Controlled direct electron transfer kinetics of fructose dehydrogenase at cup-stacked carbon nanofibers.

Graphene edge sites not only facilitate heterogeneous electron transfer reactions of redox species because of localization of electrons, but also allow sensitivities and selectivities to be tuned by controlling the atomic oxygen/carbon (O/C) ratio. Here, we immobilized fructose dehydrogenase (FDH) onto the surface of cup-stacked carbon nanofibers (CSCNFs), which provide highly ordered graphene edges with a controlled O/C ratio, and investigated the direct electron communication with FDH. As the O/C ratio decreased at the CSCNF surface, the negative zeta potential was mitigated and the electrochemical communication with FDH was facilitated. This is likely due to improved orientation of FDH molecules on the CSCNF surface. CSCNFs with a controlled O/C ratio could be applied to FDH-based d-fructose biosensors with tunable dynamic range and fructose biofuel cells with a controlled maximum current.

Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells.

In macroautophagy, cytoplasmic components are delivered to lysosomes for degradation via autophagosomes that are formed by closure of cup-shaped isolation membranes. However, how the isolation membranes are formed is poorly understood. We recently found in yeast that a novel ubiquitin-like system, the Apg12-Apg5 conjugation system, is essential for autophagy. Here we show that mouse Apg12-Apg5 conjugate localizes to the isolation membranes in mouse embryonic stem cells. Using green fluorescent protein-tagged Apg5, we revealed that the cup-shaped isolation membrane is developed from a small crescent-shaped compartment. Apg5 localizes on the isolation membrane throughout its elongation process. To examine the role of Apg5, we generated Apg5-deficient embryonic stem cells, which showed defects in autophagosome formation. The covalent modification of Apg5 with Apg12 is not required for its membrane targeting, but is essential for involvement of Apg5 in elongation of the isolation membranes. We also show that Apg12-Apg5 is required for targeting of a mammalian Aut7/Apg8 homologue, LC3, to the isolation membranes. These results suggest that the Apg12-Apg5 conjugate plays essential roles in isolation membrane development.

The reversible modification regulates the membrane-binding state of Apg8/Aut7 essential for autophagy and the cytoplasm to vacuole targeting pathway.

Autophagy and the Cvt pathway are examples of nonclassical vesicular transport from the cytoplasm to the vacuole via double-membrane vesicles. Apg8/Aut7, which plays an important role in the formation of such vesicles, tends to bind to membranes in spite of its hydrophilic nature. We show here that the nature of the association of Apg8 with membranes changes depending on a series of modifications of the protein itself. First, the carboxy-terminal Arg residue of newly synthesized Apg8 is removed by Apg4/Aut2, a novel cysteine protease, and a Gly residue becomes the carboxy-terminal residue of the protein that is now designated Apg8FG. Subsequently, Apg8FG forms a conjugate with an unidentified molecule "X" and thereby binds tightly to membranes. This modification requires the carboxy-terminal Gly residue of Apg8FG and Apg7, a ubiquitin E1-like enzyme. Finally, the adduct Apg8FG-X is reversed to soluble or loosely membrane-bound Apg8FG by cleavage by Apg4. The mode of action of Apg4, which cleaves both newly synthesized Apg8 and modified Apg8FG, resembles that of deubiquitinating enzymes. A reaction similar to ubiquitination is probably involved in the second modification. The reversible modification of Apg8 appears to be coupled to the membrane dynamics of autophagy and the Cvt pathway.

Genetic control of T cell receptor BJ gene expression in peripheral lymphocytes of normal and rheumatoid arthritis monozygotic twins.

The amino acids encoded at the junctions of T cell receptor (TCR) V and J genes directly interact with MHC bound peptides. However, the regulation of the human TCRBJ gene repertoire has been difficult to analyze, because of the potentially complex number of BJ gene rearrangements. To overcome this problem, we developed a PCR-ELISA method to study BJ gene expression, and compared peripheral T lymphocytes from 12 pairs of monozygotic twins, including 6 rheumatoid arthritis (RA) discordant pairs, and 5 normals. Analyses of the TCRBV5, 13 and 17 gene families, which have been reported to be increased in RA patients, showed: (a) the three TCRBV transcripts have common features of BJ gene usage; (b) TCR transcripts from each TCRBV family display a distinctive BJ gene profile, which is displayed better by CD4+ than CD8+ lymphocytes; (c) the BJ gene repertoires of monozygotic twins are more similar than those of unrelated individuals; and (d) the inflammation of RA does not induce specific changes in the genetically determined pattern of BJ expression. These results indicate that the frequency of expression particular TCRBV-TCRBJ recombinants in human lymphocytes is controlled genetically, and is maintained despite the presence of a chronic inflammatory disease.

Apg5p functions in the sequestration step in the cytoplasm-to-vacuole targeting and macroautophagy pathways.

The cytoplasm-to-vacuole targeting (Cvt) pathway and macroautophagy are dynamic events involving the rearrangement of membrane to form a sequestering vesicle in the cytosol, which subsequently delivers its cargo to the vacuole. This process requires the concerted action of various proteins, including Apg5p. Recently, it was shown that another protein required for the import of aminopeptidase I (API) and autophagy, Apg12p, is covalently attached to Apg5p through the action of an E1-like enzyme, Apg7p. We have undertaken an analysis of Apg5p function to gain a better understanding of the role of this novel nonubiquitin conjugation reaction in these import pathways. We have generated the first temperature-sensitive mutant in the Cvt pathway, designated apg5(ts). Biochemical analysis of API import in the apg5(ts) strain confirmed that Apg5p is directly required for the import of API via the Cvt pathway. By analyzing the stage of API import that is blocked in the apg5(ts) mutant, we have determined that Apg5p is involved in the sequestration step and is required for vesicle formation and/or completion.

Apg7p/Cvt2p: A novel protein-activating enzyme essential for autophagy.

In the yeast Saccharomyces cerevisiae, the Apg12p-Apg5p conjugating system is essential for autophagy. Apg7p is required for the conjugation reaction, because Apg12p is unable to form a conjugate with Apg5p in the apg7/cvt2 mutant. Apg7p shows a significant similarity to a ubiquitin-activating enzyme, Uba1p. In this article, we investigated the function of Apg7p as an Apg12p-activating enzyme. Hemagglutinin-tagged Apg12p was coimmunoprecipitated with c-myc-tagged Apg7p. A two-hybrid experiment confirmed the interaction. The coimmunoprecipitation was sensitive to a thiol-reducing reagent. Furthermore, a thioester conjugate of Apg7p was detected in a lysate of cells overexpressing both Apg7p and Apg12p. These results indicated that Apg12p interacts with Apg7p via a thioester bond. Mutational analyses of Apg7p suggested that Cys507 of Apg7p is an active site cysteine and that both the ATP-binding domain and the cysteine residue are essential for the conjugation of Apg7p with Apg12p to form the Apg12p-Apg5p conjugate. Cells expressing mutant Apg7ps, Apg7pG333A, or Apg7pC507A showed defects in autophagy and cytoplasm-to-vacuole targeting of aminopeptidase I. These results indicated that Apg7p functions as a novel protein-activating enzyme necessary for Apg12p-Apg5p conjugation.

The mouse SKD1, a homologue of yeast Vps4p, is required for normal endosomal trafficking and morphology in mammalian cells.

The mouse SKD1 is an AAA-type ATPase homologous to the yeast Vps4p implicated in transport from endosomes to the vacuole. To elucidate a possible role of SKD1 in mammalian endocytosis, we generated a mutant SKD1, harboring a mutation (E235Q) that is equivalent to the dominant negative mutation (E233Q) in Vps4p. Overexpression of the mutant SKD1 in cultured mammalian cells caused defect in uptake of transferrin and low-density lipoprotein. This was due to loss of their receptors from the cell surface. The decrease of the surface transferrin receptor (TfR) was correlated with expression levels of the mutant protein. The mutant protein displayed a perinuclear punctate distribution in contrast to a diffuse pattern of the wild-type SKD1. TfR, the lysosomal protein lamp-1, endocytosed dextran, and epidermal growth factor but not markers for the secretory pathway were accumulated in the mutant SKD1-localized compartments. Degradation of epidermal growth factor was inhibited. Electron microscopy revealed that the compartments were exaggerated multivesicular vacuoles with numerous tubulo-vesicular extensions containing TfR and endocytosed horseradish peroxidase. The early endosome antigen EEA1 was also redistributed to these aberrant membranes. Taken together, our findings suggest that SKD1 regulates morphology of endosomes and membrane traffic through them.

The pleiotropic role of autophagy: from protein metabolism to bactericide.

Autophagy is in principle a nonselective, bulk degradation system within cells, with a contribution to intracellular protein degradation estimated to be as large as that of the ubiquitin--proteasome system. The primary roles of autophagy are baseline turnover of intracellular proteins and organelles, production of amino acids in nutrient emergency, and regression of retired tissues. These functions guarantee rejuvenation and adaptation to adverse conditions, and even underlie dynamic processes such as development/metamorphosis. In addition, several other roles for autophagy have recently been discovered, such as presentation of endogenous antigens and degradation of invasive bacteria. This review will discuss the biological significance of autophagy from yeast to higher eukaryotes.

Prolactin and growth hormone responses to psychological stress in normal and neurotic subjects.

In order to study the response of plasma prolactin (PRL) to acute psychological stress and to compare it with that of growth hormone (GH), the mirror drawing test (MDT) was performed in 20 normal controls (11 male, 9 female) and 22 neurotic patients (12 male, 10 female). Plasma PRL and GH were measured serially before, during and after the test. In controls, the test caused no significant change in plasma levels of either hormone. In neurotic males, the response of PRL to the test was not consistent, whereas, in neurotic females, plasma PRL level rose significantly following the test. Increase of GH, on the other hand, was apparent in the neurotics of both sexes. The correlation between the responses of the two hormones in the neurotics was low and non-significant. The results indicate that although the psychoendocrine coping mechanism in the neurotics works less effectively for both PRL and GH, the two hormones may have different psychological correlates.

Growth hormone and cortisol responses to psychological stress: comparison of normal and neurotic subjects.

The mirror drawing test (MDT) was performed to induce acute psychological stress in 9 normal controls and 10 neurotic subjects. Plasma growth hormone (GH) and cortisol were determined serially before, during, and after the test. In controls the MDT caused no significant change in plasma GH level, while in neurotics plasma GH increased progressively following the test. The increase of cortisol also tended to be greater in neurotics as a group, but there was considerable overlap in individual responses. The maximum increments of GH in neurotics correlated inversely with those of cortisol. The results indicate: 1) effective psychological coping mechanisms operate in normal man to keep the hormonal response minimum. 2) GH response is a more adequate indicator than cortisol response to psychological stress in neurotics. 3) GH and cortisol may have different psychological correlates in neurotics.

Ceramide induces apoptosis via CPP32 activation.

Although both ceramide and interleukin-1beta converting enzyme (ICE) family proteases are key molecules during apoptosis, their relationship remains to be elucidated. We report here that cell-permeable ceramide induced cleavage and activation of CPP32, a Ced-3/ICE-like protease, but not ICE. Ceramide-induced apoptosis of Jurkat cells was blocked by the CPP32-specific tetrapeptide inhibitor DEVD-CHO, but not by the ICE inhibitor YVAD-CHO. Furthermore, variant Jurkat cells with defective CPP32 activation were resistant to both anti-Fas- and ceramide-induced apoptosis. These results indicate that CPP32 activation is required for ceramide-induced apoptosis, and suggest sphingomyelin-ceramide pathway functions upstream of CPP32.

Surface markers of NK cells in peripheral blood of patients with cirrhosis and hepatocellular carcinoma.

The surface markers and NK activity of blood lymphocytes in 38 normal controls, 57 cirrhotic patients and 22 cirrhotics with hepatocellular carcinoma were studied by the flow cytofluorometry using monoclonal antibodies. A reduction of OKM1+ cells was remarkable in cirrhotics, and a further decrease in HNK-1+ cells as well as OKM1+ cells was observed in cirrhotics with hepatocellular carcinoma, accompanied by depression of NK activity. We postulate that the decreased NK activity in these patients may result from the disturbed maturation of NK cells.

Lipid microsphere preparation of a lipophilic ceramide derivative suppresses colony formation in a murine experimental pulmonary metastasis model.

Ceramide is a well-known regulator of apoptosis and cell growth. In this study, we synthesized lipophilic ceramide derivatives to incorporate into lipid microspheres (LM) and their activity was evaluated in vivo. Cera 03, a lipophilic ceramide derivative synthesized from membrane-permeable C2-ceramide, caused potent growth inhibition and DNA fragmentation of Meth A-T tumor cells in vitro. Its potency was similar to that of C2-ceramide. Both compounds increased the proportion of apoptotic cells. Cera 02, the diacetylated form of natural ceramide (Cer), also suppressed in vitro cell growth with a similar or higher potency to that of Cer, but both were far less potent than C2-ceramide and Cera 03. LM containing Cera 03 (Lipo-Cera 03) could not totally prevent metastatic incidence of Meth A-T cells, but reduced pulmonary metastatic nodules in number. Intravenous injection of Lipo-Cera 03 (1 mg/kg of Cera 03) produced about 35% inhibition, while Lipo-Cera 02 had no significant effect. In conclusion, Lipo-Cera 03 may have potential as an antimetastatic drug and may also be a useful tool for researching the role of ceramides in vivo.

Deafness following mumps: the possible pathogenesis and incidence of deafness.

Mumps is thought to be the most common cause of unilateral acquired sensorineural deafness in children. Mumps deafness is usually sudden in onset, profound or complete, and may be associated with vestibular symptoms. The authors' clinical survey of 55 patients with unilateral deafness which could reasonably be ascribed to mumps indicates that the hearing loss is exclusively unilateral, severe or total and permanent, and that approximately 45% of the patients experienced dysequilibrium of vestibular origin. An analysis of the present series of mumps deafness also suggests that the primary route of invasion of the virus is hematogenous, and thus the term "viral endolymphatic labyrinthitis" is proposed as the possible pathogenesis of the deafness, since both tympanogenic and meningogenic routes of viral invasion to the labyrinth can be excluded on the basis of the clinical and cerebrospinal fluid studies. This view of the pathogenesis, particularly that mumps meningitis is not associated with deafness, is supported by several reports including those of Vuori et al. (1962), Azimi et al., (1969), Lindsay (1973), Nadol (1978), etc. The incidence of deafness following mumps appears to be extremely low, approximately 1:20,000, as estimated by Everberg (1957).

Release and rectal absorption of aminophylline suppositories prepared in a hospital pharmacy.

In vitro release and in vivo rectal absorption of suppositories containing aminophylline and various bases prepared in our hospital pharmacy were investigated. The release tests were conducted using three methods: the cylindrical filter paper method, the Sartorius solubility simulator method and the disintegration test in J.P. IX. In all cases, release from PEG-containing suppositories was better than that from Witepsol-containing products. However, this tendency was not reflected in the blood level of theophylline after rectal absorption of these suppositories.

Evaluation of aminophylline suppositories prepared in a hospital pharmacy.

Physical and chemical stability of suppositories containing aminophylline and various bases prepared in our hospital pharmacy was investigated. Ethylenediamine in aminophylline decreased, the melting points of the suppositories rose, and the disintegration and liquefaction times were prolonged in suppositories with Witepsol base when stored at room temperature. Thin-layer chromatography produced evidence supporting the assumption that ethylenediamine may react with Witepsol constituents to form an acid amide linkage. However, the suppositories were stable at lower temperatures. Suppositories prepared with PEG base were found to be satisfactorily stable at room temperature.

A quantitative tear fluids determination of therapeutic efficacy for allergic conjunctivitis.

A provocation test was conducted with volunteers in 10 cases with seasonal cedar pollinosis during a quiescent stage to determine quantitatively the therapeutic effect of 0.08% Ketotifen eye drops. The efficacy rate of this preparation was found to be 80%.

[Two cases of lupus nephritis having a high titer of anti-(histone-DNA) complex antibody without IgG anti-dsDNA antibody].

IgG antibody specific to double-stranded DNA (dsDNA) has been recognized as a predictive indicator of the renal involvement in systemic lupus erythematosus. However, we recently experienced two cases of overt lupus nephritis without IgG anti-dsDNA antibody. In both cases, high titers of antibody to the histone dimer (H2A-H2B)-dsDNA complex were detected. They responded well to the corticosteroid therapy, with a cytotoxic agent in one case, and the titers of anti-(histone-DNA) antibody was decreased along with the improvement of proteinuria. Based on the recent reports suggesting a pathogenic role of this antibody in lupus nephritis, we suggest that measurement of the anti-(histone-DNA) antibody is necessary in lupus nephritis patients, especially when IgG anti-dsDNA antibody is not detectable.

Therapeutic effects of a new, anti-allergic ophthalmic preparation.

A 0.1% Ketotifen ophthalmic preparation was evaluated in 11 cases of conjunctivitis due to Japanese cedar pollinoisis (three cases also associated with vernal catarrh). The following results were obtained concerning the safety and the therapeutic efficacy of the preparation. (1) In the computation of the overall effects of the preparation, a marked therapeutic effect was noted in three out of 11 cases studied while one case remained unaffected. The overall effective rate was computed to be 91%. (2) The time required for the preparation to take effect was found to be 3 days or less in seven cases (70.0%) out of 10 in which therapeutic effects were noted. (3) In observations of the adverse effects of the preparation, transient irritation at the site of application was noted in seven cases. No other serious side effects were recorded. From the above results, it was concluded that the present preparation is an effective therapeutic agent for conjunctivitis caused by pollinosis.

[A case report of multiple intracranial tuberculoma associated with miliary tuberculosis and review of the literature].

We reported a case of multiple intracranial tuberculoma associated with miliary tuberculosis and reviewed the cases reported as intracranial tuberculoma in the past 11 years. A 41-year-old diabetic man was admitted to our hospital for the treatment of miliary tuberculosis and respiratory insufficiency. On admissIon, he had no neurological deficits except mild consciousness disturbance due to respiratory failure. He developed headache and mental confusion three weeks after the beginning of antituberculous therapy with isoniazid, streptomycin, rifampicin, and ethambutol. Neurological examination revealed that he had progressive right hemiparesis and was in a confusional state. Enhanced CT showed multiple intracranial nodular lesions. During 6 weeks, he had progressive neurological manifestations in spite of his initial antituberculous treatment. He responded well, however, to the chemotherapy with combination of isoniazid, kanamycin, pyrazinamide and ethionamide that were sensitive to tuberculous bacilli separated from his sputum. He became minimally right-hemiparetic by 6 weeks after the change of antituberculous medication. Serial enhanced CT scan proved to be of great value in the diagnosis and follow-up study of intracranial tuberculoma. From 1978 to 1988, there were 72 reported cases of intracranial tuberculoma in Japan; 37 were male, 32 were female and 3 were uncertain because of no detailed document. The age of onset was distributed from 6 month to 81 years in age and 2 peaks were seen in the second decade and fifth to seventh decade. Thirty-three (48%) out of 69 cases had multiple intracranial lesions. A few reports commented that neurological complications tended to appear even if they were under antituberculous therapy.(ABSTRACT TRUNCATED AT 250 WORDS)