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Ocular candidiasis is a major complication of candidemia that is sometimes sight-threatening. Although prompt ophthalmologic consultation and antifungal medication have been emphasized, recent changes in the causative species and drug susceptibilities make the picture unclear. This study aimed to determine whether there are trends among patients with ocular candidiasis and included 80 patients with candidemia who underwent ophthalmological screening at our hospital between 2010 and 2020. Data on the clinical characteristics, comorbidities, biochemical test results, causative Candida species, treatment, outcomes, visual acuity, and antifungal susceptibility were collected and analyzed. Statistical analyses were performed by comparing two groups, namely, the ocular candidiasis (n = 29) and non-ocular candidiasis (n = 51) groups. In the ocular candidiasis group, there were significantly more cases of central venous catheter insertion (82.8%, p = 0.026) and Candida albicans candidemia (72.4%, p < 0.001). Regarding ocular involvement, the majority of patients were asymptomatic. Most cases improved with antifungal therapy, but one case underwent vitrectomy. Between 2016 and 2020, there was a diversification of species, with a decrease in Candida parapsilosis and the emergence of Candida glabrata and Candida tropicalis. Regarding drug susceptibility, the minimum inhibitory concentrations of echinocandin and 5-fluorocytosine against Candida albicans, Candida parapsilosis, and Candida glabrata were slightly increased. In conclusion, in addition to appropriately performing ophthalmologic examinations, it is beneficial to select antifungal agents according to the diversity of species and drug susceptibilities., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases, and Japanese Society for Infection Prevention and Control. Published by Elsevier Ltd. All rights reserved.)

Purpose: To assess the dependency of the Time to enhancement (TTE) of breast lesions and normal breast parenchyma from menopausal status and menstrual cycle using ultrafast compressed sensing (CS) -accelerated dynamic contrast-enhanced (DCE) MRI., Methods: This institutional review board approved retrospective study included 89 breast cancers, 22 benign lesions and 131 normal breast parenchymal foci. A prototypical ultrafast DCE sequence obtained 30 phases with 2.9 s temporal resolution. Mean and median TTE of all breast cancers, benign lesions and normal breast parenchymal foci were assessed. we also assessed whether there were any differences in TTE regarding the menopausal status and menstrual cycle., Results: The TTE of breast cancer was significantly shorter than that of benign lesions and normal breast parenchymal foci in both the premenopausal status (5.8 vs. 8.7 and 8.7 s, respectively) (p = 0.0028 and < 0.0001, respectively) and postmenopausal status (5.8 vs. 11.6 and 11.6 s, respectively) (p < 0.0001 in both). The TTE of parenchymal foci in the premenopausal status was significantly shorter than that in the postmenopausal status (p = 0.0025). Although the TTE interval between cancer and parenchymal foci in premenopausal status is shorter than that in postmenopausal status, the AUCs in the pre- and postmenopausal status for differentiating breast cancer and parenchymal foci were comparable with using different cutoff TTE values. There were no differences in TTE regarding the menstrual cycle., Conclusions: The TTE derived from ultrafast CS-accelerated DCE MRI was useful to differentiate breast cancer from benign lesions and normal breast parenchymal foci in both pre- and postmenopausal status., Competing Interests: Declaration of Competing Interest Katsuya Maruyama: Employee of Siemens Healthcare K.K. Dominik Nickel: Employee of Siemens Healthcare GmbH. The all other authors of this manuscript declare no relationships with any companies whose products or services may be related to the subject matter of the article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Nitrous oxide (N 2 O) emissions from soils applied with livestock excreta have been widely reported previously. The highest N 2 O emissions from soils are also often reported during thawing periods in cold regions where soil freezing is common. However, the combined effects of cow urine application and freeze-thaw events on N 2 O emissions and the related enzyme activities are still not clear. Thus, we simulated a freeze-thaw event at -3 °C for 7 days, and then increased to 3 °C for 46 days using intact soil cores with cow urine (392 kg N ha -1 ). We compared the factors influencing the magnitudes of N 2 O emissions through soil microbial processes with and without the freeze-thaw event. Dicyandiamide (DCD), an inhibitor of nitrification, was added to investigate the significance of nitrification on N 2 O emissions. The N 2 O emission rates from the urine-applied soils peaked to approximately 1000 μg N 2 O-N m -2  h -1 immediately after the soils thawed. Soil freezing with urine application was significantly higher cumulative N 2 O emissions (537 mg N 2 O-N m -2 ), compared to non-frozen soils with urine (247 mg N 2 O-N m -2 ) during the incubation period (54 days). The effect of DCD application on N 2 O emissions was not clear during the freeze-thaw event, although nitrate production rates were reduced. After the freezing event, soil moisture (water-filled pore space) was significantly higher in the non-frozen soils compared to the frozen soils, due to a 9% decline in bulk density of frozen soils. Additionally, the impact of thawing on urease and denitrification enzyme activities was influenced by the urine application. Urine application increased the urease activity, while the freezing event decreased the magnitudes. The physical changes in the soils were also important controlling factors of the N 2 O emissions from cow urine-applied soils in cold regions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Re-emerging multidrug-resistant typhoid fever is becoming a worldwide threat, especially in East Africa. At the beginning of 2015, an outbreak of typhoid fever started in the capital city of Uganda, and 1940 suspected cases were reported by 5 March 2015. In this report, we describe a case of typhoid fever caused by a MDR strain with HIV infection and hemoglobin S-syndrome thalassemia in an Ugandan from Kampala City. It is essential to consider MDR strains of Salmonella enterica serovar Typhi infections, including fluoroquinolone-resistant strains, in patients from Africa and Southeast Asia., (Copyright © 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Acinetobacter calcoaceticus-Acinetobacter baumannii complex, especially A. baumannii, Acinetobacter pittii and Acinetobacter nosocomialis, constitutes an important group of nosocomial pathogens; however, epidemiological or clinical characteristics and prognosis is limited in Japan. From 2009 to 2013, 47 blood stream infection cases resulting from A. baumannii group were reviewed at the National Defense Medical College, an 800-bed tertiary hospital. To determine the genospecies, further comparative nucleotide sequence analyses of the RNA polymerase b-subunit (rpoB) gene were performed. Sequence analysis of rpoB gene showed that 25 (49.0%), 17 (33.3%) and 5 (9.8%) cases were caused by A. baumannii, A. pittii and A. nosocomialis, respectively. The 30-day and in-hospital mortality rates of A. baumannii were 8.5% and 25.5%, respectively, and there were no significant differences between Acinetobacter species. Clinical characteristics were statistically insignificant. Multidrug-resistant Acinetobacter species were detected in 3 cases (5.9%) with same pulsed-field gel electrophoresis (PFGE) pattern and A. baumannii was less susceptible to amikacin and levofloxacin. In this study, the mortality and clinical characteristics were similar among A. baumannii group isolate cases despite some showing drug resistance. However, identification of Acinetobacter species helps to initiate appropriate antibiotic therapy in earlier treatment phase, because A. baumannii shows some drug resistance., (Copyright © 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Onconeural immunity, a cancer-stimulated immune reaction that cross-reacts with neural tissues, is considered to be the principal pathological mechanism for paraneoplastic neurological syndromes (PNS). A common PNS is paraneoplastic cerebellar degeneration (PCD). We had encountered a PCD patient with urothelial carcinomas (UC) of the urinary bladder who was negative for the well-characterized PNS-related onconeural antibodies. In the present study, we aimed to identify a new PCD-related onconeural antibody, capable of recognizing both cerebellar neurons and cancer tissues from the patient, and applied a proteomic approach using mass spectrometry. We identified anti-creatine kinase, brain-type (CKB) antibody as a new autoantibody in the serum and cerebrospinal fluid from the patient. Immunohistochemistry indicated that anti-CKB antibody reacted with both cerebellar neurons and UC of the urinary bladder tissues. However, anti-CKB antibody was not detected in sera from over 30 donors, including bladder cancer patients without PCD, indicating that anti-CKB antibody is required for onset of PCD. We also detected anti-CKB antibody in sera from three other PCD patients. Our study demonstrated that anti-CKB antibody may be added to the list of PCD-related autoantibodies and may be useful for diagnosis of PCD., (© 2013.)

Background: Lymphangioleiomyomatosis (LAM) is a rare cystic lung disease predominantly affecting young women. Some of these patients develop lymphedema of the lower extremities and buttocks; however, neither the exact frequency of LAM-associated lymphedema nor the clinical features of such patients is well delineated., Objectives: To document the frequency, features, and treatment of LAM-associated lymphedema., Methods: We reviewed all medical records of patients listed in the Juntendo University LAM registry for the 30 years preceding August 2010., Results: Of 228 patients registered with a diagnosis of LAM, eight (3.5%) had LAM-associated lymphedema of the lower extremities. All were females with sporadic LAM, and their mean age when diagnosed was 32.5 years (range 23-44). Lymphedema of the lower extremities was the chief or a prominent presenting feature in five of these LAM patients. CT scans showed that all eight patients had enlarged lymph nodes (lymphangioleiomyomas) in the retroperitoneum and/or pelvic cavity. Yet, cystic destruction of the lungs was mild in four patients, moderate in two and severe only in two. Seven of these patients were treated by administering a fat-restricted diet and complex decongestive physiotherapy, and four received a gonadotropin-releasing hormone analog. With this combined protocol, all eight patients benefitted from complete relief or good control of the lymphedema., Conclusions: Lymphedema is a rare complication of LAM and may be associated with axial lymphatic involvement or dysfunction rather than severe cystic lung destruction. The combined multimodal treatments used here effectively resolved or controlled LAM-associated lymphedema., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Tissue factor pathway inhibitor (TFPI) is a physiological protease inhibitor that inhibits the initial reactions of the extrinsic blood coagulation pathway. Most TFPI in human plasma is associated with lipoproteins; however, the most functionally active form is thought to be the free, full-length form (f-pTFPI). Cell culture derived TFPI and recombinant TFPI (rTFPI) exhibit variations in their respective anticoagulant activity, which may be caused by post-translational modifications, such as the frequent differences in sugar chain structures among recombinant proteins. Sugar chain structures in rTFPI expressed in Chinese hamster ovary (CHO) cells have been reported previously, but those of plasma TFPI have not been., Objectives: To purify f-pTFPI and analyze the sugar chain structures., Results and Conclusion: f-pTFPI was purified to homogeneity from blood plasma using a combination of anion-exchange, heparin affinity, immunoaffinity, and reversed-phase chromatographies, resulting in a yield of 76%. f-pTFPI showed a partially phosphorylated glycoprotein comprising a total of 276 amino acids by peptide mapping. The sugar chain structures were analyzed by two-dimensional sugar mapping combined with exoglycosidase digestion of the pyridylamino sugar chains and the following results were obtained. (Sialyl) Galbeta1-3GalNAc was linked to Thr(175), partially to Thr(14) and Ser(174); sialyl complex-type sugar chains to Asn(117) and Asn(167), whereas Asn(228) was not glycosylated. Neuraminidase-resistant acidic sugar chains including sulfated sugar chains were not observed significantly. The protease inhibitory activities of f-pTFPI towards activated factor (F) X and tissue factor-activated FVII complex were identical to those of full-length rTFPI expressed in CHO cells.

Damaged DNA-binding protein (DDB) is a heterodimer (DDB1 and DDB2), which is implicated in the repair of UV-irradiated DNA damage. Here we have identified four DDB2 variants from HeLa cells (D1-D4) that are generated by alternative splicing. Analysis of tissue distribution by RT-PCR indicates that D1 is the most highly expressed in human brain and heart. A DNA repair assay revealed that both D1 and D2 are dominant negative inhibitors. Electrophoresis mobility shift assays indicated that D1 and D2 are not part of the damaged DNA-protein complex. Co-immunoprecipitation studies show that DDB2-WT interacts with D1 and itself. Nuclear import of DDB1 was less induced by transfection with D1 than WT. Based on these results, D1 and D2 are dominant negative inhibitors of DNA repair, which is probably due to disruption of complex formation between DDB1 and DDB2-WT and of DDB1 nuclear import.

The accumulation of D-isomers of aspartic acid (D-Asp) in proteins during aging has been implicated in the pathogenesis of Alzheimer's disease, cataracts, and arteriosclerosis. Here, we identified a specific lactacystin-sensitive endopeptidase that cleaves the D-Asp-containing protein and named it D-aspartyl endopeptidase (DAEP). DAEP has a multi-complex structure (MW: 600kDa) and is localized in the inner mitochondrial membrane of mouse and rabbit, but DAEP activity was not detected in Escherichia coli, Saccharomyces cerevisiae, and Caenorhabditis elegans. A specific inhibitor for DAEP was newly synthesized, and inhibited DAEP activity (IC(50), 3microM), a factor of 10 greater than lactacystin on DAEP. On the other hand, the inhibitor did not inhibit either the 20S or 26S proteasome.

OPA1 is a cause gene for autosomal dominant optic atrophy and possesses eight alternative splicing variants. Here, we identified two isoforms of OPA1 proteins in HeLa cells and examined their submitochondrial localization and complex formations. RT-PCR shows that HeLa cells mainly express isoforms 7 and 1 of OPA1. Since the third cleavage site is mainly utilized in HeLa cells, the predicted molecular masses of their processed proteins are consistent with the 93- and 88-kDa proteins. Biochemical examinations indicate that both of the OPA1 isoforms are present in the intermembrane space. Submitochondrial fractionation by sucrose density-gradient centrifugation shows that the 88-kDa protein predominantly associates with the mitochondrial outer membrane, on the contrary, the 93-kDa protein associates with the inner membrane. Gel filtration analysis indicates that they compose the different molecular mass complexes in mitochondria. These differences between two isoforms of OPA1 would suggest their crucial role involved in the mitochondrial membrane formation.

Neural RNA recognition motif (RRM)-type RNA-binding proteins play essential roles in neural development. To search for a new member of neural RRM-type RNA-binding protein, we screened rat cerebral expression library with polyclonal antibody against consensus RRM sequences. We have cloned and characterized a rat cDNA that belongs to RRM-type RNA-binding protein family, which we designate as drb1. Orthologs of drb1 exist in human and mouse. The predicted amino acid sequence reveals an open reading frame of 476 residues with a corresponding molecular mass of 53kDa and consists of four RNA-binding domains. drb1 gene is specifically expressed in fetal (E12, E16) rat brain and gradually reduced during development. In situ hybridization demonstrated neuron-specific signals in fetal rat brain. RNA-binding assay indicated that human Drb1 protein possesses binding preference on poly(C)RNA. These results indicate that Drb1 is a new member of neural RNA-binding proteins, which expresses under spatiotemporal control.

Colocalization of mitochondria is the first step of intermitochondrial interaction or fusion in a cell. Here, we showed colocalization between exogenous mitochondria and endogenous ones or between exogenous proteoliposomes and endogenous mitochondria in mouse fertilized eggs by confocal laser microscopy. Isolated mitochondria from mouse liver and proteoliposomes containing mitochondrial membrane were directly labeled with red fluorescent aliphatic marker, PKH26, which is incorporated into lipid membrane, and then were microinjected into fertilized mouse eggs. Exogenous mitochondria appeared to be almost colocalized with endogenous mitochondria at the 4- and 8-cell stages, when mitochondria were stained with Rhodamine 123 (green fluorescent marker). On the contrary, when liposomes consisted of soy bean phospholipid were microinjected into the eggs as a control, their localization was different from that of endogenous mitochondria. Next, the submitochondrial particles and proteoliposomes were microinjected. Both the proteoliposomes and the submitochondrial particles appeared to colocalize with endogenous mitochondria at the 4-cell stage. These results suggest the existence of a factor that makes liposomes colocalize with mitochondria. Such a proteoliposome would be useful for the development of mitochondrial gene transfer techniques., (Copyright 2000 Academic Press.)

Ganglioside GD3 induced the release of cytochrome c from isolated rat liver mitochondria. This process was completely prevented by cyclosporin A and partially prevented by a cysteine protease inhibitor, n-acetyl-leu-leu-norleucinal. Cyclosporin A is a potent inhibitor of the permeability transition pore, whereas n-acetyl-leu-leu-norleucinal has no effect on this pore. These results indicate that the release of cytochrome c from mitochondria requires both the opening of the permeability transition pore and a cysteine protease inhibitor-sensitive mechanism. Gangliosides GD1a, GD1b, GT1b, and GQ1b along with the synthetic GD3 mimetics TMS-42 and CI-22, which are glycerophospholipids carrying a disialo residue, also induced cytochrome c release. In contrast, gangliosides GM1, GM2, and GM3 did not induce cytochrome c release. These results indicate that two sialo residues must play an important role in the induction of cytochrome c release by gangliosides., (Copyright 2000 Academic Press.)

The acceptor substrate specificities of ST6GalNAc I and II, which act on the synthesis of O-linked oligosaccharides, were reexamined using ovine submaxillary mucin, [Ala-Thr(GalNAc)-Ala]n polymer (n = 7-11). It has been suggested that only ST6GalNAc I can synthesize carbohydrate structures of sialyl-Tn-antigen; i.e., NeuAc alpha2-6GalNAc-O-Thr/Ser [Kurosawa et al., J. Biol. Chem. 269, 19048-19053 (1994)] based on the result that ST6GalNAc I, not ST6GalNAc II, exhibited activity toward asialoagalacto-fetuin. In this study, we present evidence that both ST6GalNAc I and II exhibit activity toward asialo-OSM (ovine submaxillary mucin) and [Ala-Thr(GalNAc)-Ala]n polymer (n = 7-11) which have only the GalNAc-O-Thr/Ser-structures. These results strongly indicate that not only ST6GalNAc I but also II are candidates for sialyl-Tn synthases.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta superfamily, play a variety of roles during mouse development. BMP type II receptor (BMPR-II) is a type II serine/threonine kinase receptor, which transduces signals for BMPs through heteromeric complexes with type I receptors, including activin receptor-like kinase 2 (ALK2), ALK3/BMPR-IA, and ALK6/BMPR-IB. To elucidate the function of BMPR-II in mammalian development, we generated BMPR-II mutant mice by gene targeting. Homozygous mutant embryos were arrested at the egg cylinder stage and could not be recovered at 9.5 days postcoitum. Histological analysis revealed that homozygous mutant embryos failed to form organized structure and lacked mesoderm. The BMPR-II mutant embryos are morphologically very similar to the ALK3/BMPR-IA mutant embryos, suggesting that BMPR-II is important for transducing BMP signals during early mouse development. Moreover, the epiblast of the BMPR-II mutant embryo exhibited an undifferentiated character, although the expression of tissue-specific genes for the visceral endoderm was essentially normal. Our results suggest that the function of BMPR-II is essential for epiblast differentiation and mesoderm induction during early mouse development., (Copyright 2000 Academic Press.)

alpha(1)-Protease inhibitor (alpha(1)PI), the most abundant serine protease inhibitor found in human plasma (at 30-60 microM), is a glycoprotein (53 kDa) having a single cysteine residue at position 232 (Cys(232)). We have found that Cys(232) of human alpha(1)PI was readily S-nitrosylated by nitric oxide (NO) without affecting inhibitory activity to trypsin or elastase. S-nitrosylated alpha(1)PI (S-NO-alpha(1)PI) not only retained inhibitory activity against these serine proteases, but also gained thiol protease inhibitory activity against a Streptococcus pyogenes protease; the parental alpha(1)PI did not have this activity. Furthermore, S-NO-alpha(1)PI exhibited bacteriostatic activity against Salmonella typhimurium at concentrations of 0.1-10 microM, which were 20- to 3000-fold stronger than those of the other NO-generating compounds or S-nitroso compounds such as S-nitrosoalbumin and S-nitrosoglutathione. NO appears to be transferred into the bacterial cells from S-NO-alpha(1)PI via transnitrosylation, as evidenced by electron spin resonance spectroscopy with an NO spin trap. Thus, we conclude that S-NO-alpha(1)PI may be generated from the reaction between alpha(1)PI and NO under inflammatory conditions, in which production of both is known to increase. As a result, new functions, i.e., antibacterial and thiol protease inhibitory activities of alpha(1)PI, were generated., (Copyright 2000 Academic Press.)

Recent advances in bioenergetics consist of discoveries related to rotational coupling in ATP synthase (FoF(1)), uncoupling proteins (UCP), reactive oxygen species (ROS) and mitochondrial DNA (mtDNA). As shown in cloned sheep, mammalian genomes are composed of both nuclear DNA (nDNA) and maternal mtDNA. Oxidative phosphorylation (oxphos) varies greatly depending on cellular activities, and is regulated by both gene expression and the electrochemical potential difference of H(+) (Delta muH(+)). The expression of both mtDNA (by mtTFA) and nDNA for oxphos and UCP (by NRFs, etc.) is coordinated by a factor called PGC-1. The Delta muH(+) rotates an axis in FoF(1) that is regulated by inhibitors and ATP-sensitive K(+)-channels. We cultured human rho(o) cells (cells without mtDNA) in synthetic media and elucidated relationships among mtDNA, nDNA, Delta muH(+), UCPs, ROS, and apoptosis. These cells lack oxphos-dependent ROS formation and survive under conditions of high O(2). Cells cultured in the absence of ROS scavengers have proliferated for 40 years. UCPs lower Delta muH(+) and prevent ROS formation and resulting apoptosis. These results were applied to diabetology and gerontology. The pancreatic rho(o) cells did not secrete insulin, and mtDNA mutations caused diabetes, owing to the deficient Delta muH(+). Insulin resistance was closely related to UCPs and other energy regulators. The resulting high-glucose environment caused glycation of proteins and ROS-mediated apoptosis in vascular cells involved in diabetic complications. Telomeres, oxphos, and ROS are determinants in cellular aging. Cell division and ROS shortened telomeres and accelerated aging. In aged cells, Delta muH(+) was reduced by the slow respiration, and this change induced apoptosis. Cybrids made from aged cytoplasts and rho(o) cells showed that both decreased expression of nDNA, and somatic mutations of mtDNA are involved in the slowing of respiration in aged cells., (Copyright 1999 Academic Press.)

The present paper describes a rapid and systematic method for semi-quantitative analysis of the expression of sialyltransferase genes. So far, fifteen sialyltransferase cDNAs have been cloned from mice. Most of these genes are expressed in developmental stage-dependent and/or tissue-specific manners, and the expression levels of some of them are too low to detect on Northern blot analysis. To resolve how each sialyltransferase contribute to synthesize sialylglycoconjugates, it is necessary to establish the method for quantification of gene expression levels of these fifteen sialyltransferases. Therefore, we developed a competitive PCR-based method for analyzing the quantitative relationship of the gene expression of fifteen sialyltransferases. Using this method, we can investigate the levels of gene expression of sialyltransferases in various cell lines and various tissues of mice, and can accurately determine their expression levels., (Copyright 1999 Academic Press.)

The cDNA encoding a new type of alpha 2,3-sialyltransferase (mST3Gal V) was cloned from mouse brain cDNA library by PCR-based cloning approach using a pair of degenerate primers deduced from the nucleotide sequence information of mouse ST3Gal III and IV. The predicted amino acid sequence of mST3Gal V showed 27.3% and 26.4% identity to mST3Gal III and IV, respectively. The recombinant soluble mST3Gal V fused with protein-A, which expressed in the culture media of COS-7 cells, showed activity toward lactosylceramide (LacCer), and synthesized GM3. The apparent Km value for LacCer was 9.3 microM. mST3Gal V did not exhibit any activity toward other substrates we tested in this study, including glycolipids, glycoproteins and disaccharides. The mST3Gal V cDNA transfected F28-7 cells, which express large amount of lactosylceramide and very small amount of GM3 at native stage, expressed a large amount of GM3. The ST3Gal V gene was strongly expressed in mouse brain and liver, which contained a large amount of ganglioside. The gene expression seemed to be coincident with ganglioside expression in mouse. Thus, we conclude that mST3Gal V is the fifth-type alpha 2,3-sialyltransferase carrying GM3 synthetic activity.

Tissue-specific alternative RNA splicing in human F1gamma pre-mRNA produces muscle- and nonmuscle-type isoforms. Muscle-specific exclusion of exon 9 of the F1gamma gene is cell-specifically induced by acidic treatment of human fibrosarcoma HT1080 and rhabdomyosarcoma KYM-1 cells. We constructed an F1gamma minigene containing parts of exon 8, intron 8, and exon 9 of the human F1gamma gene and then analyzed a negative factor that inhibited inclusion of exon 9 via an in vitro splicing assay using acid-stimulated HT1080 cell nuclear extract. In vitro splicing of the F1gamma minigene, similarly to the beta-globin minigene used as a control, was observed in HeLa cell nuclear extract. Next, we performed supplemental experiments using HeLa and HT1080 cell nuclear extracts. The splicing reaction of the F1gamma minigene was specifically inhibited by supplementation with nuclear extract from acid-stimulated HT1080 cells, whereas that of human beta-globin was not inhibited. These results indicated that acidic stimulation induced a negative factor that blocked inclusion of alternatively spliced exon in the F1gamma minigene in vitro, and a regulatory factor acted in a sequence-specific manner for muscle-specific alternative splicing in F1gamma pre-mRNA., (Copyright 1998 Academic Press.)

A single molecule of ATP synthase (F0F1) is by itself a rotary motor, the smallest ever found, and this biomotor is driven by an electrochemical potential of H+ (delta microH+). F0F1 is composed of an ion-conducting portion (F0) and a catalytic portion (F1). The major breakthroughs in studies on the mechanochemical coupling have been the direct observation of the rotation of a stable alpha 3 beta 3 gamma complex of thermophilic F1 (TF1), and X-ray crystallography of the alpha 3 beta 3 gamma portion of mitochondrial F1 (MF1) and the alpha 3 beta 3 oligomer of TF1. This review focuses on the dynamics of TF1, demonstrated by a crucial experiment. The torque of the rotation was estimated to be 42 pN.nm from the delta microH+ and frictional force. Important unsolved problems are the crystallography of F0, elastic energy conversion, and the stator and rotor of this biomotor.

DNA-coated Au particles were accelerated by pressurized He gas to supersonic velocities for introduction of a gene into cells. Experimental and theoretical analyses both revealed a heterogeneous distribution of the particles per shot (1 mg Au = 2.4 x 10(7) particles with 2 microg [32P] DNA = 2.5 x 10(11) moles). For introduction of genes into the liver of living rats, the best results were obtained with a newly developed hand-held gene delivery system. The beta-galactosidase gene introduced into rat liver with Au particles by He at 250 psi was expressed (1.2 microunits/microg protein) in a limited area of the liver surface (8 x 8 mm, depth 0.5 mm). When the same gene gun was used on a monolayer of cultured COS7 cells (about 5 microm thick), cells were lost in the central area of heavy bombardment. Cell death caused by influx of Ca2+ was prevented by the use of the cytosol-type culture medium.

To clarify a universal mechanism of the intramolecular rotation of ATP-synthase, an operon encoding a stable, ancestral ATPase was cloned from a heterotrophic archaeum Desulfurococcus strain SY. The operon of about 7 kbp contained genes E, C, G, A, B and D encoding subunits with predicted molecular weights of 23,217, 41,659, 11,499, 65,476, 52,295, and 24,897, respectively. The sequence was compared with that of Na-ATPase of Enterococcus hirae, A-ATPase of Halobacterium salinarium, V-ATPase of Methanosarcina mazei, and ATP synthase of Methanococcus jannaschii, which are homologous. (1) The cause of hyperthermostability: The main exchanges in the amino acid residues of hyperthermophilic proteins included Asp --> Glu (11 residues of A subunit of E.h.) and, Ser --> Ala. (2) The domains needed for the intramolecular rotation: The domains similar to those established in F-type ATPases were also found in the V-type ATPases of species with a different energy metabolism.

To elucidate thermoresistance, a gene of a hyperthermophilic heat shock protein (HHSP) was isolated from the hyperthermophile Desulfurococcus strain SY which grows at 95 degrees C. The molecular weight of HHSP deduced from the open reading frame was 59,137 (545 amino acid residues). Sequence alignments of peptides reveal similarities (evolutionary distances) to the alpha (0.279) and beta (0.296) subunits of thermosome, TF55 (0.343) and human t-complex polypeptide 1. The structure of a thermophilic heat shock protein TGroEL (Tamada et al. (1991) Biochem, Biophys. Res. Commun. 179, 565) was quite different from that of HHSP. TGroEL and HSP60 have sequences identical to HHSP at its equatorial domain, while those identical to the alpha subunit of F-type ATPase are at its apical domain.

The translation of a heat shock protein (HSP), TGroEL, of thermophilic bacterium PS3 increased within 10 minutes when the culture temperature was raised from 60 degrees C to 70 degrees C. In contrast to hyperthermophilic HSPs such as Pyrodictium ATPase, TGroEL is homologous to GroEL of E. coli (Tamada et al. (1991) Biochem. Biophys. Res. Commun. 197, 565-571). The promoter of the thermophilic heat shock gene (tgrE) was sequenced to analyze the heat shock response. The sequences of the promoter were CCTACTTGCA (-35 region) and GTTCATTAATA (-10 region), which are more heat labile than those of the sigma 32 recognition sites of GroEL. The expression of this homologous tgrE in E. coli may be lethal to the cells. The ATPase alpha subunit of this thermophile is also homologous to TGroEL.

Heat shock proteins 60 (hsp60) and 10 (hsp10) are essential for the formation and restoration of many supramolecular structures. For reconstitution of these structures, we isolated stable hsps of 61kDa and 12kDa, which are similar to hsp60 and hsp10, respectively, from the supernatant fraction of thermophilic bacterium PS3 by ATP-Agarose chromatography. Using synthetic DNA of the deduced sequence, the 1.6kbp double stranded DNA encoding both proteins was obtained by the polymerase chain reaction (PCR). The complete sequence of the resulting reading frames showed high homology to those of the genes encoding GroEL (hsp60) and GroES (hsp10) of E. coli, and hsp60s and hsp10s of several other species. The genes for the 12K and 61K were present in the same operon. 61K was also partially similar to the F1 alpha subunit of thermophilic ATP synthase, which is highly reconstitutable to form the alpha beta complex.

Culture media for mammalian cells contain electrolytes at concentrations that approximate those of the extracellular fluid (ECF). However, we observed that HeLa strain cells can grow in a cytosol type, serum-free, high amino acid medium (KN13) in which the concentrations of K+, Mg2+ and Pi were increased to about 10 times those in ECF by replacing NaCl. The Ca2+ concentration of KN13 is 10% of that in ECF. The additions of Mg2+ and Pi are essential to prevent the harmful effect of a high concentration of K+ in the medium. Even an increase in the K/Na ratio to 7 and 9 permitted growth for at least 2 weeks and 4 days, respectively. HeLaP3 cells adapted to KN13 (HeLaK cells) have proliferated (4 fold/week) for 4 years in this medium. The Na,K-ATPase of HeLaK cells was the alpha 1 isoform. HeLaK cells in KN13 medium have a lower membrane potential (-3.0 +/- 6.1 mV) than that of the original HeLaP3 cells in DM160 (-26 +/- 10 mV).

A Clostridium cellulovorans lambda gt11 gene bank was screened for endo-1,4-beta-glucanase [EC 3.2.1.4, EGase, Carboxy Methyl Cellulase (CMCase)] genes using a chromogenic substrate. Three genes (engA, engB, and engC) were isolated. The engB expressed the most active CMCase. The engA encoded a bifunctional enzyme that displayed endo-1,4-beta-glucanase and beta-glucosidase activities. The three recombinant glucanases, when expressed in Escherichia coli, were partially degraded into multiform active enzymes as evidenced by their SDS-PAGE-CMC zymograms. None of the clones could degrade crystalline cellulose, thus supporting the hypothesis that the integrity of the C. cellulovorans cellulase complex was essential for its 'true cellulase' activity.

Collagenase-like (CL) peptidase activity in serum, which was measured using a newly synthesized substrate, (succinyl-Gly-Pro-Leu-Gly-Pro)-4-methylcoumaryl-7-amide, was significantly lower in patients with advanced rheumatoid arthritis or with systemic lupus erythematosus than that in normal controls. Decrease of the serum enzyme activity was more pronounced in systemic lupus erythematosus. No significant change in serum CL-peptidase activity was found in other connective tissue diseases such as mixed connective tissue disease and Sjögren's syndrome.

Voltage-dependent calcium channels are vital to cardiac muscle contraction. Therefore it is very important to isolate physiologically active channel proteins, however there have been few reports on their solubilization and reconstitution. Highly purified sarcolemmal membranes from bovine cardiac muscle were solubilized with octylglucoside, partially purified by gel filtration, and reconstituted into planar lipid bilayer by the direct insertion method. At least, two cation channel activities were observed: one with about 4.2 pS and the other with about 28 pS in conductance. From the reversal potential, it was concluded that Ba2+ ions are the current carrier through these two channels.