Anti-bacterial antibodies in Epstein-Barr virus (EBV)-transformed oligoclonal B-cell lines established from normal persons and autoimmune disease patients

We have established 950 and 430 oligoclonal B-lymphoblastoid cell lines (LCL) from two normal persons and eight autoimmune disease patients, respectively by using Epstein-Barr virus (EBV)-induced transformation. To re-evaluate the EBV technique for production of human monoclonal antibodies (mAb) related to infectious disease, we screened these oligoclonal LCLs for antibodies against 31 bacterial strains systematically. A total of 74 cultures out of 1380 were reactive to a total of 18 strains out of 31. Among these, eight cultures showed 10^-3 antibody (Ab) titers to Pseudomonas aeruginosa serotypes C, E, F and I, Staphylococcus aureus, Serratia marcescens and Bacillus cereus. Ten cultures showed 10^-2 Ab titers to Ps. aeruginosa serotypes D, E, F and I, Ps. maltophilia, Staph, epidermidis, Klebsiella ozaenae, Ser. marcescens and B. subtilis. The results reveal the further possibilities for the EBV technique to produce various infectious disease-related human mAbs.     

(2S)-2-Amino-5-chloro-4-hydroxy-5-hexenoic acid,a new chloroamino acid,and related compounds fromAmanita gymnopus

Combining different chromatography systems, unusual nonprotein amino acids were isolated and unequivocally identified from a small amount (less than 100 g fresh weight) ofAmanita gymnopus fruit body. Without obtaining crystals of these amino acids, on the basis of¹H-NMR determination, high resolution mass spectrometry, chlorine analysis and oxidation with L-amino acid oxidase, one of them proved to be a new chloroamino acid, (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid (G2). The other three were (2S)-2-amino-5-hexenoic acid (G1), (2S)-2-amino-4,5-hexadienoic acid (G3) and (2S)-2-amino-5-hexynoic acid (G4). Amino acid (G1) was also encountered for the first time in natural products. Amino acid (G3) has been reported from several kinds of fungi belonging toAmanita, subgenusLepidella. The occurrence of amino acid (G4) was already reported fromCortinarius claricolor.Part 23 in the series Biochemical studies of nitrogen compounds in fungi. Part 22, Hatanaka, S. I. et al. 1985. Trans. Mycol. Soc. Japan26: 61–68.     

Determination of a new immunosuppressant, mycophenolate mofetil, and its active metabolite, mycophenolic acid, in rat and human body fluids by high-performance liquid chromatography

Mycophenolate mofetil, a new immunosuppressant, is a morpholinoethyl ester of mycophenolic acid. A new selective, sensitive and simple high-performance liquid chromatographic method was developed for the determination of mycophenolic acid and mycophenolate mofetil in biological samples. The preparation of samples was based on liquid—liquid extraction. The compounds were separated on a CN column using acetonitrile—0.01 M phosphate buffer (1:4, v/v) as the mobile phase. UV detection was used at wavelengths 215 and 304 nm. The detection limit was 5 ng per injection volume. This method enabled pharmacokinetic and pharmacodynamic studies in humans and rats.     

Effect of ATP on the Release of hsp 70 and hsp 40 from the Nucleus in Heat-Shocked HeLa Cells

We have recently found a novel 40-kDa heat-shock protein (hsp 40) in mammalian and avian cells and reported that the N-terminal amino acid sequence of mammalian hsp 40 has homology with the bacterial DnaJ heat-shock protein. Also, hsp 40 has been shown to be translocated from the cytoplasm into the nuclei/nucleoli by heat shock and colocalized with hsc 70 (p73) in the nucleoli of exactly the same cells. We here investigated the effect of ATP on the release of hsp 70 (both constitutive p73 and inducible p72) and hsp 40 from the nuclei/nucleoli of heat-shocked HeLa cells which were permeabilized with Nonidet-P40 using immunoflourescence and immunoblotting. Hsp 70 in the nucleoli was released by the addition of ATP but not by ADP, GTP, nonhydrolyzable ATP, nor high salt buffer. In contrast, hsp 40 was not released from the nucleoli with any of these treatments or any combination of these treatments. Thus, hsp 40 might dissociate spontaneously from the nucleoli after hsp 70 has been released in an ATP-dependent manner. Using cell fractionation methods, we showed that while the majority of hsp 40 is localized in the cytoplasm, a small portion of it is located in the microsome fraction in non-heat-shocked control cells and in cells which recovered from heat shock.     

A model for feature linking via collective oscillations in the primary visual cortex

A neural network model for explaining experimentally observed neuronal responses in cat primary visual cortex is proposed. In our model, the basic functional unit is an orientation column which is represented by a large homogeneous population of neurons modeled as integrate-and-fire type excitable elements. The orientation column exhibits spontaneous collective oscillations in activity in response to suitable visual stimuli. Such oscillations are caused by mutual synchronization among the neurons within the column. Numerical simulation for various stimulus patterns shows that as a result of activity correlations between different columns, the amplitude and the phase of the oscillation in each column depend strongly on the global feature of the stimulus pattern. These results satisfactorily account for experimental observations.     

Nupr1 deficiency downregulates HtrA1, enhances SMAD1 signaling,and suppresses age-related bone loss in male mice

Nuclear protein 1 (NUPR1) is a stress-induced protein activated by various stresses, such as inflammation and oxidative stress. We previously reported that Nupr1 deficiency increased bone volume by enhancing bone formation in 11-week-old mice. Analysis of differentially expressed genes between wild-type (WT) and Nupr1-knockout (Nupr1-KO) osteocytes revealed that high temperature requirement A 1 (HTRA1), a serine protease implicated in osteogenesis and transforming growth factor-β signaling was markedly downregulated in Nupr1-KO osteocytes. Nupr1 deficiency also markedly reduced HtrA1 expression, but enhanced SMAD1 signaling in in vitro-cultured primary osteoblasts. In contrast, Nupr1 overexpression enhanced HtrA1 expression in osteoblasts, suggesting that Nupr1 regulates HtrA1 expression, thereby suppressing osteoblastogenesis. Since HtrA1 is also involved in cellular senescence and age-related diseases, we analyzed aging-related bone loss in Nupr1-KO mice. Significant spine trabecular bone loss was noted in WT male and female mice during 6−19 months of age, whereas aging-related trabecular bone loss was attenuated, especially in Nupr1-KO male mice. Moreover, cellular senescence-related markers were upregulated in the osteocytes of 6−19-month-old WT male mice but markedly downregulated in the osteocytes of 19-month-old Nupr1-KO male mice. Oxidative stress-induced cellular senescence stimulated Nupr1 and HtrA1 expression in in vitro-cultured primary osteoblasts, and Nupr1 overexpression enhanced p16^ink4a expression in osteoblasts. Finally, NUPR1 expression in osteocytes isolated from the bones of patients with osteoarthritis was correlated with age. Collectively, these results indicate that Nupr1 regulates HtrA1-mediated osteoblast differentiation and senescence. Our findings unveil a novel Nupr1/HtrA1 axis, which may play pivotal roles in bone formation and age-related bone loss.     

Accumulation on the Cytoplasmic Membrane of the Precursor to Dimethyl Sulfoxide Reductase in Molybdenum Cofactor-Deficient Mutants of Rhodobacter sphaeroides f. sp. denitrificans

The role of the molybdenum cofactor (Mo cofactor) in the translocationof dimethyl sulfoxide (DMSO) reductase to the periplasmic spacewas studied in vivo by isolating chlorate-resistant mutantsof Rhodobacter sphaeroides f. sp. denitrificans. More than 50%of the chlorate-resistant mutants isolated were defective inthe biosynthesis of the Mo cofactor and all of these mutantsaccumulated the precursor form of the enzyme. About 45% of themutants contained the same level of Mo cofactor as the parentstrain and exhibited normal levels of DMSO reductase and nitratereductase activities when chlorate was absent from the medium,but the activities of these enzymes were depressed when chloratewas present. Much of the accumulated precursor form of the enzymein a Mo cofactor-deficient mutant was bound to the cytoplasmicmembrane and was sensitive to treatment with proteinase K fromthe periplasmic side of the membrane, an indication that theprecursor was exposed on the periplasmic surface of the membrane.The precursor accumulated on the membrane of the parent strainwhen molybdate was removed from the medium or upon additionof tungstate and this precursor was also sensitive to the treatmentwith proteinase K from the periplasmic side. These results suggestthat the Mo cofactor is necessary for proteolytic processingof the precursor to the mature enzyme on the periplasmic sideof the membrane, whereas binding of the precursor to the membraneand translocation across it can occur in the absence of thecofactor. Almost all of the Mo cofactor available for directreconstitution in vitro of nitrate reductase activity from thenit-l mutant of Neurospora crassa was present in the cytoplasmicfractions. (Received December 11, 1991; Accepted March 25, 1992)     

ANALYSIS OF POLYPEPTIDES ASSOCIATED WITH SHOOT FORMATION IN TOBACCO CALLUS CULTURES

Callus lines of Nicotiana tabacum were selected for competence and lack of competence in shoot formation. Changes in total and chromosomal polypeptides in these shoot-forming and nonshoot-forming tobacco cultures were examined by twodimensional polyacrylamide gel electrophoresis. Qualitative and quantitative differences in total, nonhistone chromosomal, and basic chromosomal polypeptides were evident throughout the 7-d test period. The analysis of total proteins identified polypeptides specific to shoot-forming and nonshoot-forming tissue during the 7-d sampling period. A small number of basic chromosomal proteins were found solely in shoot-forming or nonshoot-forming tissue. One basic chromosomal protein was detected in only nonshoot-forming tissue at all sampling times. Two proteins, although present in shoot-forming tissue, were present at elevated levels in the nonshoot-forming cultures. No temporal changes in basic proteins over the 7-d incubation period were observed. Qualitative differences in total nonhistone chromosomal polypeptides in the shoot-forming and nonshoot-forming tissue were also observed. Differences in chromosomal polypeptides were observed. In contrast to the basic chromosomal proteins, temporal variation in the nonhistone chromosomal polypeptides was demonstrated. Throughout the 7-d sampling period, 29 and 12 nonhistone chromosomal polypeptides varied qualitatively in shoot-forming and nonshoot-forming callus cultures, respectively. In vitro labeling with ³²P-orthophosphate indicated that approximately 1.0% and 0.3% of the nonhistone chromosomal proteins were phosphorylated in the shoot-forming and nonshoot-forming cultures. Of these phosphorylated polypeptides, one was present in nonshoot-forming tissue and three were detected only in the shoot-forming tissue. Phosphorylation occurred at serine or threonine residues.     

Changes in contractile properties and neuromuscular propagation evaluated by simultaneous mechanomyogram and electromyogram during experimentally induced hypothermia.

The present study examined, whether or not mechanomyogram (MMG) amplitude and frequency component could reflect the contractile properties of the triceps surae muscles, composed of relatively slow soleus (SOL) and fast medial gastrocnemius (MG), during experimentally induced hypothermia condition. In eight male subjects, lying in prone position, supramaximal single twitch and repetitive electrical stimulations at 10 Hz were applied at the intramuscular temperatures of control (34 degrees C), 15, 20, and 25 degrees C, respectively. The hypothermia induced substantial reduction in muscle contractile properties, e.g. prolonged twitch contraction and half relaxation times, resulted in a highly significant reduction in the fluctuation of force signal during the repetitive stimulations. These changes were almost mirrored by the similar and significant reductions in the MMG amplitude in both SOL and MG. Power spectrum analysis revealed that peak frequency components of MMG and fluctuation of force were almost matched with the applied stimulation frequencies, independent of the temperature condition. These results strongly suggest that MMG analysis could be employed to study muscle contractile properties varying across different physiological conditions.     

An Improved Enrichment Broth for Isolation of Escherichia coli O157, with Specific Reference to Starved Cells, from Radish Sprouts

An enrichment broth was developed for the efficient isolation of Escherichia coli O157 from radish sprouts. The broth was buffered peptone water containing 0.5% sodium thioglycolate (STG-BPW), which was designed to allow growth of E. coli O157 in starved and unstarved states. However, this medium suppressed the growth of non-carbohydrate-fermenting obligate aerobes whose colonial appearance on sorbitol MacConkey agar containing cefixime and tellurite (CT-SMAC) resembled that of E. coli O157. Both starved and unstarved cells of E. coli O157 experimentally inoculated into radish sprouts were successfully recovered with STG-BPW enrichment in all cases, most of which showed marked disappearance of E. coli O157-like colonies on CT-SMAC.