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Biomedical subjects

M Kataoka

Publications and source records attributed to M Kataoka.

At least 253 records · Page 14Linked to original sources
PubMed1994

Energy coupling in an ion pump. The reprotonation switch of bacteriorhodopsin.

The active site of an ion pump must communicate alternately with the two opposite membrane surfaces. In the light-driven proton pump, bacteriorhodopsin, the retinal Schiff base is first the proton donor to D85 (with access to the extracellular side), and then it becomes the acceptor of the proton of D96 (with access to the cytoplasmic side). This "reprotonation switch" has been associated with a protein conformation change observed during the photocycle. When D85 is replaced with asparagine, the pKa value of the Schiff base is lowered from above 13 to about 9. We determined the direction of the loss or gain of the Schiff base proton in unphotolyzed and in photoexcited D85N, and the D85N/D96N and D85N/D96A double mutants, in order to understand the intrinsic and the induced connectivities of the Schiff base to the two membrane surfaces. The influence of D96 mutations on proton exchange and on acceleration of proton shuttling to the surface by azide indicated that in either case the access of the Schiff base on D85N mutants is to the cytoplasmic side. In the wild-type protein (but with the pKa of the Schiff base lowered by 13-trifluoromethyl retinal substitution) the results suggested that the Schiff base can communicate also with the extracellular side. Raising the pH without illumination of D85N so as to deprotonate the Schiff base caused the same, or nearly the same, change of X-ray scattering as observed when the Schiff base deprotonates during the wild-type photocycle. The results link the charge state of the active site to the global protein conformation and to the connectivity of the Schiff base proton to the membrane surfaces. Their relationship suggests that the conformation of the unphotolyzed wild-type protein is stabilized by coulombic interaction of the Schiff base with its counter-ion. A proton is translocated across the membrane after light-induced transfer of the Schiff base proton to D85, because the protein assumes an alternative conformation that separates the donor from the acceptor and opens new conduction pathways between the active site and the two membrane surfaces.

Acid-Base Equilibrium↗
PubMed1994

Cold denaturation of the molten globule states of apomyoglobin and a profile for protein folding.

Protein folding is a process in which an extended polypeptide chain acquires compact packing through the formation of specific secondary and tertiary structures and hydrophobic interactions. Although much attention has been paid to secondary and tertiary structures, there is no definitive view about the relationship between these structures, compactness, and hydrophobic interactions during the process of protein folding. We show here that the molten globule intermediates of horse apomyoglobin exhibit cold denaturation in addition to heat denaturation, which indicates that the heat capacity change upon unfolding is positive and significant. This demonstrates a small but distinct contribution of hydrophobic interactions to the stability of the molten globule state. We determined the radius of gyration of the various conformational states of horse apomyoglobin and holomyoglobin by measuring small angle X-ray scattering. By comparing the conformational states in terms of secondary structure, radius of gyration, and change in heat capacity upon unfolding, we constructed a folding profile. The profile shows that the protein becomes more compact with formation of the secondary structure, but does not form substantial hydrophobic interactions until a later rate-limiting stage when tight packing of the protein side chains occurs. A very similar profile was also obtained with horse cytochrome c. We propose that the folding profile obtained with these proteins will be common to many globular proteins.

Apoproteins↗
PubMed1994

Complete nucleotide sequence of the Streptomyces nigrifaciens plasmid, pSN22: genetic organization and correlation with genetic properties.

The complete nucleotide sequence of the multicopy, self-transmissible, broad-host-range Streptomyces plasmid pSN22, originating from Streptomyces nigrifaciens, was determined. pSN22 is a circular DNA molecule, 10,922 bp with 71.76% G + C. Computer-assisted analysis identified 10 open reading frames (ORFs); 8 of them--traA (155 amino acids [aa], traB (651 aa), traR (246 aa), spdB1 (107 aa), spdB2 (251 aa), spdB3 (70 aa), spdB4 (128 aa) and spdA (154 aa)--are involved in plasmid transfer and pock-formation. One ORF, rep (451 aa), probably encodes a replication protein similar to known replication proteins of rolling circle replicons. The four spdB genes have hydrophobic amino termini that might attach to the cytoplasmic membrane. The deduced rep proteins of pSN22 and pIJ101 are very similar, suggesting that both are derived from a recent common ancestor. The transfer regions of the two plasmids are, however, very different. The only detectable similarities between presumably analogous proteins are DNA- and NTP-binding motifs and hydrophobic regions. This suggests that two transfer regions are of separate origins.

Amino Acid Sequence↗
PubMed1994

Replication of the Streptomyces plasmid pSN22 through single-stranded intermediates.

The replication of the 11 kb conjugative multicopy Streptomyces plasmid pSN22 was analyzed. Mutation and complementation analyses indicated that the minimal region essential for plasmid replication was located on a 1.9 kb fragment of pSN22, containing a transacting element encoding a replication protein and a cis-acting sequence acting as a replication origin. Southern hybridization showed that minimal replicon plasmids accumulated much more single-stranded plasmid molecules than did wild-type pSN22. Only one strand was accumulated. A 500 bp fragment from the pSN22 transfer region was identified which reduced the relative amount of single-stranded DNA, when added in the native orientation to minimal replicon plasmids. This 500 bp DNA sequence may be an origin for second-strand synthesis. It had no effect on the efficiency of co-transformation, plasmid incompatibility, or stability. The results indicate that pSN22 replicates via single-stranded intermediates by a rolling circle mechanism.

DNA Replication↗
PubMed1994

Radioiodinated nordiazepam analog for in vivo assessment of benzodiazepine receptors by single photon emission tomography.

2'-Iodo-nordiazepam (2'-IND), a nordiazepam analog iodinated at the 2'-position of the C-5 phenyl ring, was synthesized and evaluated as a potential radiopharmaceutical for investigating brain benzodiazepine receptors by SPECT. [125I]2'-IND was synthesized by the halogen exchange reaction and purified by HPLC. In an in vitro competitive binding study using [3H]diazepam and rat cortical synaptosomol membranes, 2'-IND showed an almost equal affinity for benzodiazepine receptors as diazepam. In a saturation binding study using rat cortical synaptosomal membranes, 2'-IND displayed a Kd of 1.10 nM and a Bmax of 1.87 pmol/mg protein. Biodistribution and metabolism studies in mice showed that [125I]2'-IND exhibited rapid and high accumulation in the brain, and that the cerebral uptake and distribution of this compound occurred in the intact form. Furthermore, the administration of diazepam and flumazenil reduced cortical uptake by approx. 20%, suggesting that the uptake of 2'-IND occurred at least partly in association with benzodiazepine receptors.

Animals↗
PubMed1994

Regulation of the transfer genes of Streptomyces plasmid pSN22: in vivo and in vitro study of the interaction of TraR with promoter regions.

Expression of the tra operon, essential for conjugative transfer of the 11-kb Streptomyces nigrifaciens plasmid pSN22, is negatively regulated by traR, which is located upstream of the tra operon and transcribed in the opposite orientation. The transcriptional start points for the tra and traR mRNAs were determined by primer extension; they are 72 bp apart and have identical -10 promoter sequences. The TraR protein was overexpressed in Escherichia coli and used for gel retardation and DNase I protection experiments. It bound specifically to the bidirectional tra-traR promoter region and protected four DNA regions, each of which contains a similar 12-bp sequence. The binding was strongest to the region downstream of the tra promoter, probably ensuring that expression of the potentially lethal traB gene is turned off before traR. The efficiency of intramycelial plasmid transfer was decreased by the mutation at the downstream region.

Bacterial Outer Membrane Proteins↗
PubMed1994

Tissue residues of sulfadimethoxine following dietary administration to broiler-chickens.

Broilers were divided into four groups and the first group served as the control. The second, third and fourth groups were given feed containing 25, 50 and 100 mg/kg of sulfadimethoxine (SDM), respectively, for 21 days, and thereafter each group received the SDM free feed. On certain days during the experiment period, three broilers in each group were sacrificed and tissues, including blood, heart, liver, spleen, gizzard, thigh muscle, breast muscle and fat, were collected and residual SDM were determined by HPLC. Two days after withdrawal, SDM in each tissue had decreased to below the detection limit of 0.01 microgram/g.

Animal Feed↗
PubMed1994

Effect of 1,6-dihydro-2-[2-(2-methylpropoxy)anilino]-6-oxo-5-pyrimidinecarboxylic acid on gastric mucosal defensive factors and gastric secretion in rats.

1,6-dihydro-2-[2-(2-methylpropoxy)anilino]-6-oxo-5-pyrimidinecarbo xylic acid, (MAR-99, CAS 98772-05-5) (10-30 mg/kg i.g.) improved the reduction of gastric blood flow rate induced by the administration of 99.5% ethanol or acidified-acetylsalicylic acid (ASA). In addition, MAR-99 (3 x 10(-6)-3 x 10(-5) mol/l) protected dose-dependently the damage of epithelial cells induced by ulcerogenic agents such as ethanol or acidified-ASA. MAR-99 (1-10 mg/kg p.o.) prevented dose-dependently the reduction of hexosamine content in glandular stomach. Furthermore, MAR-99 (10-30 mg/kg i.g.) improved the decrease in gastric potential difference induced by 99.5% ethanol and acidified-ASA. MAR-99 (10-30 mg/kg p.o.) significantly inhibited the lesion formation induced by 99.5% ethanol and such effect of this compound was not attenuated by the pretreatment with indomethacin. Furthermore MAR-99 (10 and 30 mg/kg p.o.) had no effect on the prostaglandins (PGE2 and I2) contents in the stomach of normal rats. In pylorus-ligated rats, MAR-99 (3-100 mg/kg i.d.) showed a weak or no effect on acidity and pepsin activity in gastric juice, although this compound decreased dose-dependently the volume of gastric juice. In perfused stomachs, MAR-99 (30-100 mg/kg i.d.) slightly prevented the acid secretion induced by carbachol and pentagastrin. However, MAR-99 did not affect the acid secretion stimulated by histamine. These results indicated that anti-ulcer effect of MAR-99 was mainly due to maintenance of the gastric mucosal resistance.

Animals↗
PubMed1994

[Our surgical indication for breast-conserving therapy for breast cancer--special reference to extensive intraductal component and mammographic calcifications].

To ascertain accurate indications for breast-conserving therapy, we clinicopathologically examined 597 primary breast cancers. From July 1989 to October 1993, 118 of 597 (19.8%) patients were treated with lumpectomy plus breast radiation therapy. Among 118 of these primary breast cancers, 79 tumors were histologically further investigated with deep cut sections. In 20 of 79 (25.3%) breast cancers, microscopic tumor involvement was observed at resection margins. Most of them showed an extensive intraductal component (EIC). In order to predict EIC, intraductal lesions were histologically examined and classified either as comedo type or non-comedo (e.g., papillary type, cribriform type and solid type). Among 118 primary breast cancer tissues derived from lumpectomy, 66 breast tissues were histologically classified as comedo type. Sixty of 66 (90.9%) of these comedo types showed specific microcalcifications by light microscopic examination. These irregular shaped and relatively large granulated microcalcifications were localized in intraductal lesions with central necrotic ducts. With retrospective mammographic findings, these histological calcifications of comedo type reflected specific mammographic calcifications with columnar and specular formation. These evidences suggested that detection of mammographic calcifications in comedo type breast cancers may be helpful to predict EIC, and this prediction can possibly increase the accuracy of the indication for breast-conserving therapy.

Adult↗
PubMed1994

[Sarcoidosis and Propionibacterium acnes].

Propionibacterium acnes (P. acnes) was isolated in high rates and in high concentrations from lymph nodes in patients with sarcoidosis. However, the precise mechanism of granuloma formation and immunomodulation by P. acnes has not been elucidated yet. In patients with sarcoidosis, it was found that the high levels of interleukin-2 released from alveolar lymphocytes as well as interleukin-1, tumor necrosis factor and interleukin-6 released from alveolar macrophages were stimulated by P. acnes. These cytokines (mainly IL-2), released by P. acnes in large quantities, play a major role in the compartmentalization of the T-cell population in the lung and lead to the formation of alveolitis and granuloma in the lung parenchyma of patients with sarcoidosis.

Animals↗
PubMed1994

Changes in manganese content of mononuclear blood cells in patients receiving total parenteral nutrition.

We measured the manganese content of mononuclear blood cells and the manganese concentrations in whole blood and plasma from 31 healthy volunteers and 25 patients receiving total parenteral nutrition (TPN) (i.e., no dietary manganese). The manganese content (mean +/- 2 SD) of mononuclear blood cells and the manganese concentration in whole blood and plasma in the healthy volunteers were 8.84 +/- 4.18 ng/10(8) cells, 11.21 +/- 4.68 micrograms/L, and 1.21 +/- 0.72 micrograms/L, respectively. The manganese content of mononuclear blood cells from patients receiving TPN gradually decreased, falling below the lowest limit of the normal range by 21 weeks (95% confidence limits, 13-30 weeks) after starting TPN. Manganese concentrations in whole blood and plasma were within normal limits during our study period. These results suggest that the manganese content of mononuclear blood cells may better indicate manganese nutritional status than whole-blood or plasma concentrations.

Adult↗
PubMed1993

Trimeric mutant bacteriorhodopsin, D85N, shows a monophasic CD spectrum.

The structure of mutant bacteriorhodopsin (bR), D85N, was examined by CD and X-ray diffraction at pH 7. The absorption maximum of D85N at pH 7 is located at 605 nm, which is similar to the acid-blue form of wild-type bR. D85N shows a monophasic CD band, the maximum of which is at 575 nm, although the crystalline arrangement and the trimeric structure is maintained. The acid-blue form of wild-type bR shows a biphasic CD despite the similarity in absorption spectra.

Bacteriorhodopsins↗
PubMed1993

Intermediate conformational states of apocytochrome c.

Horse apocytochrome c has been assumed to be a typical unfolded protein. At low ionic strength, the far- and near-UV circular dichroism spectra are typical of an unfolded protein at all pH values between 2 and 9. On the other hand, in the presence of high concentrations of salt, substantial secondary structure is present at both neutral and acidic pH. At low pH, perchlorate anion, either from salt or from acid, stabilizes an intermediate state (the A state) with secondary structure similar to that previously observed in the molten globule state of holocytochrome c. To further characterize the conformational states of apocytochrome c as a function of pH and salt, a fluorescence-labeled derivative was prepared, in which the two cysteine residues were labeled with N-(iodoacetyl)-N'-(5-sulfo-1- naphthyl)ethylenediamine(IAEDANS). The conformational transitions of the fluorescence-labeled apocytochrome c measured by circular dichroism were similar to those of unmodified apocytochrome c, indicating that the effects of the modification on the conformation and stability are small. Fluorescence energy transfer from tryptophan to the fluorescence label revealed several salt- and pH-dependent transitions. At very low ionic strength, apocytochrome c became compact as the pH was increased with a transition midpoint at pH 4.5. At acidic pH, increasing concentration of perchlorate induced a more compact state with a transition midpoint similar to that observed by circular dichroism. In contrast, at neutral pH, increasing perchlorate concentration had little effect on the compactness, as determined by a lack of change in the energy-transfer efficiency (but did increase the amount of secondary structure).(ABSTRACT TRUNCATED AT 250 WORDS)

Apoproteins↗
PubMed1993

Mutations can cause large changes in the conformation of a denatured protein.

Deletion of 13 amino acids from the carboxyl terminus of staphylococcal nuclease (WTSNase delta) results in a denatured, partially unfolded molecule that lacks significant persistent secondary structure but is relatively compact and monomeric under physiological conditions [Shortle & Meeker (1989) Biochemistry 28, 936-944; Flanagan et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 748-752]. Because of these and other properties of the SNase delta polypeptide, it is a useful model system for investigating the conformation of the denatured state of a protein without using extreme temperature or solvent conditions. Moreover, since the modification is a carboxyl-terminal deletion, SNase delta may also resemble a transient state of the polypeptide chain as it emerges from a ribosome prior to its folding. In the present study, we have examined the sizes and conformations of mutated forms of SNase delta, using small-angle X-ray scattering and circular dichroism spectroscopy. Seven mutated forms were studied: four with single substitutions, two with double substitutions, and one triple substitution. When present in the full-length SNase, each of these mutated forms exhibited unusual behavior upon solvent or thermal denaturation. In the case of the truncated form (SNase delta), the small-angle scattering curves of the mutated forms fall into two classes: one resembling the scattering curve of compact native nuclease and the other having features consistent with those expected for an expanded coil-like polymer. In contrast, the scattering curve of WT SNase delta exhibits features intermediate between those observed for globular proteins and random polymers. The amino acid substitutions that gave rise to compact, native-like versions of SNase delta were all of the m--type (m-substitutions are predicted to decrease the size of the denatured state). Those which gave rise to versions of SNase delta that were more extended and coil-like than WT SNase delta were of the m+ type (m+ substitutions are predicted to increase the size of the denatured state). Estimates of the residual secondary structure present in WT SNase delta, as well as both the m+ and m-substituted versions of SNase delta, as determined by CD, suggest that the formation of secondary structure and compaction of the polypeptide chain occur concurrently. Our results show that single amino acid substitutions can radically alter the conformational distribution of a partially condensed polypeptide chain.(ABSTRACT TRUNCATED AT 400 WORDS)

Algorithms↗