Disseminated toxoplasmosis with atypical symptoms which developed with exacerbation of systemic lupus erythematosus.

Toxoplasma is a common parasite worldwide that mainly affects the brain, lungs and eyes. Although toxoplasmic encephalitis is a lethal disease without treatment, past case reports show most patients with systemic lupus erythematosus who developed toxoplasmic encephalitis were misdiagnosed and treated as neuropsychiatric systemic lupus erythematosus, which led to unfavorable outcomes. We herein describe a case of disseminated toxoplasmosis affecting all the above organs with atypical symptoms, which developed with exacerbation of systemic lupus erythematosus. She had initially manifested with retinochoroiditis without vitritis, mild cognitive impairment and an isolated lung mass. These are completely different from the classic symptoms of toxoplasmosis that have been reported in patients with HIV infection and/or those after hematopoietic transplantation. Our case, together with previously reported cases, suggests the manifestation of toxoplasmosis that develops in systemic lupus erythematosus patients can be different from that seen in conventional cases and varies between individual patients. Our case highlights both the difficulty in and the importance of diagnosing toxoplasmosis in patients with systemic lupus erythematosus and provides helpful information to identify this rare, devastating, yet treatable disease.

Development of mixed connective tissue disease and Sjögren's syndrome in a patient with trisomy X.

Increased risk of developing systemic lupus erythematosus (SLE) has been reported in patients with Klinefelter syndrome. Here, we describe a 16-year-old Japanese patient with trisomy X (47,XXX) who developed mixed connective tissue disease (MCTD) and Sjögren's syndrome. She had polyarthritis, edematous fingers with Raynaud's phenomenon, sicca syndrome, interstitial lung disease, possible myositis, and was positive for anti-nuclear antibody, anti-nRNP antibody and rheumatoid factor. This is the first report in the literature of a case of MCTD with female polysomy X, which further supports the link between the presence of extra X chromosome(s) and the development of autoimmune diseases.

Brca1 heterozygous mice have shortened life span and are prone to ovarian tumorigenesis with haploinsufficiency upon ionizing irradiation.

BRCA1 mutation carriers have an 85% lifetime risk of breast cancer and 60% for ovarian cancer. BRCA1 facilitates DNA double-strand break repair, and dysfunction of BRCA1 leads to hypersensitivity to DNA damaging agents and consequently genomic instability of cells. In this communication, we have examined the tumor incidence and survival of Brca1 heterozygous female mice. Brca1 heterozygotes appear to have a shortened life span with 70% tumor incidence. Lymphoma, but not ovarian and mammary gland tumors, occurs commonly in these mice. After a whole-body exposure to ionizing radiation, Brca1 heterozygous mice have a 3-5-fold higher incidence specific to ovarian tumors, but not lymphoma, when compared with the Brca1+/+ mice. All the tumors from heterozygous mice examined retain the wild-type allele and the cancer cells express Brca1 protein, precluding the chromosomal mechanism for loss of heterozygosity of Brca1 locus. Although the manifestation of BRCA1 haploinsufficiency may be different between human and mouse, this study suggests that women carrying Brca1 mutations may be more prone to ovarian tumor formation after IR exposure than nonmutation carriers.

Inhibition of NADPH oxidase 4 activates apoptosis via the AKT/apoptosis signal-regulating kinase 1 pathway in pancreatic cancer PANC-1 cells.

Pancreatic adenocarcinoma is an aggressive human malignancy and is characterized by resistance to apoptosis. Recently, NADPH oxidase (Nox) 4-mediated generation of intracellular reactive oxygen species (ROS) was proposed to confer antiapoptotic activity and thus a growth advantage to pancreatic cancer cells. The signaling mechanism by which Nox4 transmits cell survival signals remains unclear. Here, we show that both a flavoprotein inhibitor, diphenylene iodonium (DPI), and small interfering RNAs designed to target Nox4 mRNA (siNox4RNAs) inhibited superoxide production in PANC-1 pancreatic cancer cells, and depletion of ROS by DPI or siNox4RNAs induced apoptosis. Parallely, DPI treatment and siNox4RNA transfection blocked activation of the cell survival kinase AKT by attenuating phosphorylation of AKT. Furthermore, AKT phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) on Ser-83 was reduced by DPI and siNox4RNAs. When ASK1Ser83Ala (an AKT phosphorylation-defective ASK1 mutant) was introduced into PANC-1 cells, this mutant alone induced apoptosis. But, addition of DPI or co-transfection of siNox4RNA had no additive effect, indicating that the mutant can substitute for these reagents in apoptosis induction. Taken together, these findings suggest that ROS generated by Nox4, at least in part, transmit cell survival signals through the AKT-ASK1 pathway in pancreatic cancer cells and their depletion leads to apoptosis.

Heterogeneity of beta-type myosin isozymes in the human heart and regulational mechanisms in their expression. Immunohistochemical study using monoclonal antibodies.

To investigate the existence of heterogeneity of beta-type myosin isozymes (HC beta) in human hearts, immunohistochemical studies using monoclonal antibodies (MoAbs) raised against human ventricular myosin heavy chains were performed. Two types of MoAbs recognized some muscle fibers in the atrium, whereas both reacted with all ventricular muscle fibers. Since atrial muscle fibers reactive with each MoAb were found to be clearly different, the existence of two immunologically distinct HC beta (beta 1, and beta 2) was suggested in the atrium. By using affinity chromatography, two molecular variants of HC beta were isolated from the bovine atrium, and differences in the primary structure of beta 1 and beta 2 were confirmed by analysis of peptides produced by chymotryptic digestion. In pressure-overloaded human atria, myofibers containing beta 1 and/or beta 2 increased in accordance with decrement of myofibers containing alpha-type myosin isozyme (P less than 0.01). But they differed in expression during the developmental stage, since beta 2 did not exist in the early embryonic bovine heart, but beta 1 did. Thus, there are two distinct HC beta whose expression is regulated by at least two factors: pressure overload and developmental stage.

Isozymic changes in myosin of human atrial myocardium induced by overload. Immunohistochemical study using monoclonal antibodies.

An immunohistochemical study using monoclonal antibodies specific for the heavy chains of either human atrial (HC alpha) or ventricular (HC beta) myosin was performed to clarify the distribution of each isozyme in normal as well as pressure-overloaded human hearts. In normal human ventricles, all muscle fibers were stained by a monoclonal antibody (HMC14) specific for HC beta, whereas a small number of fibers reacted with a monoclonal antibody (CMA19) specific for HC alpha. In contrast, in normal human atria, almost all muscle fibers were stained by CMA19, and a relatively larger number of muscle fibers also reacted with HMC14. Furthermore, in pressure-overloaded atria, muscle fibers reactive with HMC14 were strikingly increased while those reactive with CMA19 showed a corresponding decrease. The extent of this isozymic redistribution was in good correlation with atrial pressure. These results not only confirmed the existence of isoforms of myosin heavy chain in human hearts, but also demonstrated that redistribution of iso-myosins could occur as an adaptation to pressure overload.

Identification and characterization of a 500-kb homozygously deleted region at 1p36.2-p36.3 in a neuroblastoma cell line.

Loss of heterozygosity of the distal region of chromosome 1p where tumor suppressor gene(s) might harbor is frequently observed in many human cancers including neuroblastoma (NBL) with MYCN amplification and poor prognosis. We have identified for the first time a homozygously deleted region at the marker D1S244 within the smallest region of overlap at 1p36.2-p36.3 in two NBL cell lines, NB-1 and NB-C201 (MASS-NB-SCH1), although our genotyping has suggested the possibility that both lines are derived from the same origin. The 800-kb PAC contig covering the entire region of homozygous deletion was made and partially sequenced (about 60%). The estimated length of the deleted region was 500 kb. We have, thus far, identified six genes within the region which include three known genes (DFF45, PGD, and CORT) as well as three other genes which have been reported during processing our present project for the last 3(1/2) years (HDNB1/UFD2, KIAA0591F/KIF1B-beta, and PEX14). They include the genes related to apoptosis, glucose metabolism, ubiquitin-proteasome pathway, a neuronal microtubule-associated motor molecule and biogenesis of peroxisome. At least three genes (HDNB1/UFD2, KIAA0591F/KIF1B-beta, and PEX14) were differentially expressed at high levels in favorable and at low levels in unfavorable subsets of primary neuroblastoma. Since the 1p distal region is reported to be imprinted, those differentially expressed genes could be the new members of the candidate NBL suppressor, although RT-PCR-SSCP analysis has demonstrated infrequent mutation of the genes so far identified. Full-sequencing and gene prediction for the region of homozygous deletion would elucidate more detailed structure of this region and might lead to discovery of additional candidate genes. Oncogene (2000) 19, 4302 - 4307

Dietary vitamin A modulates lecithin-retinol acyltransferase activity in developing chick intestine.

Retinol absorbed and generated from beta-carotene requires to be esterified by lecithin-retinol acyltransferase (LRAT) in intestinal absorptive cells. To characterize developmental changes in retinol absorptive capability in intestine, we determined LRAT activity and the amount of its retinol donor, cellular retinol-binding protein, type two (CRBP(II)) in the duodenum of developing chicks. The LRAT activity in duodenal microsomes was very low at 18- and 20-day chick embryo, but exhibited a rapid (15-fold) increase during 48 h around hatching, which occurred in parallel with the abrupt elevation of the content of CRBP(II) in chick duodenum. To examine whether dietary vitamin A affects the developmental change in LRAT activity and CRBP(II) content, 1-day-old chicks were pair-fed vitamin A-depleted or vitamin A-supplemented diet for 14 days. The chicks fed vitamin A-depleted diet showed significantly reduced LRAT activity and CRBP(II) in duodenum as early as 3 days after the start of the vitamin A-depleted diet. Changing the diet from vitamin A-depleted to vitamin A-supplemented diet led to an increase in duodenal LRAT activity within 24 h, while serum retinol concentration remained unchanged. These results suggest that duodenal LRAT activity and CRBP(II) are modulated by dietary vitamin A during the perinatal period.

Effects of metal chelating agents on the oxidation of lipids induced by copper and iron.

The non-enzymatic oxidations of soybean phosphatidylcholine liposomes, methyl linoleate micelles and low-density lipoprotein in aqueous dispersions induced by copper and iron have been studied aiming specifically at elucidating the action of the metal chelating agents such as ethylenediaminetetraacetic acid disodium salt (EDTA), nitrilotriacetic acid (NTA), adenosine-5'-diphosphate disodium salt (ADP), desferrioxamine (DFO), penicillamine (PCM), and triethylene tetramine (TTM). The effects of chelators on chemiluminescence emitted from its probe luminol in the decomposition of tert-butyl hydroperoxide by metal ion were also studied. The effects of chelators on the oxidations depended both on the metal ion and the substrate. Namely, in the oxidations of both liposomes and micelles, EDTA and NTA suppressed the copper-induced oxidations, whereas they enhanced the oxidations induced by iron. ADP had little effect, while PCM and TTM had accelerating effect for both metal ions. On the other hand, in the oxidation of LDL, none of these chelators enhanced the oxidation. Especially, TTM and PCM suppressed the copper-induced oxidation of LDL, suggesting that the chelating agents blocked the access of the metal ion to the hydroperoxide within LDL. The effects of chelators on chemiluminescence emission were similar to those on the oxidations of liposomes and micelles. The cyclic voltammograms of metal complexes were also measured. The multiple effects of chelators on the rate of non-enzymatic, metal-catalyzed oxidations of lipids were interpreted by their influence on redox-potential and accessibility to hydroperoxide of the metal-chelator complex.

Reduction of beta-oxidation capacity of rat liver mitochondria by feeding orotic acid.

Rats were maintained on fat-free high carbohydrate diets either with or without orotic acid (1%, w/w), pantethine (1%, w/w), adenine (0.25%, w/w), and/or p-chlorophenoxyisobutyrate (0.25%, w/w). Oxidation of fatty acid by liver mitochondria was inhibited to less than half that of the control after administration of orotic acid. Activities of acyl-CoA dehydrogenases were markedly decreased by orotic acid administration, but the following enzyme activities were not, or only slightly decreased: acyl-CoA synthetase, carnitine acyltransferases, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase. Simultaneous addition of pantethine in the orotic acid-containing diet prevented induction of fatty liver. It also prevented decreases in fatty acid oxidation capacity and acyl-CoA dehydrogenase activity. Introduction of adenine or p-chlorophenoxyisobutyrate, which reverse orotic acid-induced fatty liver, reversed oxidation and acyl-CoA dehydrogenase activities to control levels. The oxidation capacity of the peroxisomal system remained unchanged after administration of orotic acid.

Multiple-flash development of thermoluminescence bands in dark-grown spruce leaves.

Spruce leaves greened in darkness were devoid of three of the five thermoluminescence bands found for mature leaves. These bands were developed rapidly by exposure of the dark-grown leaves to continuous light. The development of these bands was studied by illumination with repetitive flashes at varied intervals. Flashes at intervals of 1 s were the most effective in inducing these bands. Those at shorter or longer intervals were less effective. It was deduced from these data that the development of these bands is a multiquantum process which involves at least two photo-events with a dark reaction between them.

Turnover of enzymes of peroxisomal beta-oxidation in rat liver.

Male Wistar rats were given a diet containing 2% (w/w) di-ethylhexyl)-phthalate (DEHP), a peroxisomal proliferator, for 4 weeks. The activities of enzymes of peroxisomal beta-oxidation and of catalase were markedly increased by the DEHP administration. The time required to reach halfway to the maximal induction for enzymes of peroxisomal beta-oxidation was 5--7 days, whereas that for catalase was 3 days. A separate DEHP group was placed on the control diet after 14 days of feeding with the DEHP diet. On the withdrawal of DEHP, activities of enzymes of the beta-oxidation system and of catalase decreased to the control levels with a half-life of 2--3 days. Responses of some mitochondrial enzymes involved in fatty acid oxidation are also described.

Light-dependent development of thermoluminescence, delayed emission and fluorescence variation in dark-grown spruce leaves.

Thermoluminescence profiles of spruce leaves grown under various light or dark conditions were measured after excitation at a low temperature (-70 to -20 degrees C) by 1-min illumination with red light, and the following results were obtained. Mature spruce leaves showed five thermoluminescence bands at -30, -5, +20, +40 (or +35) and +70 degrees C (denoted as Zv, A, B1, B2 and C bands, respectively), but dark-grown spruce leaves with a similar chlorophyll content showed only two bands, at -30 and +70 degrees C (the Zv and C bands) and were devoid of the three other bands (the A, B1, and B2 bands). On exposure of the dark-grown leaves to continuous red light, the A, B1and B2 bands were rapidly developed, and the development was accompanied by enhancement of delayed emission, fluorescence variation and the Hill activity (photoreduction of 2,6-dichlorophenolindophenol with water as electron donor). It was demonstrated that the dark-grown spruce leaves are devoid of the water-splitting system in Photosystem II, and that the latent water-splitting activity is rapidly photoactivated by exposure of the leaves to continuous red light. These results on the gymnosperm spruce leaves, in which greening proceeds in complete darkness, being independent of the development of the water-splitting system in light, were discussed in relation to previous observations on angiosperm leaves, in which both greening and the activity generation proceed in the light.

Cloning and expression of cDNA for a newly identified isozyme of bovine liver 3-hydroxyacyl-CoA dehydrogenase and its import into mitochondria.

cDNA for a heretofore undescribed mitochondrial 3-hydroxyacyl-CoA dehydrogenase, designated as the type II enzyme with different molecular and catalytic properties, compared to those of the classical mitochondrial beta-oxidation enzyme (type I enzyme), was cloned from a bovine liver cDNA library. Nucleotide sequence of the cDNA encoded 261 amino acids with a subunit molecular weight of 27,140. The deduced primary structure of the type II enzyme showed no significant homology to the reported amino acid sequence of the classical 3-hydroxyacyl-CoA dehydrogenases. On SDS-PAGE, no differences in subunit molecular weights were observed among the in vitro translation products, the recombinant type II enzyme produced in Escherichia coli and the purified enzyme. NH2-terminal and COOH-terminal amino acid sequence analysis of the purified type II enzyme revealed that the mature enzyme had not been proteolytically processed. The in vitro translation products of the type II enzyme were efficiently incorporated into isolated rat liver mitochondria, without changes in size, thereby suggesting that unlike other mitochondrial enzymes of fatty acid beta-oxidation, the type II enzyme had no cleavable signal peptide upon import into mitochondria.

Detection of hepatitis C virus markers and hepatitis C virus genomic-RNA after needlestick accidents.

BACKGROUND: Needlestick accidents are a problem among health care workers. Using sensitive new assays, we evaluated the prevalence and features of hepatitis C virus (HCV) infection following a needlestick accident. METHOD: The clinical outcome and evolution of serum HCV markers were assessed in 90 hospital employees (recipients) who sustained needlestick injuries (selected from 146 episodes) involving 92 patients with clinical non-A, non-B hepatitis (donors). RESULTS: Of the 92 patient donors, 62 (67%) and 88 (96%) were anti-C100-3 and second-generation anti-HCV positive, respectively, at the time of the needlestick accident. During the follow-up period (> or = 6 months), acute non-A, non-B hepatitis developed in three of 90 recipients about 1 month after the accident. The three respective donors were positive for serum HCV-RNA at the time of the accident. Two of the three recipients became HCV-RNA positive just after the onset of hepatitis, and subsequently, HCV antibodies developed. None of the remaining 87 recipients had any clinical or laboratory evidence of hepatitis during follow-up, or experienced seroconversion for anti-C100-3 or second-generation anti-HCV. We measured additional HCV markers in 20 of the 89 donors; 16 had evidence of HCV infection (HCV-RNA). However, none of the respective recipients of any of these 20 became positive for HCV markers during follow-up. CONCLUSION: Although transmission of HCV infection by needlestick injury may be infrequent, such transmission does occur. Appropriate precautions should be taken to protect health care workers.

[Nifekalant hydrochloride as an effective treatment for postoperative ischemic heart disease].

We experienced 2 effective cases of nifekalant hydrochloride. One patient was 76-year-old female who underwent emergent coronary artery bypass grafting (CABG) because of unstable angina pectoris (AP) and ventricular fibrillation (Vf). Her cardiac function had been decreased preoperatively due to old myocardial infarction (OMI). One day after CABG, she revealed sustained ventricular tachycardia (VT) and Vf. Although administrations of neither lidocaine hydrochloride nor magnesium sulfate were effective, nifekalant hydrochloride finally stopped the life-threatening arrhythmia without hypotension. Another patient was 77-year-old male who underwent CABG and Dor operation. His cardiac function also had been decreased due to OMI. He revealed VT attack at midnight 3 days after operation. VT attack still appeared at next 2 midnight under lidocaine hydrochloride infusion, but finally it has disappeared after starting a drip infusion of nifekalant hydrochloride. Nifekalant hydrochloride is quite useful as a new therapeutic strategy for uncontrollable VT and Vf and for the patient who has a reduced left ventricular function because it has an inotropic effect.

Improvement in local cerebral blood flow measurement in gerbil brains by prevention of postmortem diffusion of [14C]iodoantipyrine.

Postmortem diffusion of [14C]iodoantipyrine, which distorts the image of cerebral blood flow, can occur in at least three steps; from decapitation until the brain is frozen, while frozen sections on coverslips are thawed, and when dried sections are applied to x-ray film. In the present study, the first two steps were modified to reduce the time during which brain tissue was wet. When the brains of gerbils were taken out of the skulls and immersed in chilled isopentane (-45 degrees C), 90-95 s elapsed between decapitation until the brain tissues were frozen. However, immersion of whole heads in liquid nitrogen after decapitation decreased the time to 25 s. The autoradiograms had better contrast after the freezing procedure with liquid nitrogen than after that with chilled isopentane. The drying time of the thawed sections was markedly reduced by blowing hot air across the sections on a hot plate, and this resulted in clearer images on autoradiograms. In most of the tissues with values of cerebral blood flow over 100 ml 100 g-1 min-1 as measured using the conventional drying method, the corresponding values were higher if the modified method was used. In contrast, in tissues with values less than 100 ml 100 g-1 min-1, the corresponding values were lower. Moreover, the differences between flows in adjacent small brain structures were less clear if the sections were dried by the conventional method. Reducing the time during which postmortem brains or sections are wet can help prevent [14C]iodoantipyrine diffusion artifacts.

Calcium movement in ischemia-tolerant hippocampal CA1 neurons after transient forebrain ischemia in gerbils.

Hippocampal CA1 neurons exposed to a nonlethal period (2 min) of ischemia, acquired tolerance to a subsequent lethal 5-min period of ischemia, which usually causes delayed-type neuronal death. Intracellular Ca2+ movements before and after the 5 min of forebrain ischemia were evaluated in gerbil hippocampal CA1 pyramidal neurons, had acquired tolerance in comparison with nonischemia-tolerant CA1 neurons. Evaluation was performed by observing the ultrastructural intracellular Ca2+ distribution and the Ca2+ adenosine triphosphatase (Ca(2+)-ATPase) activity using electron microscopic cytochemistry. In comparison with nonischemia-tolerant CA1 neurons, mitochondria of ischemia-tolerant CA1 neurons sequestered more Ca2+ from the cytosomal fraction 15 min after the 5-min period of ischemia, and Ca2+ deposits in these mitochondria were rapidly decreased. Plasma membrane Ca(2+)-ATPase activities were already significantly elevated before the 5 min of ischemia, and remained at a higher level subsequently compared to nonischemia-tolerant CA1 neurons. Changes in the mitochondrial Ca2+ distribution and Ca(2+)-ATPase activities in ischemia-tolerant CA1 neurons after the 5-min period of ischemia showed a strong resemblance to those in CA3 neurons, which originally possess resistance to such periods of ischemia. These findings suggest that enhanced or maintained activities of mitochondrial Ca2+ sequenstration and plasma membrane Ca(2+)-ATPase reduced Ca2+ toxicity following 5-min ischemia in terms of time, resulting in escape from delayed neuronal death.

Development of a sensitive assay to detect reversibly oxidized protein cysteine sulfhydryl groups.

Protein sulfhydryl groups can undergo reversible oxidation reactions in response to reactive oxygen and reactive nitrogen species. Sensitive detection of sulfhydryl group oxidation in specific proteins is required to further our understanding of protein redox changes in biological systems. In general, to detect reversible oxidation reactions the oxidized sulfur atom is reduced to a sulfhydryl group followed by a reaction with a quantifiable agent. Our aim was to develop a sensitive method to detect reversibly oxidized protein sulfhydryl groups in a Western blot format. Conjugation of methoxypolyethylene glycol-maleimide (MAL-PEG) to protein sulfhydryl groups was optimized. Once MAL-PEG forms a covalent bond with the protein, the MAL-PEG-protein conjugate can be detected as a band shift by western analysis. The efficiency of MAL-PEG conjugation to protein was determined with creatine kinase. MAL-PEG conjugated to approximately 100% of the available sulfhydryl groups on creatine kinase within 30 min. Band shift detection sensitivity was measured using the redox-regulated protein p53. MAL-PEG conjugation coupled to western analysis detected a minimum of 0.23 pmol of oxidized p53. The MAL-PEG conjugation method described in this communication can be used to assess the reversible sulfhydryl oxidation status of proteins for which antibodies suitable for western analysis are available.

An alternative method of arterial reconstruction after hepatic arterial thrombosis following living-related liver transplantation.

BACKGROUND: Hepatic artery thrombosis (HAT) remains an important cause of graft loss after liver transplantation. Emergency rearterialization methods are limited in cases of living-related liver transplantation in which the graft hepatic artery is thin and short. CASE: A 19-year-old woman who underwent living-related liver transplantation for biliary atresia developed HAT on the 4th postoperative day. During the emergency laparotomy the recipient hepatic artery was found to be too short to anastomose, so the recipient's right gastroepiploic artery was anastomosed to the graft hepatic artery. The patient is now alive and well 6 months after reoperation, and she has experienced no further episode of HAT. CONCLUSION: The right gastroepiploic artery can be used easily and safely for hepatic graft revascularization without causing ischemia of the stomach. An additional skin incision is not required, and the artery is long enough to anastomose to the graft artery directly. The method of hepatic graft rearterialization described here is an important option for patients who undergo living-related or split liver transplantation.