dwarf virus (BYDV). In addition, genome-specific random amplified polymorphic DNA (RAPD) markers were used to ascertain the genomic sources of theTh. intermedium chromosomes carrying the WSMV or BYDV resistance. CI17766, a WSMV-resistant wheat germplasm line derived from induced homoeologous pairing by using theph1b mutant, had a translocation chromosome composed of the complete 4AL and about 45% of proximal 4AS from wheat, and the entire 4ES ofTh. intermedium. The BYDV-resistant translocation line, TC14, derived from tissue culture, had a very short distal segment of 7StL fromTh. intermedium terminally attached to 56% of the proximal 7DL. These observations indicate that translocations in these wheat germplasm lines did not involve centromeric breaks and fusion but were a result of homoeologous chromosome recombination.accepted for publication by J. S. (Pat) Heslop-Harrison     

Shb null allele is inherited with a transmission ratio distortion and causes reduced viability in utero.

SHB is an Src homology 2 domain-containing adapter protein that has been found to be involved in numerous cellular responses. We have generated an Shb knockout mouse. No Shb-/- pups or embryos were obtained on the C57Bl6 background, indicating an early defect as a consequence of Shb- gene inactivation on this genetic background. Breeding heterozygotes for Shb gene inactivation (Shb+/-) on a mixed genetic background (FVB/C57Bl6/129Sv) reveals a distorted transmission ratio of the null allele with reduced numbers of Shb+/+ and Shb-/- animals, but increased number of Shb+/- animals. The Shb- allele is associated with various forms of malformations, explaining the relative reduction in the number of Shb-/- offspring. Shb-/- animals that were born were viable, fertile, and showed no obvious defects. However, Shb+/- female mice ovulated preferentially Shb- oocytes explaining the reduced frequency of Shb+/+ mice. Our study suggests a role of SHB during reproduction and development.     

T cells from atopic individuals produce IgE-inducing activity incompletely blocked by anti-interleukin-4 antibody.

We investigated peripheral blood B and T lymphocyte functions in atopic individuals. B cells were co-cultured with mutant EL4 thymoma cells in the presence of a standard T cell supernatant (T-SN) with or without exogenous interleukin (IL)-4. IgE secretion in this assay was found to be IL-4 dependent, but not significantly different for atopic patients (n = 25) vs. normal controls (n = 25). Phytohemagglutinin plus phorbol 12-myristate 13-acetate (PHA+PMA)- induced T-SN from patients or controls was tested on normal B cells in the same assay system (in the absence of exogenous IL-4). Compared to the controls, the IgE-inducing activity was significantly increased for patients with asthma or allergic rhinitis (n = 12; p less than 0.005) but not for patients with atopic dermatitis (n = 13). The difference between the asthma or allergic rhinitis vs. the atopic dermatitis groups was significant (p greater than 0.05). Since the assay was not inhibited by interferon (IFN)-gamma, this difference can not be attributed to IFN-gamma concentrations. Other T cell activities may be different between the patient groups or atopic T cells from the respiratory mucosa may recirculate more than those from the skin. In any case, the T cells rather than the B cells were found to be abnormal in atopic individuals. If atopic T cells were stimulated with PHA+PMA not as immediately but after a resting period of 48 h in culture medium alone, the IgE-inducing activity, but not the total Ig-inducing activity or the IL-2 secretion, disappeared. In addition, a mean of 37% of the IgE-inducing activity (range of 13% to 79% for five very active T-SN) was not inhibited by an anti-IL-4 antibody which neutralized exogenous IL-4, indicating a participation of factors capable of bypassing the requirement for IL-4 for the IgE response.     

足内侧皮瓣的应用解剖

解剖观测了30侧成人下肢足内侧区皮肤血管.足底内侧动脉深支、浅支、内踝前动脉和跗内侧动脉,分别发4.5(3～8)支、7.4(3～12)支、5.9(2～12)支和2.8(1～6)支,外径在0.2～0.8mm之间的皮支分布足内侧区,动脉间吻合恒定.经选择性动脉注射显示,皮瓣面积约为9×6cm～10×8cm.以上述动脉为蒂的足内侧皮瓣,可转位修复内踝、跟腱、足跟和足底远侧区的软组织缺损,临床应用了12例均获得成功.     

Different applications of polymerases with and without proofreading activity in single-nucleotide polymorphism analysis

With the completion of the human genome project, single-nucleotide polymorphisms (SNPs) have become the focus of intense study in biomedical research. Polymerase-mediated primer extension has been employed in a variety of SNP assays. However, these SNP assays using polymerase without proofreading function are compromised by their low reliability. Using a newly developed short amplicon harboring restriction enzyme site, EcoR-I, we were able to compare the single-base discrimination abilities of polymerases with and without proofreading function in primer extension in a broad range of annealing temperatures. Thermodynamic analysis demonstrated a striking single-nucleotide discrimination ability of polymerases with proofreading function. Using unmodified 3'-end allele-specific primers, only template-dependent products were generated by polymerase with proofreading activity. This powerful single-base discrimination ability of exo(+) polymerases was further evaluated in primer extension using three types of 3' terminally modified allele-specific primers. As compared with the poor fidelity in primer extension of polymerases lacking 3' exonuclease activity, this study provides convincing evidence that the use of proofreading polymerases in combination with 3'-end modified allele-specific primers can be a powerful new strategy for the development of SNP assays.     

Homology-mediated recombination between type I collagen gene exons results in an internal tandem duplication and lethal osteogenesis imperfecta

It has been proposed that the structure of the exons that encode the triple helical domain of the fibrillar collagen genes arose by repeated tandem duplication of an ancestral unit exon. Because these exons encode a repeat motif [(Gly-X-Y)_n], sequence homology between exons may have driven the recombinational process. We have characterized a tandem duplication mutation within a COL1A1 allele of type I collagen from an infant with the lethal form of osteogenesis imperfecta. The structure of the mutation is consistent with the occurrence of an unequal crossover within a 15 base pair region of sequence identity between exons 14 and 17 of the COL1A1 gene. The recombination produced a new 81 base pair 17/14 hybrid exon and complete duplication of exons 15 and 16. The sequence implies duplication of 60 amino acid residues within the triple helical domain with preservation of the Gly-X-Y repeat. These data suggest that a recombinational mechanism that explains the hypothetical evolutionary process is active in cells, but the lethal effect of this mutation raises questions about the role of these events in creating new structures for polymeric proteins. © 1993 Wiley-Liss, Inc.     

Large-scale molecular and tissue microarray analysis of mesothelin expression in common human carcinomas

The 40-kilodalton processed glycoprotein, mesothelin, is highly expressed in epithelial mesotheliomas and adenocarcinomas of the ovary (serous papillary) and pancreas, but its expression in a large series of other common carcinomas has not been completely explored. In the present study, we used oligonucleotide and tissue microarrays to profile the expression of the mesothelin gene (MSLN) and encoded protein, respectively. Among 150 carcinomas of multiple anatomic sites, we found the highest average expression of MSLN in serous carcinomas of the ovary and adenocarcinomas of the pancreas, consistent with previous reports, as well as measurable but less-striking expression in pulmonary, gastric/esophageal, and colorectal adenocarcinomas. On tissue microarrays containing 621 carcinomas derived from the same and additional sites as those profiled by gene expression, mesothelin immunoreactivity was highest in cancers of the ovary (serous papillary, endometrioid, and undifferentiated) and pancreas, with less frequent staining seen in adenocarcinomas of the endometrium, lung, and stomach/esophagus. Some immunopositivity was observed in 42% of pulmonary adenocarcinomas, including 18% that had >50% of tumor cells that were immunoreactive. Some 14% of breast and 30% of colorectal adenocarcinomas showed immunopositivity, but no case contained >50% tumor cells that were immunoreactive. Mesothelin was either entirely absent or present in <5% of carcinomas of the prostate, bladder/ureter, liver, kidney, and thyroid. Overall, we observed good concordance between the results obtained by oligonucleotide and tissue microarrays. This large study of the MSLN gene and protein expression in common carcinomas provides data for future investigations that evaluate the utility of mesothelin/megakaryocyte potentiating factor as a potential serum tumor marker or target of immunotoxin-based therapy in human cancers.