Molecular characterization of Escherichia coli NAD kinase.

NAD kinase was purified to homogeneity from Escherichia coli MG1655. The enzyme was a hexamer consisting of 30 kDa subunits and utilized ATP or other nucleoside triphosphates as phosphoryl donors for the phosphorylation of NAD, most efficiently at pH 7.5 and 60 degrees C. The enzyme could not use inorganic polyphosphates as phosphoryl donors and was designated as ATP-NAD kinase. The N-terminal amino-acid sequence of the purified enzyme was encoded by yfjB, which had been deposited as a gene of unknown function in the E. coli whole genomic DNA sequence database. yfjB was cloned and expressed in E. coli BL21(DE3)pLysS. The purified product (YfjB) showed NAD kinase activity, and was identical to ATP-NAD kinase purified from E. coli MG1655 in molecular structure and other enzymatic properties. The deduced amino-acid sequence of YfjB exhibited homology with that of Mycobacterium tuberculosis inorganic polyphosphate/ATP-NAD kinase [Kawai, S., Mori, S., Mukai, T., Suzuki, S., Hashimoto, W., Takeshi, Y. & Murata, K. (2000) Biochem. Biophys. Res. Commun. 276, 57-63], and those of many hypothetical proteins for which functions have not yet been revealed. The YfjB homologues were considered to be NAD kinases and alignment of their sequences revealed highly conserved regions, XXX-XGGDG-XL and DGXXX-TPTGSTAY, where X represents a hydrophobic amino-acid residue.     

Transient local overexpression of human vascular endothelial growth factor (VEGF) in mouse feto-maternal interface during mid-term pregnancy lowers systemic maternal blood pressure.

The effect on maternal circulation of transient human vascular endothelial growth factor (VEGF) (165) cDNA transfection into the mouse feto-maternal interface at day 14.5 post coitus (p.c.) using a hemagglutinating virus of Japan-envelope (HVJ-E) vector system is reported. On day 15.5 p.c., Western blotting clearly showed overexpression of 18 kD VEGF protein in the uterus. After VEGF transfection, the blood pressure was significantly lowered for 48 hours. On day 17.5 p.c., the blood pressure returned to the control level. Proteinuria was not observed after VEGF transfection. No preterm birth was observed during the course of pregnancy after the transfection procedure. After 24 hours of transfection, human VEGF was not detectable and the mouse VEGF level was similar to that in peripheral blood. However, the soluble fms-like tyrosine kinase (Flt)-1 concentration was significantly lower in VEGF-transfected mice. These results suggest that extraamniotic VEGF overexpression lowered the systemic blood pressure without altering the VEGF concentration in the peripheral blood. Local overexpression of VEGF may become a novel treatment for pregnancy-related disorders such as hypertension complicated-pregnancy and preeclampsia.     

A novel mutant mouse, joggle, with inherited ataxia.

While establishing a new mouse strain, we discovered a novel mutant mouse that exhibited ataxia. Mating experiments showed that the mutant phenotype was due to a single autosomal recessive gene, which we have termed joggle (gene symbol: jog). The ataxia becomes apparent around postnatal day 12, when the mice first attempt to walk, and worsens thereafter. The life span of the mutant mouse is comparable to that of the wild-type mouse. After 21 days of age, the cerebellum weights of the jog/jog mice are significantly lower than those of the wild-type mice. These observations indicate that jog/jog mutant mice could be useful models for biomedical research.     

The third egg envelope subunit in fish: cDNA cloning and analysis, and gene expression

The inner layer of the egg envelope of a teleost fish, the medaka, Oryzias latipes, consists of two major subunit groups, Zl-1,2 and Zl-3. On SDS-PAGE, the Zl-1,2 group presents three glycoprotein bands that were considered to be composed of a common polypeptide moiety derived from their precursor, choriogenin H (Chg H). Zl-3 is a single glycoprotein derived from the precursor, choriogenin L (Chg L). In the present study, a fraction of a novel subunit protein was found in the V8 protease digest of Zl-1,2 that was partially purified from oocyte envelopes. This protein fraction was not present in the purified precursor, Chg H. By RT-PCR employing the primers based on the amino acid sequence of this fraction, a cDNA for the novel subunit was amplified, and a full-length clone of the cDNA was obtained by screening a cDNA library constructed from the spawning female liver. The clone consisted of 2025 b.p. and contained an open reading frame encoding the novel protein of 634 amino acids. This protein included Pro-X-Y repeat sequences in two-fifths of the whole length from its N-terminus. Northern blot analysis revealed that the gene expression for this protein occurred in the liver but not in the ovary of spawning female fish. This protein is considered as the third major subunit of the inner layer of the egg envelope of medaka.     

Congenital hydrocephalus caused by exposure to low level X-radiation at early gestational stage in mice.

Hydrocephalus is a severe and often lethal birth defect in humans resulting from excess accumulation off cerebrospinal fluid (CSF) in the cranial vault, accompanied by enlargement of the head, prominence of the forehead and atrophy of the brain. A wide variety of teratogenic procedures have been used to obtain congenital hydrocephalus in laboratory animals. Radiation, infections, trypan blue, hypervitaminosis A, salisylates and nutritional deficiencies were considered as the teratogens. Several investigators induced congenital hydrocephalus following irradiation of pregnant animals. The purpose off this study is to elucidate the pathogenesis off congenital hydrocephalus induced by X-irradiation in mice. Since the sensitive period for malformations produced by X-radiation in mice ranges from gestational day 7 (G7) to Gl3.     

The use of BRM-activated killer cells in adoptive immunotherapy: A pilot study with nine advanced cancer patients

Adoptive immunotherapy using MHC-nonrestricted-lymphocytes, peripheral blood T cells and NK cells was devised. Peripheral blood mononuclear cells (3 x 107) were selected by immobilization to anti-CD3 monoclonal antibody for 4 days and cultured for 2 weeks in the presence of IL-2. Thereafter they were reactivated by 500 U/ml of IFN- and 1000 U/ml of IL-2 for 1 hour. Enhancement of NK and LAK activities was confirmed. Peripheral blood T cells proliferated in response to immobilized anti-CD3 antibody (3% to 30%). Approximately 6 x 109 BRM-activated killer (BAK) cells composed of CD56+ T cells and CD56+ NK cells, were dispensed to cancer patients via intravenous drip infusion. Nine patients were treated with BAK cells every 2 weeks or every month on an outpatient basis. During the course of adoptive immunotherapy, the crossed affinity immunoelectrophoresis (CAIE) pattern of serum immunosuppressive acidic protein (IAP) was analysed. Both the production and glycosylation pattern of IAP is changed in response to tumor enlargement and may therefore act as a marker of the disease progression. During the course of BAK therapy, the glycosylation IAP pattern of 6 patients changed from tumor (T) to normal (N). In addition, the performance status of all patients was maintained at 90–100% of the Karnofsky scale and any side effects including fever were not observed during treatments with BAK cells. Moreover, the overall quality of life (QOL) of the patients, scored at the Face scale was favorable. In addition, blood levels of activated T cells producing IFN- were assayed as an indication marker of BAK therapy. The normal range of IFN- producing T cells comprised 6.9 ± 0.9% of peripheral blood mononuclear cells (PBMC), according to a single cell FACScan analyses of PBMCs derived from normal individuals. IFN- producing T cells of Patients No. 8 and 9, who received extensive chemotherapy before initiation of BAK therapy, comprised only 0.2% and 2% of PBMC, respectively. These patients died 3 and 6 months after beginning BAK therapy. Peripheral blood T cells of Patients Nos. 1–7 proliferated in response to immobilized anti-CD3 antibody and the frequency of IFN- producing T cells in PBMC preparation of these patients were over 3% before initiation of BAK therapy. Since our data show a positive correlation between survival time and initial T cell counts, a low frequency of these cells may contraindicate BAK therapy.     

Sulfated glycosaminoglycans in guinea pig basophils studied by means of cationic colloidal gold

 Bone marrow embedding in the hydrophilic resin, Lowicryl K4M, followed by cationic colloidal gold (CCG, pH 1.0) staining was used to study the sulfated glycosaminoglycans (GAGs) and their sites of sulfation ultrastructurally in various maturational stages of both basophil granulocytes and basophil granules in the guinea pig. CCG at pH 1.0 is specific for sulfated GAG staining. Basophil granulocytes and granules reacted positively to CCG with a variety of staining according to the stage of maturation. The formation of basophil granules takes place throughout the myelocyte stage. Early basophil myelocytes contain a large Golgi apparatus with active granulogenesis, while late myelocytes contain a small and less active Golgi apparatus as judged by CCG staining. All the immature granules and some of the granules with characteristic ultrastructure stained positively. However, some of the mature granules had lost their affinity for CCG upon maturation. Interestingly, strongly positive CCG staining was also observed in the trans to transmost Golgi apparatus. This indicates that sulfation of GAGs occurs in the trans to transmost Golgi apparatus in all maturational stages of basophil granulocytes. Treatment with chondroitinase ABC or heparinase I abolished the majority of CCG staining. Accepted: 17 July 1997     

Reduced Permeability to K+ and Na+ Ions of K+ Channels in the Plasma Membrane of Tobacco Cells in Suspension after Adaptation to 50 mM NaCl

The whole-cell patch-clamp technique was used to study and comparethe characteristics of K⁺-and Na⁺-transport processes acrossthe plasma membrane in two types of protoplast isolated fromNaCl-adapted and -unadapted cells of tobacco (Nicotiana tabacumL. cv. Bright Yellow-2) in suspension culture. In both typesof protoplast, with 100 mM KCl in the bathing solution and inthe pipette solution, depolarization of the plasma membranefrom the holding potential of 0 mV to a positive potential resultedin a relatively large outward current which increased with increasingpositive potential, whereas hyperpolarization to negative potentialsup to –100 mV resulted in only a small inward current.The outward current activated by depolarization was predominantlycarried by K⁺ ions through K⁺ channels. Na⁺ ions also had afinite ability to pass through these K⁺ channels. The outwardK⁺ and Na⁺ currents of the NaCl-adapted cells were considerablysmaller than those of the NaCl-unadapted cells. These resultssuggest that adaptation to salinity results in reduced permeabilityof the plasma membrane to both K⁺ and Na⁺ ions. ¹Present address: Research Laboratory of Applied Biochemistry,Tanabe Seiyaku Co., Ltd., 16-89, Kashima 3-chome, Yodogawa-ku,Osaka, 532 Japan     

Increased Phosphodiesters in Lipopolysaccharide Prepared from the Polymyxin B-Resistant Isolate of Klebsiella pneumoniae

A polymyxin B (PXB)-resistant mutant of Klebsiella pneumoniae O3 was isolated. Lipopolysaccharide (LPS) extracted from the PXB-resistant isolate bound little PXB, although LPS from the parental strain did. The ³¹P nuclear magnetic resonance (NMR) spectrum of PXB-resistant type LPS showed that it contained much less of the phosphomonoesters and the pyrophosphate esters, and an increased amount of the phosphodiesters, compared to the parental type LPS. The decrease in the binding of PXB might be due to altered phosphate groups on the PXB-resistant type LPS, suggesting that it might explain the PXB-resistance of the mutant.