Novel mutations of the peripheral myelin protein22 gene in two pedigrees with Dejerine-Sottas disease

Peripheral myelin protein22 (PMP22), a membrane glycoprotein, plays a significant role in the formation and/or maintenance of compact myelin in the peripheral nervous system. We studied two pedigrees with Dejerine-Sottas disease and identified two novel mutations in the PMP22 gene: one a 2-bp deletional mutation at nucleotide positions426 and 427 of exon4 (this is predicted to alter the reading frame at leucine80 and thus to lead to frame-shifted translation), and the other a guanine to thymine substitution at nucleotide position636 leading to a cysteine substitution for glycine150. Both mutations were located in the putative transmembrane domains reported in many cases of Charcot-Marie-Tooth neuropathy, Dejerine-Sottas disease, and hereditary neuropathy with liability to pressure palsies. The results suggest an important role for the putative transmembrane domains of PMP22 in its function. Received: 1 September 1997 / Accepted: 4 November 1997     

Establishment of a novel anti-TROP2 monoclonal antibody TrMab-29 for immunohistochemical analysis

TROP2 is a type I transmembrane glycoprotein originally identified in human trophoblast cells that is overexpressed in several types of cancer. To better understand the role of TROP2 in cancer, we herein aimed to develop a sensitive and specific anti-TROP2 monoclonal antibody (mAb) for use in flow cytometry, Western blot, and immunohistochemistry using a Cell-Based Immunization and Screening (CBIS) method. Two mice were immunized with N-terminal PA-tagged and C-terminal RAP/MAP-tagged TROP2-overexpressed Chinese hamster ovary (CHO)–K1 cells (CHO/PA-TROP2-RAP-MAP), and hybridomas showing strong signals from PA-tagged TROP2-overexpressed CHO–K1 cells (CHO/TROP2-PA) and weak-to-no signals from CHO–K1 cells were selected using flow cytometry. We demonstrated using flow cytometry that the established anti-TROP2 mAb, TrMab-29 (mouse IgG₁ kappa), detected TROP2 in MCF7 breast cancer cell line as well as CHO/TROP2-PA cells. Western blot analysis showed a 40 kDa band in lysates prepared from both CHO/TROP2-PA and MCF7 cells. Furthermore, TROP2 was strongly detected by immunohistochemical analysis using TrMab-29, indicating that TrMab-29 may be a valuable tool for the detection of TROP2 in cancer.     

Metabolism and Translocation of Gibberellins in Seedlings of Pharbitis nil. (I) Effect of Photoperiod on Stem Elongation and Endogenous Gibberellins in Cotyledons and Their Phloem Exudates

Gibberellin A₁, (GA₁), GA₁₉, and GA₂₀ in phloem exudates andcotyledons of seedlings of Pharbitis nil cv. Violet, grown underdifferent photoperiodic conditions, were qualitatively and semi-quantitativelyanalyzed by a combination of high performance-liquid chromatography(HPLC) and radioimmunoassays (RIA). The levels of GA₁₉ and GA₂₀were higher in cotyledons from plants grown under dark treatment(DT) conditons of 16 h-light/8 h-dark for 6 days followed by8 h-light/16 h-dark for 3 days than in those grown under continuouslight (CL) for 9 days. This relationship was also observed forthe GAs in phloem exudates, although the levels were much lowerthan in the cotyledons. When GAs were applied to the cotyledons,elongation of the epicotyl was promoted more by GA₂₀ than byGA₁ or GA₁₉, especially under the CL treatment. The relativeeffect of GA₁ and GA₂₀ on the epicotyl elongation was reversedwhen these GAs were applied to epicotyls pre-treated with prohexadione,an inhibitor of 2-oxoglutarate-dependent dioxygenases. ³Present address: Frontier Research Program, The Institute ofPhysical and Chemical Research (RIKEN), 2-1 Hirosawa, Wakoshi,Saitama, 351-01 Japan ⁴Present address: Laboratory of Horticulture, Faculty of Agriculture,Nagoya University, Nagoya, 464-01 Japan     

Changes observed in hemolymph proteins of the lawn armyworm,Spodoptera mauritia acronyctoides,during growth,development, and exposures to a nuclear polyhedrosis virus

Vertical electrophoresis with acrylamide gel was used to study the effects of a nuclear polyhedrosis virus (NPV) on the hemolymph proteins of Spodoptera mauritia acronyctoides. An electrophoretic pattern consisting of 20 basic bands of proteins was separated in hemolymph of normal larvae which were older than 17 days. These hemolymph proteins increased quantitatively during growth. All 20 proteins could not be detected in hemolymph of younger larvae by the techniques utilized. Additional proteins were separated with metamorphosis. Lethal doses of NPV resulted in a general reduction of hemolymph proteins (hypoproteinemia) in infected larvae. Sublethal doses of NPV elicited an increase in certain hemolymph proteins. Similar increases in proteins were also observed in larvae surviving ostensibly lethal levels of NPV, in larvae subjected to physical stress, and in larvae reared axenically without formaldehyde in their diets. These same proteins, however, were present in approximately the same quantities in mature larvae. Physiopathology of NPV in S. mauritia appears to involve stress factors, host reactions, and the host endocrine system.     

Detection of varicella-zoster virus DNA by field-inversion gel electrophoresis

A new method for detection of varicella-zoster virus (VZV) DNA using field-inversion gel electrophoresis (FIGE) was devised. VZV-genomic DNA could be differentiated from the host cell DNA of human embryonic lung (HEL) fibroblasts infected with VZV under electrophoretic conditions allowing resolution of linear and double-stranded DNAs in the 49-230 kilobase pairs (Kb) range. The detection of VZV-genomic DNA from infected HEL cells was successful regardless of whether the VZV was a laboratory strain, live vaccine strain, or fresh isolate. Under the same electrophoretic conditions, DNA of VZV-infected HEL cells could be clearly differentiated from DNA obtained from HEL cells infected with herpes simplex virus type 1 (HSV-1), type 2 (HSV-2), or human cytomegalovirus (HCMV). Furthermore, VZV genomic DNA could be detected from as small a sample as 1.9 x 10(4) VZV-infected HEL cells. Finally, we could detect VZV genomic DNA from 10 samples of vesicle tissue (blister lids, each about 1-4 mm2) and one sample of vesicle fluid (about 5 microliters) obtained from patients diagnosed as having herpes-zoster. The results of this study indicate that FIGE is a simple and promising method for the detection of VZV from clinical materials as well as infected in vitro cultured cells.     

The expression of glutathione reductase in the male reproductive system of rats supports the enzymatic basis of glutathione function in spermatogenesis.

Glutathione reductase (GR) recycles oxidized glutathione (GSSG) by converting it to the reduced form (GSH) using an NADPH as the electron source. The function of GR in the male genital tract of the rat was examined by measuring its enzymatic activity and examining the gene expression and localization of the protein. Levels of GR activity, the protein, and the corresponding mRNA were the highest in epididymis among testes, vas deferens, seminal vesicle, and prostate gland. The localization of GR, as evidenced by immunohistochemical techniques, reveals that it exists at high levels in the epithelia of the genital tract. In testis, GR is mainly localized in Sertoli cells. The enzymatic activity and protein expression of GR in primary cultured testicular cells confirmed its predominant expression in Sertoli cells. Intracellular GSH levels, expressed as mol per mg protein, was higher in spermatogenic cells than in Sertoli cells. As a result of these findings, the effects of buthionine sulfoximine (BSO), an inhibitor for GSH synthesis, and 1,3-bis(2-chlorethyl)-1-nitrosourea (BCNU), an inhibitor for GR, on cultured testicular cells were examined. Sertoli cells were prone to die as the result of BCNU, but not BSO treatment, although intracellular levels of GSH declined more severely with BSO treatment. Spermatogenic cells were less sensitive to these agents than Sertoli cells, which indicates that the contribution of these enzymes is less significant in spermatogenic cells. The results herein suggest that the GR system in Sertoli cells is involved in the supplementation of GSH to spermatogenic cells in which high levels of cysteine are required for protamine synthesis. In turn, the genital tract, the epithelia of which are rich in GR, functions in an antioxidative manner to protect sulfhydryl groups and unsaturated fatty acids in spermatozoa from oxidation during the maturation process and storage.