The enzyme CoA transferase from porcine heart (EC 2.8.3.5) is a homodimer; each subunit consists of two domains linked by a hydrophilic "hinge" region. We have prepared separate DNA segments encoding each of these domains. Incorporation of these two DNA segments within an operon or within two separate transcription units does not preclude the synthesis and assembly of CoA transferase in Escherichia coli. When the two domain fragments are produced and purified individually from separate cultures and subsequently mixed, enzyme activity accumulates to near wild-type levels only after a lengthy incubation. Each domain is more susceptible to aggregation than wild-type CoA transferase. Circular dichroism shows that, prior to mixing, the domains possess a different secondary structural profile compared to their counterparts in the native enzyme. However, mixing and incubation of the domains produces a complex with far-UV CD, fluorescence, and ultracentrifugation properties similar to those of wild-type CoA transferase. Finally, we show that the intact hydrophilic peptide which links the two domains is essential for the recovery of activity observed upon refolding of the denatured enzyme in vitro. These results indicate that the folding and assembly of pig heart CoA transferase require a productive interaction between its two domains, involving a substantial conformational rearrangement.

Animals↗

PubMed1997

Long-term fasting of donors deteriorates mitochondrial adenosine triphosphate synthesis in liver grafts during cold preservation.

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Adenosine Triphosphatases↗

PubMed1996

Fluorescence studies of lipid association-induced conformational adaptations of an exchangeable amphipathic apolipoprotein.

The conformational adaptability of Manduca sexta apolipophorin III (apoLp-III) has been evaluated by monitoring the spectroscopic properties of its sole tyrosine residue, Tyr145, present in the fifth helical segment of the protein. M. sexta apoLp-III adopts a globular five-helix bundle structure in solution and has been postulated to undergo an opening at putative hinge domains upon interaction with lipid surfaces. Previous results have shown that the intrinsic fluorescence of Tyr145 is highly quenched in the closed, water-soluble conformation but is dramatically enhanced upon lipid association. We have carried out a spectroscopic characterization of Tyr145 and its microenvironment, to enable its use as a structural probe of lipid-induced conformational changes of apoLp-III. The pKa of Tyr145 in lipid-free apoLp-III was found to be 10.5, as determined from uv-spectrophotometry, indicating that, in the ground state, the tyrosyl phenolic group is not ionized under physiological conditions. Compared to free tyrosine in aqueous buffer (pH 7.0), a red shift (77 nm) in the (lambda)max of absorbance of Tyr145 was observed, suggesting that an H-bonding interaction is responsible for the quenched state of tyrosine fluorescence. In an effort to explain the observed quenching phenomenon, the quantum yield and lifetimes of Tyr145 fluorescence emission were investigated as a function of pH and lipid binding. The quantum yield of Tyr145 in lipid-free apoLp-III was enhanced fivefold upon decreasing the pH, with a half-maximal point around pH 5.5. Time-resolved fluorescence decay analysis showed that Tyr145 exhibits nonexponential emission decay with two components having lifetimes of 3.3 ns (76%) and 0.89 ns (24%) in the lipid-free state. The lifetime and amplitude of Tyr145 remain essentially unaltered upon lipid association or decreasing the pH. This is consistent with the hypothesis that, in the lipid-free helix bundle conformation, a quenching residue exists within H-bonding distance of the phenolic side chain of Tyr145 which, at physiological pH, is responsible for the observed fluorescence quenching. Opening of the helix bundle repositions this acceptor base, possibly a carboxylate or an imidazole side chain, making it unavailable for quenching. Using differential polarized phase and modulation fluorometry, it was seen that the segmental motion of Tyr145 is also altered considerably upon lipid interaction. These spectroscopic and motional properties of Tyr145 distinguish this unique residue as a useful probe to monitor structural flexibility of apoLp-III.

Animals↗

PubMed1996

Nuclear encoding of a chloroplast RNA polymerase sigma subunit in a red alga.

A chloroplast RNA polymerase sigma factor is encoded by a nuclear gene, sigA, in the red alga Cyanidium caldarium RK-1. The encoded protein functions as an RNA polymerase sigma factor in vitro and it is localized to the chloroplast in vivo. SigA shows high sequence similarity to the sigma factors of cyanobacteria, which is indicative of the ancestral endosymbiotic event and subsequent transfer of the sigA gene to the nuclear genome.

Amino Acid Sequence↗

PubMed1996

In vivo EPR detection and imaging of endogenous nitric oxide in lipopolysaccharide-treated mice.

Nitric oxide (NO), a simple diatomic free radical, is known to play a critical physiological role in diverse organisms. An iron complex, with N-(dithiocarboxy)sarcosine (Fe-DTCS), has a high affinity for endogenous NO and can trap, stabilize, and accumulate it. The stable NO adduct thus formed is detectable at room temperature with electron paramagnetic resonance (EPR) spectrometry. We report in vivo EPR imaging of endogenous NO, trapped by an Fe-DTCS complex, in the abdomen of a live mouse. To our knowledge, this is the first report on EPR imaging of endogenous free radicals produced in vivo. This EPR imaging method will be useful for the noninvasive investigation of the spatial distribution of NO in pathologic organs or tissues.

Animals↗

PubMed1996

The consequences of engineering an extra disulfide bond in the Penicillium camembertii mono- and diglyceride specific lipase.

The extracellular lipase from Penicillium camembertii has unique substrate specificity restricted to mono- and diglycerides. The enzyme is a member of a homologous family of lipases from filamentous fungi. Four of these proteins, from the fungi Rhizomucor miehei, Humicola lanuginosa, Rhizopus delemar and P. camembertii, have had their structures elucidated by X-ray crystallography. In spite of pronounced sequence similarities the enzymes exhibit significant differences. For example, the thermostability of the P. camembertii lipase is considerably lower than that of the H. lanuginosa enzyme. Since only the P. camembertii enzyme lacks the characteristic long disulfide bridge, corresponding to Cys22-Cys268 in the H. lanuginosa lipase, we have engineered this disulfide into the former enzyme in the hope of obtaining a significantly more stable fold. The properties of the double mutant (Y22C and G269C) were assessed by a variety of biophysical techniques. The extra disulfide link was found to increase the melting temperature of the protein from 51 to 63 degrees C. However, no difference is observed under reducing conditions, indicating an intrinsic instability of the new disulfide. The optimal temperature for catalytic activity decreased by 10 degrees C and the optimum pH was shifted by 0.7 units to more acidic.

Base Sequence↗

PubMed1996

Leakage of superoxide radicals from mitochondrial electron transport system after cold ischemia in liver grafts.

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Animals↗

PubMed1996

Usefulness of the endotoxin test for assessing CAPD peritonitis by gram-negative organisms.

The endotoxin (ET) in cloudy peritoneal fluid was measured, and the usefulness of the method was examined. Thirteen continuous ambulatory peritoneal dialysis (CAPD) patients with an episode of peritonitis from December, 1993 to December, 1995 were studied. The peritoneal fluid was collected without contamination from the bags and divided into two batches. The ET was measured in one of them with the limulus amebocyte lysate (LAL) assay, and the other was cultured for bacteria. Among the 13 patients ET was positive in 7 Gram-negative bacteria were identified in all 7 cases. Two of these patients were transferred to hemodialysis. On the other hand, gram-positive bacteria were cultured in only four episodes of the 6 ET-negative patients, all of which were treated successfully with vancomycin. Microbiological culture of the dialysate is still necessary for the treatment of peritonitis. But it takes 24-48 hours to obtain microbiological results. The result of the ET test can be given within one and one half hours (and it has 100% sensitivity and specificity for gram-negative peritonitis) at presentation in the episodes. Therefore, the ET test of the dialysate is very effective for the therapy of peritonitis in CAPD.

Adult↗

PubMed1996

[Assistance to enhance the use of home helper services for the cancer patient].

For the end-stage cancer patient to live at home with his or her quality of life (QOL) sustained, Home Helper Services are considered effective. In fact, however, many do not avail themselves of such services. Since the disease situation is apt to change dramatically, it is difficult to predict the length of time of care at home, and the patient's family does not tend to utilize such services unless the patient is completely bedridden. Short-term use or the difficulty or rapid response of such services has been among the reasons given of this. Given this background, there appears to be a need for suitable assessment in dealing quickly with possible disabilities which might occur and a number of occupations. Also, when considering home medical care for the cancer patient, an administrative system capable of a rapid response when problems of this kind occur is indispensable. Working on the administration to fulfill this need is also the task of the professionals in this field.

Adolescent↗

PubMed1996

[Psychosocial support to improve quality of life (QOL) in home terminal carcinoma patients].

There are numerous psychosocial problems with home care for terminal cancer patients. We studied one case from the viewpoint of providing support for cancer patients and their families. Psychosocial problems are important for the life of cancer patients and their families. The cancer diagnosis and the terminal stage have different levels of psychosocial concerns. We must consider effective intervention with such psychosocial support. (1) The team delivery of health care in oncology is more crucial than ever. (2) Social work plays a key role. (3) We should strive to understand the roles of other members of teams. (4) Members of a support team should take turns in providing care according to the psychosocial conditions of the patients. (5) Sympathy and a broad social outlook are required of medical staff members.

Adult↗

PubMed1995

Free radical imaging by electron spin resonance computed tomography in rat brain.

Images of nitroxide radicals were obtained from the brains of living rats following intracarotid injection of imaging agent, [2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinylox y] (16 DS) using L-band electron spin resonance computed tomography (ESR-CT). The image patterns obtained from the 3-carbamoyl-2,2,5,5-tetramethyl-1-pyrrolinyloxy injected rats showed the agent in tissues of the cranium but not within the brain. The uptake of 16 DS was found in the cortex, hippocampus, striatum, midbrain, pons medulla oblongata and cerebellum, and nuclei, mitochondria, synaptosomes of the cerebrum after intracarotid injection of 16 DS using an x-band ESR spectrometer. These results suggest that 16 DS penetrates the blood brain barrier.

Animals↗

PubMed1995

Zn2+ binding to cardiac calsequestrin.

Zn2+ binding to canine cardiac calsequestrin was investigated using the Zn2+ specific fluorescence dye salicylcarbohydrazone (SACH), 65Zn2+ overlay and Zn(2+)-IDA chromatography. Cardiac calsequestrin binds approximately 200 moles of Zn2+/mole of protein with the Kd = 300 microM. Zn2+ binding to calsequestrin was further confirmed by 65Zn2+ overlay and Zn(2+)-dependent aggregation of the protein. However, calsequestrin did not bind to a Zn(2+)-IDA-agarose column, indicating that histidine residues may not be involved in Zn2+ binding to the protein. Circular dichroism revealed only minor Zn(2+)-dependent conformational changes in calsequestrin. We conclude that calsequestrin is a Ca(2+)- and Zn(2+)-binding protein and that Zn2+ may modulate the structure and function of the protein.

Animals↗

PubMed1995

Modulation of substrate selectivity in plasma lipid transfer protein reaction over structural variation of lipid particle.

The modulation of substrate selectivity of human plasma LTP reaction is the subject of the present investigation. The moderate selectivity by a factor of 5 to 6 was observed in the LTP-catalyzed transfer of cholesteryl ester over triacylglycerol between plasma lipoproteins. On the other hand, the transfer of cholesteryl ester by LTP was highly selective over the negligible transfer of triacylglycerol, by a factor of 60 to 500, between the microemulsions with LDL size, regardless of the activators such as human and pig apolipoprotein (apo) A-I, human apo C-III and apo E that bound to the surface of the emulsion in equilibrium. The presence of free cholesterol in these microemulsions reduced slightly the rate of cholesteryl ester transfer but had no effect on triacylglycerol transfer. Other surface-active reagents such as cholic acid, Triton X-100 and Tween-20, did not have an effect on the triacylglycerol transfer either. Triacylglycerol transfer by LTP became measurable between such lipid particles as prepared by co-sonication of lipid with pig apo A-I and isolated as the mixed-microemulsions in the density of LDL and HDL. In these conditions, the substrate selectivity for cholesteryl ester over triacylglycerol was a factor of 6 to 16 mimicking the ratio in plasma lipoproteins. The conformation of pig apo A-I estimated by circular dichroism showed that its apparent helical content was further more induced when apo A-I was integrated into the mixed-microemulsion by co-sonication than the lipid-bound apo A-I in equilibrium. Apo A-I, thus integrated into lipid particles, was highly resistant to the denaturation by guanidine hydrochloride while the lipid-bound apo A-I in equilibrium was denatured as readily as the lipid-free protein. Thus, triacylglycerol transfer by LTP was induced by structural modulation of substrate-carrying lipid particles such as higher integration of apolipoproteins.

Animals↗

PubMed1995

Hepatitis E probably contracted via a Chinese herbal medicine, demonstrated by nucleotide sequencing.

Hepatitis E is endemic in developing countries and may occur as imported hepatitis in industrialized countries. A 46-year-old Japanese man developed immunoserologically diagnosed acute hepatitis E in Japan 4 months after he had made a trip to China. He had bought a Chinese herbal medicine there, taking it occasionally until approximately 6 weeks prior to the onset of acute hepatitis. Nucleotide sequencing of the 3' terminal region of the viral cDNA amplified from the patient's serum by polymerase chain reaction revealed a high degree of homology (99.8% of 752 nucleotides) with the Chinese strain. Thus, the results of sequencing suggest that his hepatitis E was caused by infection with the Chinese strain, via the Chinese herbal medicine.

Acute Disease↗