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Biomedical subjects

M Furuya

Publications and source records attributed to M Furuya.

12 recordsLinked to original sources

Structural requirements of C-type natriuretic peptide for elevation of cyclic GMP in cultured vascular smooth muscle cells.

C-type natriuretic peptide (CNP), which was recently found to be a selective ligand for one of the two known natriuretic peptide receptor guanylyl cyclases (NPR-B), potently stimulates cGMP production in cultured rat vascular smooth muscle cells (VSMC) and exerts potent antiproliferative effects on the cells. To investigate the structural requirements of CNP for stimulation of cGMP accumulation via NPR-B, we prepared CNP analogs and tested them on cultured rat VSMC. Our results indicate that only the ring portion of CNP with a disulfide bond (CNP(6-22)) participates in stimulation of cGMP accumulation, especially the sequence Leu9-Lys10-Leu11 in the ring portion executes essential roles for both elevation of cGMP and selectivity of the ligand for NPR-B. We also found a good correlation between the activities of the CNP analogs for stimulation of cGMP accumulation and inhibition of DNA synthesis.

Amino Acid Sequence

Serine-to-alanine substitutions at the amino-terminal region of phytochrome A result in an increase in biological activity.

We have used a tobacco transgenic plant system to assay the structure/function relationship of phytochrome A (phyA), a plant photoreceptor. The amino terminus of phyA from different plant species is very rich in serine residues. To investigate whether these serine residues are required for phytochrome function, the first 10 serine codons encoding amino acid residues 2-4, 10-14, 19, and 20 in the amino-terminal domain of the rice phyA gene (phyA) were changed to alanine codons. The mutant (S/A phyA), as well as the wild-type phyA cDNA, was placed under the control of the 35S promoter, and the chimeric genes were transferred into the tobacco genome by Agrobacterium-mediated transformation. Transgenic tobacco plants expressing either wild-type or S/A phyA showed similar phenotypic alterations, including dwarfism and dark-green leaves. However, hypocotyl elongation experiments revealed that transgenic seedlings expressing S/A phyA showed a higher amplitude of the red light response with respect to the inhibition of hypocotyl elongation. The observed difference is not correlated with expression levels of the transgene. The chromophore is attached to the mutant phyA apoprotein (PHY A), and the mutant photoreceptor is photoreversible, giving a difference spectrum indistinguishable from that of the rice phyA. Our results indicate that the S/A mutant has a higher biological activity as compared with the wild-type rice phyA.

Alanine

Detection of antibodies against streptococcal peptidoglycan and the peptide subunit (synthetic tetra-D-alanyl-bovine serum albumin complex) in rheumatic-diseases.

Serum antibodies reactive with streptococcal cell wall peptidoglycan (PG) and its peptide subunit (synthetic tetra-D-alanine) were measured by enzyme-linked immunosorbent assay (ELISA) in patients with rheumatoid arthritis (RA), juvenile rheumatoid arthritis (JRA), osteoarthritis and acute rheumatic fever (RF) compared with healthy subjects. Using 'checkerboard' titrations, anti-PG antibody in human serum was detected at a concentration of PG antigen at 10 micrograms per well with serum dilutions of 1:1,000. For measurement of anti-tetra-D-alanine antibody, the antigen, (D-Ala4)31 was used at 0.5 micrograms per well and sera were diluted to 1:200. When the IgG antibody levels to the PG and the tetra-D-alanine of the sera of patients with RA, JRA and RF were compared with sera from healthy subjects, the sera of the patients had significantly higher levels than did healthy subjects. Antibody that reacted with the PG in serum was absorbed with purified group-specific C-carbohydrate (A-CHO), but A-CHO was not capable of absorbing anti-(D-Ala4)31 antibodies. Therefore, the peptide subunit should be used as antigen in order to measure the specific antibody to PG. Both anti-PG and anti-tetra-D-alanine antibody in human sera primarily belonged to the IgG2 subclass.

Adolescent

Xeroderma pigmentosum variant associated with multiple skin cancers and a lung cancer.

A 65-year-old Japanese man with a xeroderma pigmentosum (XP) variant, XP127TO, is described. The XP127TO skin fibroblasts exhibited the typical XP variant characteristics of a 1.5-fold higher sensitivity than normal cells to the lethal effect of 254 nm ultraviolet (UV) light and the normal level of unscheduled DNA synthesis induced by 254 nm UV. Caffeine dose-dependently increased the cytotoxic effect of 254 nm UV on XP127TO cells. Clinically, the patient developed not only 3 cutaneous squamous cell carcinomas on sun-exposed areas but also an adenocarcinoma of the upper lobe of the right lung. A review of the 14 documented Japanese XP patients with nonskin malignancies indicates that the incidence of nonskin malignancy in XP patients is much lower than that of skin cancer in XP but higher than that in the general population.

Adenocarcinoma, Papillary

The cucumber long hypocotyl mutant lacks a light-stable PHYB-like phytochrome.

A novel cDNA sequence homologous to a phytochrome B (phyB) gene that was isolated in a library from tobacco tissue has been used in an Escherichia coli expression system to raise anti-phytochrome B (anti-PHYB) polypeptide-specific monoclonal antibodies. The specificity of these antibodies has been tested by cross-reactivity against purified pea light-labile type 1 and light-stable type 2 phytochromes, with some antibodies reacting with the type 2 and none with the type 1 phytochromes. One such antibody, monoclonal mAT1, has been employed to analyze the phytochrome molecular species present in a photomorphogenic long hypocotyl (lh) mutant of cucumber. The results indicated that the mutant contains wild-type levels of the light-labile type 1 phytochrome polypeptide (PHYA), which has an apparent molecular mass of approximately 120 kD, but shows less than 1% (detection limit) of a light-stable polypeptide recognized by mAT1 in wild-type seedlings. This protein, not detectable in the lh mutant, has the properties of light-stable type 2 phytochrome, has an apparent molecular mass of 116 to 117 kD, and remains at constant levels under continuous low-fluence-rate red light. Therefore, we conclude that the lh mutant lacks at least one type 2 phytochrome-like polypeptide, most probably a phyB gene product. The correlation between the lack of this protein and the deficiency or absence of physiological responses to a light-stable phytochrome species in this mutant helps to identify the physiological roles played by the products of different subfamilies within the phytochrome gene family.

Antibodies, Monoclonal

C-type natriuretic peptide is a growth inhibitor of rat vascular smooth muscle cells.

C-type natriuretic peptide (CNP) which potently stimulates particulate guanylate cyclase activity in cultured rat vascular smooth muscle cells (VSMC) inhibited serum-induced DNA synthesis of the cells 10-fold more effectively than alpha-human atrial natriuretic peptide (alpha-hANP). The inhibitory effect of CNP was mimicked by 8-bromo-cGMP. The proliferation of VSMC was also suppressed by CNP more potently than alpha-hANP, while the peptide was less active for cGMP augmentation and for vasorelaxation than alpha-hANP in isolated rat aorta. These results suggest that CNP may be a growth regulating factor of VSMC rather than a vasodilator.

Animals

Effect of bradykinin to cyclic AMP levels and response of murine lymphocytes.

No information is available on the pharmacological effect of bradykinin to lymphocytes and immunological responses of them. In this study it was clarified that bradykinin as well as histamine elevated cyclic adenosine 3',5' monophosphate (cAMP) levels of murine splenic or lymph node lymphocytes and mature thymocytes (cortisone-resistant thymus), but did not increase cAMP levels of immature thymocytes as well as histamine. The increased cAMP ratios in T cell-enriched splenic lymphocytes by the impulse of bradykinin were higher than that in splenic and lymph node lymphoid cells by the stimulation of bradykinin. It was also demonstrated that bradykinin as well as histamine suppressed DNA synthesis by mitogenic (PHA-P, Con-A) stimulation of splenic lymphocytes, but not a effect to the response of lymphocytes by mitogenic (LPS) stimulation was observed. These facts suggest that bradykinin may play an important role in the regulation of immunologic lymphocyte responses.

Animals

Protective immune response in mice immunized with antigens from Trypanosoma gambiense-infected mouse blood.

Immunogenicity and property of antigens obtained from Trypanosoma gambiense-infected mouse blood (IMP) were examined. A strong vaccine effect against intravenous challenges with 3 x10(3) parasites given on study day 3, 5, or 14 (day 0 = immunization) was observed in mice immunized with a combination of IMP (2 mg protein/mouse) and Freund's complete adjuvant (FCA). But when the challenge was given on day 21 or 30, per cent survival in mice dropped to the 20- and 40-per cent level, respectively. Among fractioned components of IMP, IMP-1, IMP-2, and imp-3, by gel filtration with Sephadex G-200, all of the mice immunized with IMP-1 antigen alone or together with FCA and challenged on day 5 were able to conquer intraperitoneal challenges with 1 x10(2) parasites. Mice immunized with IMP-2 or IMP-3 died within 6 days after challenge. Moreover, protection efficacy shown by IMP-1p (144,000 xg sediment of IMP-1) antigen in mice was similar to that by IMP and IMP-1 antigens. IMP-3 yielded a single precipitin line against mouse anti-IMP serum by Ouchterlony double diffusion method but this response was eliminated when the antiserum was absorbed by IMP-1p. No precipitin line was identified between mouse anti-IMP serum and IMP-1 or IMP-2. From electron microscopic observations, elements of IMP-1 and IMP-1p are possibly corresponded to the fragments of filopodia of the parasites.

Agglutination Tests

In situ microspectrofluorometry of nuclear and kinetoplast DNA in Trypanosoma gambiense.

Using a spectrofluorometer with the Zeiss Universal Micro-Spectrophotometer 1 (UMSP 1), both nuclear and kinetoplast DNA (N-DNA and K-DNA) in the trypomastigote form of Trypanosoma gambiense (Strain Wellcome) were measured in situ without being extracted. As the fluorescent dye, ethidium bromide was preferably employed because there was a very marked increase in the ethidium fluorescence when the dye was intercalated between the base paires of the DNA helix. According to this method, it became possible to demonstrate the existence of double-stranded DNA in both nucleus and kinetoplast clearer than before.

DNA

Characteristic synthesis and redistribution of 70 kd heat shock protein in thermotolerant Chinese hamster V79 cells.

Upon exposure to heat shock the increased rate of hsp70 synthesis decreased more rapidly in thermotolerant V79 cells than in the non-thermotolerant cells. However, the levels of hsp70 in the thermotolerant cells at 12 h after a heat shock were almost the same as those in the non-thermotolerant cells. On the other hand, the migration of hsp70 from cytoplasm to nucleoli after a heat shock was very rapid in both thermotolerant and non-thermotolerant cells, but hsp70 in the nucleoli disappeared faster in the thermotolerant cells than in the non-thermotolerant cells, and this coincided with the faster decline of hsp70 synthesis in the thermotolerant cells. For the characteristic distribution of hsp70, protein synthesis was not required. Furthermore, the induction and expression of thermotolerance by the cells were little affected by the inhibition of protein synthesis. Thus, the synthesis of hsp70 itself seemed not to be essential for the induction and expression of thermotolerance of the cells, although hsp70 may be essential for thermoresistance of cells. The rapid decrease of hsp70 synthesis and the rapid disappearance of hsp70 from the nucleoli after a heat shock may be essential for the expression of thermotolerance of the cells.

Animals