Isolation and mapping of 88 new RFLP markers on human chromosome 8.

To obtain new RFLP markers for construction of a high-resolution map of human chromosome 8, a cosmid library was constructed from a somatic hybrid cell that contained chromosome 8 as the only human component in mouse genomic background. Eighty-eight new RFLP markers were isolated and characterized, and 71 of them were sublocalized to chromosomal bands by fluorescent in situ hybridization (FISH). Of these, 36 were localized to the short arm, 34 to the long arm, and 1 to the centromeric region. Five markers defined VNTR loci. This work represents the first extensive isolation and physical mapping of RFLP markers on human chromosome 8. These new markers will serve as useful resources for linkage mapping of loci for inherited diseases and for efforts to identify a putative tumor suppressor gene(s) on chromosome 8.     

Structure and Chromosomal Assignment of the Human S1-5 Gene (FBNL) That Is Highly Homologous to Fibrillin

The human S1-5 gene (fibrillin-like; FBNL) was originally isolated from a subtractively enriched cDNA library established from a subject with Werner syndrome (WS). We isolated genomic clones containing the entire S1-5 gene and determined its genomic structure including the exon–intron organization. The gene spanned approximately 18 kb of genomic DNA and consisted of 12 exons. Its expression was abundant in all tissues examined except brain and peripheral leukocytes, where it was undetectable. In addition, we have mapped S1-5 by fluorescencein situhybridization to chromosome 2p16, a position that excludes it as a candidate for WS. Our data should facilitate an understanding of the function and regulation of S1-5 in human tissues.     

Successful adjuvant bi-weekly gemcitabine chemotherapy for pancreatic cancer without impairing patients’ quality of life

Background

Although adjuvant gemcitabine (GEM) chemotherapy for pancreatic cancer is standard, the quality of life (QOL) in those patients is still impaired by the standard regimen of GEM. Therefore, we studied whether mild dose-intensity adjuvant chemotherapy with bi-weekly GEM administration could provide a survival benefit with acceptable QOL to the patients with pancreatic cancer.

Methods

After a phase I trial, an adjuvant bi-weekly 1,000 mg/m² of GEM chemotherapy was performed in 58 patients with pancreatic cancer for at least 12 courses (Group A). In contrast, 36 patients who declined the adjuvant bi-weekly GEM chemotherapy underwent traditional adjuvant 5FU-based chemotherapy (Group B). Careful periodical follow-ups for side effects of GEM and disease recurrence, and assessment of patients’ QOL using the EORTC QOL questionnaire (QLQ-C30) and pancreatic cancer-specific supplemental module (QLQ-PAN26) were performed. Retrospectively, the degree of side effects, patients’ QOL, compliance rate, disease-free survival (DFS), and overall survival (OS) in Group A were compared with those in Group B.

Results

No severe side effects (higher than Grade 2 according to the common toxicity criteria of ECOG) were observed, except for patients in Group B, who were switched to the standard GEM chemotherapy. Patients’ QOL was better in Group A than B (fatigue: 48.9 ± 32.1 versus 68.1 ± 36.3, nausea and vomiting: 26.8 ± 20.4 versus 53.7 ± 32.6, diarrhea: 21.0 ± 22.6 versus 53.9 ± 38.5, difficulty gaining weight: 49.5 ± 34.4 versus 67.7 ± 40.5, P < 0.05). Compliance rates in Groups A and B were 93% and 47%. There was a significant difference in the median DFS between both groups (Group A : B =12.5 : 6.6 months, P < 0.001). The median OS of Group A was prolonged markedly compared with Group B (20.2 versus 11.9 months, P < 0.005). For OS between both groups, univariate analysis revealed no statistical difference in 69-year-old or under females, and T1–2 factors, moreover, multivariate analysis indicated three factors, such as bi-weekly adjuvant GEM chemotherapy, T2 or less, and R0.

Conclusions

Adjuvant chemotherapy with bi-weekly GEM offered not only the advantage of survival benefits but the excellent compliance with acceptable QOL for postoperative pancreatic cancer patients.     

Inhibition of the Growth Factor MDK/Midkine by a Novel Small Molecule Compound to Treat Non-Small Cell Lung Cancer

Midkine (MDK) is a heparin-binding growth factor that is highly expressed in many malignant tumors, including lung cancers. MDK activates the PI3K pathway and induces anti-apoptotic activity, in turn enhancing the survival of tumors. Therefore, the inhibition of MDK is considered a potential strategy for cancer therapy. In the present study, we demonstrate a novel small molecule compound (iMDK) that targets MDK. iMDK inhibited the cell growth of MDK-positive H441 lung adenocarcinoma cells that harbor an oncogenic KRAS mutation and H520 squamous cell lung cancer cells, both of which are types of untreatable lung cancer. However, iMDK did not reduce the cell viability of MDK-negative A549 lung adenocarcinoma cells or normal human lung fibroblast (NHLF) cells indicating its specificity. iMDK suppressed the endogenous expression of MDK but not that of other growth factors such as PTN or VEGF. iMDK suppressed the growth of H441 cells by inhibiting the PI3K pathway and inducing apoptosis. Systemic administration of iMDK significantly inhibited tumor growth in a xenograft mouse model in vivo. Inhibition of MDK with iMDK provides a potential therapeutic approach for the treatment of lung cancers that are driven by MDK.     

Dual subcellular localization in the endoplasmic reticulum and peroxisomes and a vital role in protecting against oxidative stress of fatty aldehyde dehydrogenase are achieved by alternative splicing

Fatty aldehyde dehydrogenase (FALDH, ALDH3A2) is thought to be involved in the degradation of phytanic acid, a saturated branched chain fatty acid derived from chlorophyll. However, the identity, subcellular distribution, and physiological roles of FALDH are unclear because several variants produced by alternative splicing are present in varying amounts at different subcellular locations. Subcellular fractionation experiments do not provide a clear-cut conclusion because of the incomplete separation of organelles. We established human cell lines heterologously expressing mouse FALDH from each cDNA without tagging under the control of an inducible promoter and detected the variant FALDH proteins using a mouse FALDH-specific antibody. One variant, FALDH-V, was exclusively detected in peroxisomal membranes. Human FALDH-V with an amino-terminal Myc sequence also localized to peroxisomes. The most dominant form, FALDH-N, and other variants examined, however, were distributed in the endoplasmic reticulum. A gas chromatography-mass spectrometry-based analysis of metabolites in FALDH-expressing cells incubated with phytol or phytanic acid showed that FALDH-V, not FALDH-N, is the key aldehyde dehydrogenase in the degradation pathway and that it protects peroxisomes from oxidative stress. In contrast, both FALDHs had a protective effect against oxidative stress induced by a model aldehyde for lipid peroxidation, dodecanal. These results suggest that FALDH variants are produced by alternative splicing and share an important role in protecting against oxidative stress in an organelle-specific manner.     

Monoclonal antibodies against the minimal DNA-binding domain in the carboxyl-terminal region of human immunodeficiency virus type 1 integrase

Integrase of human immunodeficiency virus type 1 (HIVIN) consists of 288 amino acids, and its minimum DNA-binding domain (MDBD) (amino acids [aa] 220 to 270) is required for the integration reaction. We produced and characterized four murine monoclonal antibodies (MAbs) to the MDBD of HIVIN (strain LAI). Immunoblot and enzyme-linked immunosorbent assays with truncated HIVINs showed that those MAbs recognized sequential epitopes within the MDBD (aa 228 to 236, 237 to 252, 253 to 261, and 262 to 270). Their binding to HIVIN inhibited terminal cleavage and strand transfer activities but not disintegration activity in vitro. This collection of MAbs is useful for studying the structure and function of the MDBD by complementing mutational analyses and other biochemical studies.     

Expression of APG-2 protein, a member of the heat shock protein 110 family, in developing rat brain

APG-2 protein is a member of the heat shock protein 110 family, and it is thought to play an important role in the maintenance of neuronal functions under physiological and stress conditions. However, neither the tissue-distribution of APG-2 protein nor developmental change of its expression has been studied at the protein level. Therefore, we generated an antiserum against APG-2 protein and studied expression of this protein in rat brain and other tissues by use of the Western blot method. The results showed a high expression of APG-2 protein in various regions of the central nervous system (cerebral cortex, hippocampus, striatum, midbrain, hypothalamus, cerebellum, medulla pons, and spinal cord) throughout the entire postnatal stage. Similarly, a high level of APG-2 protein was detected in the whole brain of rat embryos and in adult rat tissues such as liver, lung, spleen, and kidney. In contrast, its expression in heart was high at postnatal days 1 and 3, but thereafter drastically decreased to a low level. Furthermore, APG-2 protein was detected in neuronal primary cultures prepared from rat cerebral cortex, and its level did not change notably during neuronal differentiation. These results show that APG-2 protein is constitutively expressed in various tissues and also in neuronal cells throughout the entire embryonic and postnatal period. suggesting that it might play an important role in these tissues under non-stress conditions.     

Identification of a gene disrupted by inv(11)(q13.5;q25) in a patient with left-right axis malformation

An inv(11)(q13.5;q25) inversion was previously identified in a 9-month-old male patient with complex cyanotic heart defects, altered lung lobation, symmetric liver, and abnormally lobulated spleen (polysplenia). This chromosomal rearrangement was inherited from the phenotypically normal father. We termed these regions DHTX-A (disrupted in heterotaxy)-- A at 11q13.5 and DHTX-B at 11q25. Here, we report the isolation and characterization of the inversion breakpoints and the gene that is disrupted by the DHTX-A breakpoint. The putative DHTX is identical to the UVRAG gene, which was originally identified as a gene that complements the UV sensitivity of xeroderma pigmentosum complementation group C. The 4-kb mRNA was found to be encoded by a large gene, at least 300 kb long, composed of 15 exons. The function of the gene product remains largely unknown. However, the near central portion of the UVRAG protein is predicted to contain a coiled-coil domain, which has been implicated in mediating protein-protein interactions. Southern analyses and fluorescence in situ hybridization (FISH) revealed that the DHTX-A breakpoint in the patient and his father lies within the intron between exons 6 and 7 of UVRAG. Northern blot analysis indicated strong expression in human fetal and adult tissues and in mouse embryonic day-7 and adult tissues, respectively. Whole mount in situ hybridization also showed that the Uvrag gene is expressed in the presomite-stage embryo. Several hypotheses are discussed to explain the relationship between the chromosomal inversion and the accompanying phenotypes.     

Recovery of Bacillus thuringiensis from Marine Sediments of Japan

Marine sediments from a Japanese bay were examined for the occurrence of Bacillus thuringiensis. Of 1313 colonies belonging to the Bacillus cereus/B. thuringiensis group, 22 (1.7%) were allocated to B. thuringiensis. Marine isolates of B. thuringiensis consisted of heterogeneous multiple H serogroups; 10 isolates were assigned to the eight serovars (kurstaki, sumiyoshiensis, sotto, aizawai, darmstadiensis, thompsoni, neoleonensis, and higo); two motile isolates failed to react with the reference antisera; and the others were serologically untestable. Insecticidal activities were associated with two kurstaki isolates (toxic to both Lepidoptera and Diptera) and a higo isolate (Diptera-specific). None of the parasporal inclusion proteins of the 22 isolates exhibited in vitro cytotoxic activity against two vertebrate cells, sheep erythrocytes and HeLa cells. All B. thuringiensis isolates had no halophilism, although seawater-based medium supported their growth, sporulation, and formation of parasporal inclusions. Received: 29 November 1999 / Accepted: 10 January 2000     

Non-synonymous FGD3 Variant as Positional Candidate for Disproportional Tall Stature Accounting for a Carcass Weight QTL (CW-3) and Skeletal Dysplasia in Japanese Black Cattle

Recessive skeletal dysplasia, characterized by joint- and/or hip bone-enlargement, was mapped within the critical region for a major quantitative trait locus (QTL) influencing carcass weight; previously named CW-3 in Japanese Black cattle. The risk allele was on the same chromosome as the Q allele that increases carcass weight. Phenotypic characterization revealed that the risk allele causes disproportional tall stature and bone size that increases carcass weight in heterozygous individuals but causes disproportionately narrow chest width in homozygotes. A non-synonymous variant of FGD3 was identified as a positional candidate quantitative trait nucleotide (QTN) and the corresponding mutant protein showed reduced activity as a guanine nucleotide exchange factor for Cdc42. FGD3 is expressed in the growth plate cartilage of femurs from bovine and mouse. Thus, loss of FDG3 activity may lead to subsequent loss of Cdc42 function. This would be consistent with the columnar disorganization of proliferating chondrocytes in chondrocyte-specific inactivated Cdc42 mutant mice. This is the first report showing association of FGD3 with skeletal dysplasia.