In vivo and in vitro transcription of small mRNAs containing a leader sequence from mouse hepatitis virus strain JHM.
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Animals↗
Biomedical subjects
A Maeda
Publications and source records attributed to A Maeda.
Relationship of proton release at the extracellular surface to deprotonation of the schiff base in the bacteriorhodopsin photocycle.
The surface potential of purple membranes and the release of protons during the bacteriorhodopsin photocycle have been studied with the covalently linked pH indicator dye, fluorescein. The titration of acidic lipids appears to cause the surface potential to be pH-dependent and causes other deviations from ideal behavior. If these anomalies are neglected, the appearance of protons can be followed by measuring the absorption change of fluorescein bound to various residues at the extracellular surface. Contrary to widely held assumption, the activation enthalpies of kinetic components, deuterium isotope effects in the time constants, and the consequences of the D85E, F208R, and D212N mutations demonstrate a lack of direct correlation between proton transfer from the buried retinal Schiff base to D85 and proton release at the surface. Depending on conditions and residue replacements, the proton release can occur at any time between the protonation of D85 and the recovery of the initial state. We conclude that once D85 is protonated the proton release at the extracellular protein surface is essentially independent of the chromophore reactions that follow. This finding is consistent with the recently suggested version of the alternating access mechanism of bacteriorhodopsin, in which the change of the accessibility of the Schiff base is to and away from D85 rather than to and away from the extracellular membrane surface.
Bacteriorhodopsins↗
Nanosecond time-resolved infrared spectroscopy distinguishes two K species in the bacteriorhodopsin photocycle.
The photochemical reaction process of bacteriorhodopsin in the nanosecond time range (-120-860 ns) was measured in the 1400-900 cm-1 region with an improved time resolved dispersive-type infrared spectrometer. The system is equipped with a newly developed detection unit whose instrumental response to a 5-ns laser pulse has a full width of the half-maximum of 60 ns. It provides highly accurate data that enabled us to extract a kinetic process one order of magnitude faster than the instrumental response. The spectral changes in the 1400-900 cm-1 region were analyzed by singular value decomposition and resolved into three components. These components were separated by fitting with 10- and 1000-ns exponential functions and a step function, which were convoluted with the instrumental response function. The components with decay time constants of 10 and 1000 ns are named K and KL, respectively, on the basis of previous visible spectroscopy. The spectral shapes of K and KL are distinguishable by their hydrogen-out-of-plane (HOOP) modes, at 958 and 984 cm-1, respectively. The former corresponds to the K intermediate recorded at 77 K and the latter to a K-like photoproduct at 135 K. On the basis of published data, these bands are assigned to the 15-HOOP mode, indicating that the K and KL differ in a twist around the C14-C15 bond.
Bacteriorhodopsins↗
Effect of alpha-tocopherol on in vitro and in vivo metabolism of low-density lipoproteins in haemodialysis patients.
It has been reported that some modified low-density lipoproteins (LDLs) such as glycated LDL and malondialdehyde-rich LDL (MDA-LDL) probably exist in the circulation. The present study was undertaken to investigate the in vitro and in vivo metabolism of MDA-LDL occurring in chronic haemodialysis patients and the effects of alpha-tocopherol on these abnormalities. MDA-LDL from haemodialysis patients was degraded more rapidly by human monocyte-derived macrophages and disappeared more slowly from the circulation when compared with LDL from healthy controls. Treatment with alpha-tocopherol at doses of 600 mg/day for 2 weeks resulted in improvement of these metabolic abnormalities depending upon the degree of return to normal MDA concentrations in LDL.
Humans↗
Vitronectin and integrin vitronectin receptor localization in multiple sclerosis lesions.
Vitronectin (Vn) is a multifunctional plasma and extracellular matrix glycoprotein involved in cell attachment, coagulation, phagocytosis, and the protection of bystander cells from complement- and T cell-mediated lysis. To determine where Vn is localized and where cells expressing integrin Vn receptors may recognize it in central nervous system (CNS) lesions of multiple sclerosis (MS), CNS tissue samples were immunostained for Vn and the alphav, beta 1, and beta 3 integrin Vn receptor subunits. By light and electron microscopy, Vn was localized within dystrophic, demyelinated axons in active but not chronic lesions, normal or other neurologic disease controls. This localization is distinct from that of other plasma proteins in MS lesions and it differs from the pattern of neuron cell body localization found in other conditions. Microvascular Vn was increased and small numbers of reactive astrocytes were also Vn-positive in active plaques. Endothelial cell expression of the alpha v subunit was increased over controls and that of the beta 1 subunit was decreased whereas both the alpha v and beta 1 subunits were prominently expressed on macrophages and glia in active lesions. The beta 3 integrin subunit was expressed on platelets within and around vessels and was more prominent on endothelial cells in active plaques. The precise functions of Vn in situ are not presently known. These results indicate, however, that the regulation of expression of integrin Vn receptors is complex and that Vn may be recognized and have multiple functions in different microanatomic sites as MS lesions evolve. Intravascular Vn could participate in clotting, thereby contributing to leukocyte extravasation.(ABSTRACT TRUNCATED AT 250 WORDS)
Acute Disease↗
Lattice thermal conductivity via homogeneous nonequilibrium molecular dynamics.
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Journal Article↗
Inhibition of mouse hepatitis virus multiplication by phosphorothioate analogues of oligodeoxynucleotides complementary to the leader RNA.
Phosphorothioate oligodeoxynucleotides (PS-oligo) complementary to a leader RNA of mouse hepatitis virus (MHV) were more effective inhibitors of MHV multiplication than natural oligodeoxynucleotides (PO-oligo). Sequence-dependent inhibition of viral multiplication was shown at low concentrations (0.001-0.1 microM) of antisense PS-oligo. Phosphorothioate oligodeoxycytidine, PS-(dC)20 and PS-oligo, which has no significant homology to the MHV sequence, showed inhibitory effects on MHV multiplication at concentrations higher than 0.5 microM. These results showed that PS-oligo was more potent than PO-oligo in inhibition of MHV multiplication and that PS-oligo may inhibit MHV multiplication by two different mechanisms, that is, in sequence-dependent and -independent manners.
Animals↗
Inhibition of viral multiplication in cells chronically infected with mouse hepatitis virus by antisense RNA against the polymerase gene.
Recently, we showed that the antisense RNA containing a hammerhead ribozyme sequence against the polymerase gene of mouse hepatitis virus (MHV) inhibited viral multiplication in acute infection [10]. In the present study, we examined the inhibitory effects of an antisense RNA on viral multiplication in chronic MHV infection. In cell line LR-2, in which the 926-nucleotide (nt) antisense RNA containing a ribozyme sequence against the polymerase gene was expressed constitutively at a high level, chronic MHV infection was established through the maintenance of infection over 100 days postinfection (d.p.i.). After 200 d.p.i., no infectious progeny virus was observed in the culture medium of chronically MHV infected LR-2 cells. Our present results showed that the anitsense RNA could also inhibit viral multiplication in chronic MHV infection.
Animals↗
Characterization of DBT cell clones derived from cells persistently infected with the JHM strain of mouse hepatitis virus.
Twelve clones derived from the cells persistently infected with the JHM strain (JHMV) of mouse hepatitis virus (MHV) were established from mouse astrocytoma-derived DBT cells and characterized. All the cell clones were resistant to superinfection with MHV. Only one of the persistently infected cell clone synthesized viral RNA and proteins and produced virus particles. Viral RNA was detectable in some other cell clones without production of viral protein nor the virus. No cell clones exhibited contact fusion activity. The results suggested that such variety of cell clones might have resulted from persistent infection with JHMV.
Animals↗
[Oil mist exposure in industrial health--a review].
Many reports dealing with the toxicity of oil mist in industrial health have been published. The condition appears to be worldwide in distribution and the number of reported cases increases with increasing clinical awareness. In 158 reports published from 1965 to 1993 the following diseases were observed: Skin--contact dermatitis, oil acne and photosensitive allergic dermatitis; Scrotum--benign and malignant tumors; Respiratory system-nasal discomfort symptoms, rhinitis, nasal mucosal dysplasia, nasal mucosal tumor, laryngeal cancer, bronchitis, lipoid pneumonia, lung fibrosis, lung cancer and bronchial asthma; Others--possible carcinogenicity, high incidence of chromosomal change. This shows that oil mist appears to be involved in many industrial diseases, however, cause-and-effect relationship still remains a matter of conjecture; in which exposure dose and/or duration-dependent toxicity is highly probable. Further investigations will be required including immunotoxicological as well as environmental studies for oil mist exposure.
Dermatitis, Contact↗
[Complete remission in acute myeloblastic leukemia (M0) after treatment with rhG-CSF].
A 57-year-old man was admitted because of fever and night sweat. The bone marrow was hypercellular with 86.4% blast cells. The diagnosis of AML (M0) was made, because the blast cells were negative for peroxidase stain and had CD13 and no lymphoid antigens in marker analysis. The patient was treated with BH-AC.TMP, BH-AC.MVP and low dose Ara-C without any hematological improvement, and even additional treatment with medium dose Ara-C resulted in 66.4% blast cells in the bone marrow. Subsequent administration of rhG-CSF (150 micrograms/day) by continuous intravenous infusion resulted in the decrease of the blast cells in the bone marrow to a level that was evaluated as complete remission. He remains in complete hematological remission at present. As shown in this case, rhG-CSF might be an effective agent for the treatment of AML, even if the mechanism of its effectiveness is unclear at present. Further clinical studies should will supply useful information to analyze the pathophysiology of AML.
Granulocyte Colony-Stimulating Factor↗
Conservative treatment of isolated injuries to the posterior cruciate ligament in athletes.
We assessed arthroscopically 22 young athletes with an isolated acute posterior cruciate ligament (PCL) injury. Four had significant damage to the articular cartilage of the medial femorotibial compartment and were advised not to resume sports. Three underwent PCL reconstruction because of a reparable meniscal tear or instability. The other 15 were treated conservatively and resumed sport. At an average follow-up of 51 months, one had developed arthritic symptoms due to newly-developed severe chondral damage to the medial femoral condyle, but none of the other 14 had developed arthritic symptoms and most remained athletically active. Severe chondral damage should be seen at an early arthroscopy. Knees with an isolated injury to the PCL with concomitant articular damage may be successfully managed by conservative treatment.
Activities of Daily Living↗
Second-look arthroscopy after meniscal repair. Review of 132 menisci repaired by an arthroscopic inside-out technique.
From 1986 to 1993, we repaired 278 torn menisci in 264 patients using an arthroscopically assisted inside-out technique. A total of 132 meniscal repairs in 122 patients were evaluated by second-look arthroscopy. At review, only nine patients had meniscal symptoms, such as locking, swelling or pain. Ninety-seven menisci (73%) had healed completely at the repair site, but there were new tears in different areas of 21 menisci, some of which had complete healing at the repair site. Incomplete healing, seen in 23 menisci (17%), was frequently near the popliteus tendon, most commonly where there had been an associated anterior-cruciate-ligament injury. Arthroscopically-assisted meniscal repair seems to be a reliable procedure, but some clinically successful cases had incomplete healing at the repair site or a newly-formed tear in the meniscal body or both. These lesions may cause meniscal symptoms to appear at a later date.
Adolescent↗
[Pulmonary manifestation of collagen vascular diseases: role of cytokines in interstitial pneumonia associated with collagen vascular diseases].
Although the pathological patterns of interstitial pneumonia associated with collagen vascular disease (CVD-IP) resemble those of usual interstitial pneumonia in idiopathic interstitial pneumonia (IIP), the clinical features of CVD-IP and IIIP are quite different. We evaluated the differences between these conditions, with regard to the expression of genes in cells obtained by bronchoalveolar lavage. The reverse transcription-polymerase chain reaction was used to measure the levels of mRNA for IL-1 beta, TNF-alpha, IL-8, TGF-beta, PDGF-B, and IGF-1, and no significant differences were found between patients with CVD-IP and those with IIP. However, differential display analysis revealed a fragment that can be considered to have been derived from an unknown gene mRNA, and this was found only in patients with pulmonary fibrosis associated with progressive systemic sclerosis. Expression of specific genes may differentiate CVD-IP from IIP.
Adult↗
[Cardiovascular alpha-, beta 1- and beta 2-adrenoceptor functions in neurogenic orthostatic hypotension].
It has been believed that patients with neurogenic orthostatic hypotension (NOH) usually develop denervation supersensitivity of cardiovascular alpha-, beta 1- and beta 2-adrenoceptors, because the majority studies have shown augmented cardiovascular responses to intravenously given noradrenaline (NA) or isoprenaline (IP) in these patients. This view, however, leaves room for discussion. First, drugs were administered by means of drip infusion method in most of the previous studies. Since this method necessarily provokes baroreflex, which is buffering fluctuations in blood pressure and heart rate, the results may reflect diminished baroreflex rather than denervation supersensitivity. Second, adrenoceptor functions are modulated by multiple factors, such as ageing, thyroid hormone and chronic inflammation. It is, thus, inappropriate to explain the whole picture of functional change in adrenoceptors by the denervation mechanism only. In order to look over the cardiovascular adrenoceptor functions in NOH, we performed bolus infusion tests of NA and IP on the patients. The subjects comprised 7 cases of NOH with pre-ganglionic sympathetic deficit (NOH-I), 6 cases with post-ganglionic deficit (NOH-II) and 15 healthy controls. NOH-I group included 6 patients with multiple system atrophy and one with Parkinson disease, while NOH-II group consisted of 3 patients with idiopathic orthostatic hypotension (pure autonomic failure) and 3 with diabetic autonomic neuropathy. Both NA and IP infusion tests were carried out under the continuous measurement of blood pressure and heart rate. In NA test, different bolus doses (0.01, 0.02, 0.05 and 0.1 microgram/kg) of NA were intravenously administered, and a degree of subsequent rise in mean blood pressure was used as an index for alpha-adrenoceptor function. IP test was performed in the same manner, and an increase in heart rate and a fall in mean blood pressure in response to the drug (0.001, 0.002 and 0.005 microgram/kg) were measured as indices for beta 1- and beta 2-functions, respectively. A rise in blood pressure following the administration of any dose of NA did not statistically differ among three groups. An increase in heart rate in IP test was generally lower in both NOH groups, and a significant difference was obtained between NOH-I and control when a given dose was 0.005 microgram/kg) (p < 0.02). A fall in blood pressure in IP test was significantly greater in NOH-I compared to control when doses were 0.002 and 0.005 microgram/kg (p < 0.02 and 0.01, respectively). It was also greater in NOH-II than in control when a dose was 0.002 microgram/kg (p < 0.01). In disagreement with most of the previous studies, the present results suggest that alpha-adrenoceptor function is hardly altered, beta 1-function is suppressed, and beta 2-function is augmented in NOH.
Adult↗
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↗
The retinal Schiff base-counterion complex of bacteriorhodopsin: changed geometry during the photocycle is a cause of proton transfer to aspartate 85.
Bacteriorhodopsin contains all-trans-retinal linked via a protonated Schiff base to K216. The proton transport in this pump is initiated by all-trans to 13-cis photoisomerization of the retinal and the ensuing transfer of the Schiff base proton to D85. Changed geometrical relationship of the Schiff base and D85 after the photoisomerization is a possible reason for the proton transfer. We introduced small volume/shape changes with site-specific mutagenesis of residues V49 and A53 that contact the side chain of K216, in order to force the Schiff base into somewhat different positions relative to D85. Earlier [Zimányi, L., Váró, G., Chang, M., Ni, B., Needleman, R., & Lanyi, J. K. (1992) Biochemistry 31, 8535-8543] we had described the kinetics of absorbance changes in the microsecond to millisecond time range after photoexcitation with the scheme L<-->M1<-->M2 + H+ (where the first equilibrium is the internal proton transfer and the second is proton release on the extracellular surface). Testing it at various pH values with mutants, where selected rate constants are changed, now confirms the validity of this scheme. The kinetics of the M state thus allowed examination of the transient equilibrium that develops in the L<-->M1 reaction and represents the redistribution of the proton between the Schiff base and D85. From the structure of the protein, the V49A and V49M residue replacements were both predicted to cause decreased alignment of the Schiff base and D85, and indeed we found that they both changed the equilibrium toward the protonated Schiff base. In contrast, the residue replacements A53V and A53G were predicted to move the Schiff base in opposite directions, away from and closer to alignment with D85, respectively. The former indeed changed the equilibrium toward the protonated Schiff base and the latter toward the deprotonated Schiff base. In addition, the hydroxyl stretch band of a bound water in the L state was affected by all mutations that disfavor proton transfer to D85. We conclude that the geometry of the proton donor and acceptor in the Schiff base-D85 pair, mediated by bound water, is a determinant of the proton transfer equilibrium.
Aspartic Acid↗
Molecular cloning and characterization of yeast rho GDP dissociation inhibitor.
We have previously isolated rho GDP dissociation inhibitor (rho DGI) from bovine brain and characterized it. Bovine rho GDI is a protein of a M(r) of 23,421 with 204 amino acids. rho GDI inhibits the GDP/GTP exchange reaction of post-translationally lipid-modified small GTP-binding proteins (G proteins) of the rho family, including the rho, rac, and cdc42 subfamilies, and keeps them in the GDP-bound inactive form. In the present study, we first purified rho GDI from the cytosol fraction of the yeast Saccharomyces cerevisiae and isolated its gene. Yeast rho GDI gene had an open reading frame without introns encoding a protein of a M(r) of 23,138 with 202 amino acids. Yeast rho GDI protein was 36% identical with bovine rho GDI. Yeast rho GDI expressed in Escherichia coli was active not only on yeast rho1 but also on mammalian rho family members which were post-translationally modified. Disruption of rho GDI did not induce apparent phenotypes, whereas overexpression of yeast or bovine rho GDI resulted in the inhibition of cell growth. These results indicate that rho GDI exists and regulates the function of the rho family members in yeast.
Amino Acid Sequence↗