Light-dependent translocation of a phytochrome B-GFP fusion protein to the nucleus in transgenic Arabidopsis.

Phytochrome is a ubiquitous photoreceptor of plants and is encoded by a small multigene family. We have shown recently that a functional nuclear localization signal may reside within the COOH-terminal region of a major member of the family, phytochrome B (phyB) (Sakamoto, K., and A. Nagatani. 1996. Plant J. 10:859-868). In the present study, a fusion protein consisting of full-length phyB and the green fluorescent protein (GFP) was overexpressed in the phyB mutant of Arabidopsis to examine subcellular localization of phyB in intact tissues. The resulting transgenic lines exhibited pleiotropic phenotypes reported previously for phyB overexpressing plants, suggesting that the fusion protein is biologically active. Immunoblot analysis with anti-phyB and anti-GFP monoclonal antibodies confirmed that the fusion protein accumulated to high levels in these lines. Fluorescence microscopy of the seedlings revealed that the phyB-GFP fusion protein was localized to the nucleus in light grown tissues. Interestingly, the fusion protein formed speckles in the nucleus. Analysis of confocal optical sections confirmed that the speckles were distributed within the nucleus. In contrast, phyB-GFP fluorescence was observed throughout the cell in dark-grown seedlings. Therefore, phyB translocates to specific sites within the nucleus upon photoreceptor activation.

A family of cAMP-binding proteins that directly activate Rap1.

cAMP (3',5' cyclic adenosine monophosphate) is a second messenger that in eukaryotic cells induces physiological responses ranging from growth, differentiation, and gene expression to secretion and neurotransmission. Most of these effects have been attributed to the binding of cAMP to cAMP-dependent protein kinase A (PKA). Here, a family of cAMP-binding proteins that are differentially distributed in the mammalian brain and body organs and that exhibit both cAMP-binding and guanine nucleotide exchange factor (GEF) domains is reported. These cAMP-regulated GEFs (cAMP-GEFs) bind cAMP and selectively activate the Ras superfamily guanine nucleotide binding protein Rap1A in a cAMP-dependent but PKA-independent manner. Our findings suggest the need to reformulate concepts of cAMP-mediated signaling to include direct coupling to Ras superfamily signaling.

Regulation of myosin phosphatase by a specific interaction with cGMP- dependent protein kinase Ialpha.

Contraction and relaxation of smooth muscle are regulated by myosin light-chain kinase and myosin phosphatase through phosphorylation and dephosphorylation of myosin light chains. Cyclic guanosine monophosphate (cGMP)-dependent protein kinase Ialpha (cGKIalpha) mediates physiologic relaxation of vascular smooth muscle in response to nitric oxide and cGMP. It is shown here that cGKIalpha is targeted to the smooth muscle cell contractile apparatus by a leucine zipper interaction with the myosin-binding subunit (MBS) of myosin phosphatase. Uncoupling of the cGKIalpha-MBS interaction prevents cGMP-dependent dephosphorylation of myosin light chain, demonstrating that this interaction is essential to the regulation of vascular smooth muscle cell tone.

Involvement of adaptor protein Crk in malignant feature of human ovarian cancer cell line MCAS.

Signaling adaptor protein Crk regulates cell motility and growth through its targets Dock180 and C3G, those are the guanine-nucleotide exchange factors (GEFs) for small GTPases Rac and Rap, respectively. Recently, overexpression of Crk has been reported in various human cancers. To define the role for Crk in human cancer cells, Crk expression was targeted in the human ovarian cancer cell line MCAS through RNA interference, resulting in the establishment of three Crk knockdown cell lines. These cell lines exhibited disorganized actin fibers, reduced number of focal adhesions, and abolishment of lamellipodia formation. Decreased Rac activity was demonstrated by pull-down assay and FRET-based time-lapse microscopy, in association with suppression of both motility and invasion by phagokinetic track assay and transwell assay in these cells. Furthermore, Crk knockdown cells exhibited slow growth rates in culture and suppressed anchorage-dependent growth in soft agar. Tumor forming potential in nude mice was attenuated, and intraperitoneal dissemination was not observed when Crk knockdown cells were injected into the peritoneal cavity. These results suggest that the Crk is a key component of focal adhesion and involved in cell growth, invasion, and dissemination of human ovarian cancer cell line MCAS.

Rap2 as a slowly responding molecular switch in the Rap1 signaling cascade.

Rap2 is a member of the Ras family of GTPases and exhibits 60% identity to Rap1, but the function and regulation of Rap2 remain obscure. We found that, unlike the other Ras family proteins, the GTP-bound active form exceeded 50% of total Rap2 protein in adherent cells. Guanine nucleotide exchange factors (GEFs) for Rap1, C3G, Epac (or cyclic AMP [cAMP]-GEF), CalDAG-GEFI, PDZ-GEF1, and GFR efficiently increased the level of GTP-Rap2 both in 293T cells and in vitro. GTPase-activating proteins (GAPs) for Rap1, rap1GAPII and SPA-1, stimulated Rap2 GTPase, but with low efficiency. The half-life of GTP-Rap2 was significantly longer than that of GTP-Rap1 in 293T cells, indicating that low sensitivity to GAPs caused a high GTP/GDP ratio on Rap2. Rap2 bound to the Ras-binding domain of Raf and inhibited Ras-dependent activation of Elk1 transcription factor, as did Rap1. The level of GTP-Rap2 in rat 3Y1 fibroblasts was decreased by the expression of v-Src, and expression of a GTPase-deficient Rap2 mutant inhibited v-Src-dependent transformation of 3Y1 cells. Altogether, Rap2 is regulated by a similar set of GEFs and GAPs as Rap1 and functions as a slowly responding molecular switch in the Rap1 signaling cascade.

Inhibitor of vascular endothelial growth factor receptor tyrosine kinase attenuates cellular proliferation and differentiation to mature neurons in the hippocampal dentate gyrus after transient forebrain ischemia in the adult rat.

Neurogenesis in the adult hippocampal dentate gyrus is promoted by transient forebrain ischemia. The mechanism responsible for this ischemia-induced neurogenesis, however, remains to be determined. It has been suggested that there may be a close relationship between neurogenesis and the expression of vascular endothelial growth factor, an angiogenic factor. The purpose of the present study was to examine the relationship between vascular endothelial growth factor and cell proliferation in the dentate gyrus after transient forebrain ischemia. The mRNA expression of vascular endothelial growth factor was increased in the dentate gyrus on day 1 after ischemia. Immunohistochemical analysis on day 9 after ischemia, when a significant increase in cell proliferation was seen, showed that the cerebral vessel space in the subgranular zone of the dentate gyrus had not been affected by the ischemia. Neither were the vascular densities on days 1 and 3 after ischemia altered compared with those of non-operated naïve control rats. Furthermore, the distance from the center of the proliferative cells to the nearest cerebral vessel of ischemic rats was comparable to that of the sham-operated rats. We demonstrated that transient forebrain ischemia-induced cell proliferation and differentiation to mature neurons in the hippocampal dentate gyrus was attenuated by the i.c.v. administration of a vascular endothelial growth factor receptor tyrosine kinase inhibitor. These results suggest that vascular endothelial growth factor receptor at the early period of reperfusion may contribute to neurogenesis rather than to angiogenesis in the hippocampal dentate gyrus.

Effects of hepatic CYP3A4 activity on disposition of micafungin in liver transplant recipients with markedly small-for-size grafts.

Micafungin, the first candin antifungal drug developed in Japan, has a significant therapeutic effect against deep-seated mycoses caused by Candida or Aspergillus. Little is known, however, about the optimal dosage or disposition of micafungin in patients with severe hepatic impairment. Nine liver transplant recipients (5 males and 4 females) were enrolled in this study. In 1 recipient with a markedly small-for-size graft (ratio of graft volume to standard liver volume at the time of transplantation: 25.9%), the areas under the plasma concentration-time curves up to 12 hours postdose (AUC(0-12 h)) at doses of 50 and 100 mg/d were 79.38 and 601.17 mug.h/mL, respectively. The corresponding elimination half-life (T(1/2)) values were 16.01 and 75.75 hours, and saturated elimination was observed only at the dose of 100 mg/d. The mean urinary ratio of 6beta-hydroxycortisol to cortisol (6beta-OHF/F) in the small-for-size graft recipient was significantly (P < .05) lower than that in the other recipients. In conclusion, graft size was an important factor affecting disposition of micafungin. For liver transplant recipients with markedly small-for-size grafts, the optimal dosage of micafungin to reach and maintain therapeutic plasma levels is estimated to be 50 mg/d.

Control of membrane potential by external H+ concentration in Bacillus subtilis as determined by an ion-selective electrode.

The membrane potential of intact bacteria was monitored by measuring the tetraphenylphosphonium ion distribution across the membrane using poly--(vinyl chloride) matrix-type electrode selective to tetraphenylphosphonimum ion. It was found that the tetraphenylphosphonium ion was not countertransported against H+ movement. The membrane potential of Bacillus subtilis was estimated to be 80-120 mV inside-negative at external pH 7. The effect of the external pH on the membrane potential was studied. It varied from 30 to 40 mV/decade change in the external [H+] in the pH region of greater than 6.5, increasing pH making it more inside-negative. The addition of carbonyl cyanide m-chlorophenylhydrazone depolarized the membrane, and the membrane potential approached the H+ equilibrium potential. The addition of N,N'-dicyclohexylcarbodiimide did not abolish the pH dependence of the membrane potential. Increasing the external [K+] did not affect the pH dependence. CN- partially depolarized the membrane. A parallel conductance model for membrane potential could explain the results qualitatively.

Non-adherent cell-specific expression of DOCK2, a member of the human CDM-family proteins.

Human DOCK180, which was originally identified as a major protein bound to the Crk oncogene product, is an archetype of the CDM family of proteins, including Ced-5 of Caenorhabditis elegans and Mbc of Drosophila melanogaster. After DOCK180, at least three putative human proteins that manifest high amino acid sequence similarity to DOCK180 have been registered in the GenBank/EMBL database. We have designated one of them, KIAA0209, as DOCK2 and characterize here. DOCK2 mRNA was expressed mostly in peripheral blood cells, followed by slight expression in the spleen and thymus, whereas DOCK180 was expressed in all tissues tested except in peripheral blood cells. Immunostaining of human cadaver tissues revealed that the expression of DOCK2 was limited to the lymphocytes and macrophages of various organs. DOCK2 bound to and activated Rac1, as did DOCK180; however, DOCK2 did not bind to CrkII, which transduces signals at focal adhesions. Thus, DOCK180 and DOCK2 are regulators of Rac and function in adherent and non-adherent cells, respectively.

Phase I study on human monoclonal antibody against cytomegalovirus: pharmacokinetics and immunogenicity.

MAb C23, a human immunoglobulin G1 (IgG1) monoclonal antibody (MAb) against cytomegalovirus, was administered to 20 healthy volunteers. Sixteen of them received a single infusion of a dose ranging from 5 to 80 mg. The plasma clearance curves fit a two-compartment model, with half-lives of 31.0 +/- 23.6 h in the diffusion phase and 24.2 +/- 5.8 days in the equilibration phase. The plasma after administration had the virus neutralization activities that were equivalent to the plasma MAb C23 levels. The remaining four subjects, who received three infusions of 60, 20, and 20 mg at 1-week intervals, showed pharmacokinetics that were very consistent with those of the single infusion. No antibody response against MAb C23 was observed in any of the subjects at any time, when monitored for approximately 60 days after the single infusion or the third infusion of the three repeated doses. None of the 20 subjects showed any treatment-related clinical signs or changes. These results suggest that a human IgG MAb has the same pharmacokinetic characteristics as those of natural human serum IgG, and that it is not immunogenetic and is safe in humans.

Identification and cDNA cloning of a novel human mosaic protein, LGN, based on interaction with G alpha i2.

We have used the yeast two-hybrid system to identify proteins that interact with the alpha-subunit of the heterotrimeric GTP-binding protein, Gi2. We screened a human B cell cDNA library with full-length G alpha i2 and isolated four positive colonies, one of which expressed the 44-kDa COOH terminus of a previously unrecognized 677-amino acid (aa) protein. A full-length clone was isolated from a HeLa cell cDNA library. The deduced protein contains 10 Leu-Gly-Asn repeats, and thus we named it LGN. Computer analysis indicates that LGN is a mosaic protein with seven repeated sequences of about 40 aa in length at its N-terminal end, and four repeated sequences of about 34 aa at its C-terminal end. Each of the two repeat regions shows substantial similarity to proteins found in other organisms. RT-PCR analysis of human tissues showed that the mRNA of LGN was ubiquitously expressed. The specificity of interaction between G alpha i2 and LGN was confirmed by an in vitro binding assay using recombinant proteins. These data indicate that the yeast two-hybrid system can identify novel proteins, such as LGN, that interact with G alpha proteins. As a mosaic protein, LGN shows similarity with portions of proteins from many species and thus may define a new protein family.

Interaction of the protein nucleobindin with G alpha i2, as revealed by the yeast two-hybrid system.

The heterotrimeric G protein, G alpha i2, transduces signals from seven membrane spanning receptors to effectors such as adenylyl cyclase and ion channels. The purpose of this study was to identify these or other cellular proteins that interact with G alpha i2 by use of the yeast two-hybrid system. A human B cell cDNA library was screened by this system using full length G alpha i2. Four positive colonies were obtained. Two of the four were identified as nucleobindin, a calcium binding protein and a putative antigen to which anti-nuclear antibodies are generated in mice with a disorder that resembles systemic lupus erythematosus. Nucleobindin has a leucine zipper, EF hands, and a signal peptide sequence and is thought to localize to the nucleus as well as being secreted. The specificity of intehraction between G alpha i2 and nucleobindin was confirmed by an in vitro binding assay using recombinant proteins. Transfection of G alpha i2 and nucleobindin in COS cells increased G alpha i2 expression relative to cells transfected with G alpha i2 and mock vector. Our results indicate that the yeast two-hybrid system provides a means to identify novel proteins that interact with G alpha proteins. Nucleobindin appears to represent one of those proteins.

Characterization of three cDNA species encoding plastid RNA polymerase sigma factors in Arabidopsis thaliana: evidence for the sigma factor heterogeneity in higher plant plastids.

By database search analysis, we identified three Arabidopsis EST (Expression Sequence Tag) entries having similarity to eubacterial RNA polymerase sigma factors. cDNA clones corresponding to these partial sequences were isolated, and the complete nucleotide sequences were determined. All three sequences encode proteins highly homologous to cyanobacterial and plastid sigma factors, and the gene products have N-terminal extensions which are assumed to function as plastid-targeting transit peptides. Thus we have concluded that the gene products are RNA polymerase sigma factors of plastids, and named sigA, sigB and sigC, respectively. Expression of these genes was analyzed by RNA gel-blot analysis and shown to be induced by illumination after a short-term dark adaptation. This strongly suggests that light regulation of the nuclear encoded sigma factor genes is involved in light-dependent activation of plastid promoters.

An infectious DNA clone of HIV type 1 subtype C.

Among the 10 subtypes of the M group of human immunodeficiency virus type 1, subtype C is the most prevalent in India and may dominate worldwide in the near future; however, there has been no report on the infectious DNA clone of this subtype. We have isolated an infectious DNA clone of the 93IN101 strain of HIV-1 subtype C, which was isolated in India in 1993. MAGIC5 cells, which are derived from HeLa-CD4-LTR-beta-gal (MAGI) cells and express CCR5, were inoculated with the 93IN101 strain of HIV-1 subtype C. The genomic DNA of the infected cells was used as a template for amplification of the HIV-1 genome. The genome DNA obtained was subcloned into pBR322, and the resulting plasmid was designated as pIndie-C1. The insert of pIndie-C1 was 9680 bp in length and had an intact genomic organization with open reading frames of all structural, regulatory, and accessory proteins. Phylogenetic analysis confirmed that the nucleotide sequence of pIndie-C1 is closely related to those of HIV-1 subtype C isolated in India. Transfection of pIndie-C1 into 293T cells yielded as much virus as did pNL432, one of the most widely used HIV DNA clones. The recovered Indie-C1 virus infected MAGIC5 but not the parent MAGI cells, indicating that Indie-C1 is CCR5 tropic. Expressed Env protein was reacted efficiently with the sera of HIV-1-infected patients of India, but not of Japan. Expression of Nef and Vpr was also confirmed by immunoblotting.

A gene coding for a zinc finger protein is induced during 12-O-tetradecanoylphorbol-13-acetate-stimulated HL-60 cell differentiation.

ETR103 cDNA was cloned as an immediate early gene in the course of macrophagic differentiation of HL-60 cells stimulated by TPA (12-O-tetradecanoylphorbol-13-acetate). The induction by TPA was immediate-early (within 30 min) and transient. This gene was not induced by vitamin D3 or by retinoic acid, which stimulates differentiation of HL-60 cells to the monocytic or granulocytic lineage, respectively. The ETR103 mRNA was induced by TPA in lymphoid or myeloid leukemia cell lines of several maturation stages. The induction by TPA seems to proceed by a protein kinase C-mediated mechanism, on the basis of the results obtained by using protein kinase C inhibitor (H-7), protein kinase C activator (diC8), and an activator of protein kinase A (dibutyryl cAMP). Okadaic acid, an inhibitor of protein phosphatases, also induced the ETR103 mRNA expression. The nucleotide sequence of the ETR103 cDNA reveals that ETR103 encodes a human zinc finger-containing transcription factor identical to Egr-1 and 225, which is homologous to mouse Egr-1, Zif/268, Krox-24, and TIS8, or to rat NGFI-A.

Survival and B-cell function of neonatal pig pancreatic islet-like cell clusters in an extracellular matrix.

Islet-like cell clusters (ICCs), formed from single cells of pig pancreas in suspension culture, were embedded in an extracellular matrix. It was recently reported that nicotinamide prevented dissolution of the extracellular matrix by the ICCs. In this experiment, various conditions for embedded culture of ICCs in an extracellular matrix were studied, in an attempt to maintain the function as well as the extracted insulin content of culture specimens. The ICCs in the matrix were refed with RPMI 1640, containing 10 mM nicotinamide, 10% fetal bovine serum (FBS), 11 mM D-glucose and with or without 0.1 mM 3-isobutyl-1-methylxanthine (IBMX) or 1.0 micrograms/ml caerulein. A comparison between the different culture media showed that embedded ICCs, maintained in RPMI 1640 with caerulein, in the presence of nicotinamide, had higher insulin content accumulation than when maintained in medium containing nicotinamide alone, but had impaired glucose-stimulated insulin secretion. In the medium containing IBMX and nicotinamide, embedded ICCs showed higher insulin accumulation but lower insulin content, compared to ICCs maintained in the presence of caerulein, and also showed impaired glucose-stimulated insulin release. Thus, the effect of nicotinamide on the survival and function of B-cells is amplified by the presence of caerulein or IBMX.

Studies of body movements during night sleep in infancy.

Body movements (BMs) during night sleep of 25 neurologically normal infants, 11 premature and 14 full-term, whose ages ranged from 30 conceptional weeks to 18 months post-term, were examined to evaluate the changes of their features with age. The examinations performed during sleep periods totaled 65 times, 1 to 60 times on each subject. Through visual observation and EEG recordings, the BMs were classified into 3 types: (1) Gross movements (GM), (2) localized movements (LM), both of the above two lasting more than 0.5 second, and (3) twitch movements (TM) lasting less than 0.5 second. Total GM and LM time per hour of sleep, average duration of GM and LM and number of GM, LM and TM per hour of sleep were calculated. Percentage of 20 seconds epochs without BMs (nonbody-movement-epochs) was also estimated. These BMs parameters decreased with maturation to certain low base levels. However, each parameter showed a particular time course. TM decreased initially, then LM and lastly GM reached the base level around the age of 9 to 13 months. On the other hand, nonbody movement-epochs increased progressively until 8 months of age. These three types of BMs are considered to be controlled by the CNS with different organization levels, the simplest for TM and the most complicated for GM. They are thus correlated to the maturational process of the CNS, and could be good indicators for detecting normal and abnormal CNS developments.

Pseudoaneurysm of the subclavian artery due to Xanthomonas pneumonia in a patient with acute myeloid leukemia: its rupture treated by transcatheter coil embolization.

A 52-year-old male with acute myeloid leukemia developed pseudoaneurysm of the subclavian artery. Pneumonia due to Xanthomonas maltophilia, which was multi-drug resistant, progressed to a lung abscess even under administration of antibiotics. This lung infection contiguous to the left carotid and subclavian arteries was suggested to have caused the pseudoaneurysm of the subclavian artery. The rupture of the aneurysm by penetration to the trachea amounted to about 1,000 ml of bleeding; fortunately the bleeding ceased spontaneously. Nonetheless, an emergency transcatheter coil embolization prevented re-bleeding. Endovascular treatment should be considered especially for aneurysms which develop in patients with underlying diseases.

[Relationship between cognitive function and discharge place among stroke patients after rehabilitation].

One hundred and ninety-nine elderly stroke patients, who received rehabilitation treatment, were examined, to clarify the relationship between cognitive function and discharge place. The patients who moved to long-term care facilities showed more severe disabilities of basic activities of daily living (ADL), more frequent incontinence, and lower functional impairments (Brunnstrom stage), compared with those discharged to their home. Multivariate regression analysis was done with discharge place as the dependent variable. Independent variables were age, sex, kind of stroke, rehabilitation period, level of ADL and IQ on Kohs test, or performance IQ on the Wechsler Adult Intelligence Scale. Older age, higher levels of ADLs, and higher scores on Kohs test IQ or Wechsler Adult Intelligence Scale Performance IQ were all significantly linked with home discharge. These results suggest that non-verbal cognitive dysfunction may affect discharge place in elderly stroke patients after rehabilitation therapy.

[Normalization of hypercholesterolemia in a female stroke patient after switching from enteral tube feeding to oral feeding].

A 70-year-old female was admitted to a general hospital in a rural area due to left putamenal cerebral hemorrhage in December 1994. She had right hemiplegia and was totally aphasic. In May 1995, she was moved to Tokyo where her son lives, and she was admitted to Tokyo Metropolitan Geriatric Hospital in order to prepare a home care system. her family's support (serving her favorite dishes) allowed enteral tube feeding to be halted. After one month she could absorb enough energy to maintain her serum albumin level. The total calories ingested orally was comparable to that of enteral feeding but the fat composition was 62% of that of enteral feeding (fat was 19.6% and 31.7% of the total calories in the two diets, respectively). Her cholesterol level decreased from 286 mg/dl to 197 mg/dl. Nutrient-balanced tube feeding is useful, but may disturb lipid metabolism in patients used to having vegetable-rich diets.