Unconventional Myosin ID is Involved in Remyelination After Cuprizone-Induced Demyelination

Myelin, which is a multilamellar structure that sheathes the axon, is essential for normal neuronal function. In the central nervous system (CNS), myelin is produced by oligodendrocytes (OLs), which wrap their plasma membrane around axons. The dynamic membrane trafficking system, which relies on motor proteins, is required for myelin formation and maintenance. Previously, we reported that myosin ID (Myo1d) is distributed in rat CNS myelin and is especially enriched in the outer and inner cytoplasm-containing loops. Further, small interfering RNA (siRNA) treatment highlighted the involvement of Myo1d in the formation and maintenance of myelin in cultured OLs. Myo1d is one of the unconventional myosins, which may contribute to membrane dynamics, either in the wrapping process or transport of myelin membrane proteins during myelination. However, the function of Myo1d in myelin formation in vivo remains unclear. In the current study, to clarify the function of Myo1d in vivo, we surgically injected siRNA in the corpus callosum of a cuprizone-treated demyelination mouse model via stereotaxy. Knockdown of Myo1d expression in vivo decreased the intensities of myelin basic protein and myelin proteolipid protein immunofluorescence staining. However, neural/glial antigen 2-positive signals and adenomatous polyposis coli (APC/CC1)-positive cell numbers were unchanged by siRNA treatment. Furthermore, Myo1d knockdown treatment increased pro-inflammatory microglia and astrocytes during remyelination. In contrast, anti-inflammatory microglia were decreased. The percentage of caspase 3-positive cells in total CC1-positive OLs were also increased by Myo1d knockdown. These results indicated that Myo1d plays an important role during the regeneration process after demyelination.

     

Characterization of E. coli-derived recombinant human interferon-beta as compared with fibroblast human interferon-beta

Homogeneous E. coli-derived recombinant human interferon-beta (E. coli-rHuIFN-beta) was characterized in order to elucidate its physicochemical properties, as compared with those of fibroblast human interferon-beta (fibroblast HuIFN-beta). Purified E. coli-rHuIFN-beta and fibroblast HuIFN-beta exhibited a single band of Mr 19,000 and 23,000, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The primary structure of E. coli-rHuIFN-beta was identical to the prediction from the cDNA sequence. Furthermore, both the circular dichroism (CD) spectra and the 1H nuclear magnetic resonance (NMR) spectra of E. coli-rHuIFN-beta and fibroblast HuIFN-beta at pH 6.8 were closely similar to each other. On the other hand, on reverse-phase high-performance liquid chromatography (HPLC) using a C18 column, the retention time of E. coli-rHuIFN-beta was longer than that of fibroblast HuIFN-beta. Moreover, although the isoelectric point of E. coli-rHuIFN-beta was pH 8.9, purified fibroblast HuIFN-beta exhibited multiple isoelectric points, probably due to heterogeneity of the carbohydrate moiety. These results indicate that the E. coli-rHuIFN-beta polypeptide folds similarly to fibroblast HuIFN-beta, and the carbohydrate moiety of natural HuIFN-beta has little influence on higher-order structure but does influence the hydrophobic and the electrostatic properties of the molecule.     

Resistance of bacterial film to contamination during use as inoculum in repeated batch fermentation

Summary AnEscherichia coli strain constitutive for -galactosidase was immobilized onto cotton cloth. The resultingE.coli film was used as a resident inoculum in repeated batch fermentations for 30 days in the presence ofBrevibacterium ammoniagenes added as a contaminant. Analysis of -galactosidase production shows that contamination did not decrease the capacity of the film to generateE.coli cells, or decrease theE.coli population on the film.     

Cloning and expression of the Mycobacterium bovis BCG gene for extracellular alpha antigen.

The gene for the extracellular alpha antigen of Mycobacterium bovis BCG was cloned by using a single probe restricted to G or C in the third position. This technique should have great potential for the isolation of mycobacterial antigen genes. The gene analysis revealed that the alpha antigen gene encoded 323 amino acid residues, including 40 amino acids for signal peptide followed by 283 amino acids for mature protein. This is the first report on the structure of the mycobacterial signal peptide. The promoter-like sequence and ribosome-binding site were observed upstream of the open reading frame. In the coding region, the third position of the codon showed high G + C content (86%). The gene was expressed as an unfused protein in Escherichia coli by using an E. coli expression vector. This protein, which reacted with polyclonal antibody raised against alpha antigen from Mycobacterium tuberculosis, would be applicable to the immunodiagnosis of tuberculosis.     

Disulfide bond interchange in Escherichia coli-derived recombinant human interferon-beta 1 under denaturing conditions

Disulfide bond interchange has been pointed out as a considerable problem in preparing recombinant proteins from Escherichia coli cells. This has been reported in the system of reducing denaturation followed by a refolding process, where incorrectly folded molecules are sometimes produced. As the possibility of disulfide bond interchange may also arise in the cytoplasm of E. coli cells, the state of sulfhydryl groups of recombinant proteins obtained from a nonreducing and nondenaturing process should be examined. The state of sulfhydryl groups of E. coli-derived recombinant human interferon-beta 1, which had been purified under nonreducing and nondenaturing conditions, was examined by using the N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM) labeling technique. Among the three cysteine residues in E. coli-derived human interferon-beta 1, the 17th cysteine was identified as being unpaired, as in the natural molecule. However, it was found that three isomers of the recombinant protein could be formed when the protein was denatured with 6 M guanidine hydrochloride. These three isomers were identified as having unpaired cysteine residues at positions 17, 31, and 141, respectively. These results indicate that disulfide bond interchange occurs in E. coli-derived recombinant human interferon-beta 1 under denaturing conditions in spite of the absence of a reducing agent.     

Mechanism of interferon-gamma action. Characterization of indoleamine 2,3-dioxygenase in cultured human cells induced by interferon-gamma and evaluation of the enzyme-mediated tryptophan degradation in its anticellular activity

Induction by interferon-gamma of indoleamine 2,3-dioxygenase (a tryptophan degradation enzyme) was examined with 11 human cell lines. The enzyme induction was demonstrated in 7 of the 11 cell lines. The induced enzyme in each of the 7 cell lines was identical to the enzyme purified from human placenta, as evidenced by immunoblot analysis with a monoclonal antibody specific to the placental one. The extent of the induction varied largely with the cell line; a relatively high induction was observed with HEL (lung fibroblasts), NY (osteosarcoma), and A-431 (epidermoid carcinoma). The enzyme induction was dependent on the concentration of interferon-gamma and occurred 12-18 h after addition of interferon-gamma to the cultures. Interferon-alpha or -beta was completely ineffective in this induction. Interferon-gamma inhibited the growth of the 7 cell lines observed with the enzyme induction, and this growth inhibition was accompanied with a complete deletion of tryptophan (less than 1 microM) in the culture medium by the induction of the enzyme. For two of these cell lines, the inhibition was partially reversed by an addition of exogenous tryptophan to the medium not to be depleted. These findings indicated that the growth inhibition by interferon-gamma was in part explained by the tryptophan depletion in the medium caused by the enzyme induction.     

Scanning electron microscopy of the adoral ciliary zone of Cycloposthium bundle (Ciliophora, Entodiniomorphida)

The adoral ciliary zone of Cycloposthium spp., inhabiting the large intestine of the horse, was studied by scanning electron microscopy. It could be divided into four parts: outer, inner, left, and right zones. The outer zone, extending on the anterior periphery of the apical cone of the body, had 20 tuft-like syncilia arranged radially around the longitudinal axis. Each ciliary tuft consisted of about 170 cilia, and in cross section it had a rectangular shape. The cilia of the inner zone, situated at the top of the apical cone, were aggregated irregularly to form shorter bundles than the tufts of the outer zone. The innermost ciliar of this zone were shorter than the outermost. There was a distinct non-ciliated border between the outer and inner zones. A horseshoe-like operculum having no cilia was present at the center of the adoral ciliary zone, and the opening of the vestibulum was situated as a cleft crossing from the center to the right periphery of this zone. No cilia extended onto the vestibular wall. The left ciliary zone was situated beneath the outer zone and consisted of five short rows of barren kinetosomes of which only the central row possessed very short cilia. The right ciliary zone, consisting of a few rows of cilia situated at the bottom of the inner adoral lip, was also easily distinguished from the other ciliary zones. This zone was interpreted as an extension of the outer adoral zone passing along the right side of the apical cone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Germ cell deficiency causes testis cord differentiation in reconstituted mouse fetal ovaries

Sex-reversal in fetal ovaries was studied by using a dissociation-reconstitution technique. Gonads of 12.5 gestation-day male and female mouse fetuses were dissociated into single cells. To eliminate germ cells, the dissociated cells were cultured for 14 h, and then somatic cells attached to culture dishes were harvested and aggregated by gyratory culture for 24 h. The aggregates were then transplanted into ovarian bursa in ovary-ectomized nude mice. The recovered explants were examined histologically. Male somatic cells developed into testes containing Sertoli cells, Leidig cells, and tunica albuginea. Female somatic cells formed testis cords and differentiated into Sertoli cells, but they did not differentiate into other testis components or ovarian tissues. However, aggregates consisting of both female and male somatic cells differentiated into well-developed testes containing Leidig cells and tunica albuginea as well as Sertoli cells. Enzyme marker analysis showed significant contributions of female cells in these organized testes. In contrast, aggregates containing both female germ cells and somatic cells developed into ovaries and did not differentiate into any testicular tissues. The results indicate that female somatic cells in fetal gonads at 12.5 gestation day have the potency to form testis cords and differentiate into Sertoli cells. The subsequent steps in testis development require the contributions of male cells. The present study also suggests that testicular differentiation is independent of germ cells but ovarian development involves the interaction between germ cells and somatic cells.     

Augmentation of macrophage growth-stimulating activity of lipids by their peroxidation

Previously, we reported that some kinds of lipids (cholesterol esters, triglycerides, and some negatively charged phospholipids) that are constituents of lipoproteins or cell membranes induce growth of peripheral macrophages in vitro. In this paper, we examined the effect of peroxidation of lipids on their macrophage growth-stimulating activity because lipid peroxidation is observed in many pathological states such as inflammation. When phosphatidylserine, one of the phospholipids with growth-stimulating activity, was peroxidized by UV irradiation, its macrophage growth-stimulating activity was augmented in proportion to the extent of its peroxidation. The activity of phosphatidylethanolamine was also increased by UV irradiation. On the other hand, phosphatidylcholine or highly unsaturated free fatty acids, such as arachidonic acid and eicosapentaenoic acid, did not induce macrophage growth irrespective of whether they were peroxidized. The augmented activity of UV-irradiated phosphatidylserine was not affected by the coexistence of an antioxidant, vitamin E or BHT. These results suggest that some phospholipids included in damaged cells or denatured lipoproteins which are scavenged by macrophages in vivo may induce growth of peripheral macrophages more effectively when they are peroxidized by local pathological processes.     

Comparative studies on lipid and colony-stimulating factor-induced macrophage growth

We previously reported that lipids such as cholesterol esters, triglycerides, and some phospholipids that constitute cell membranes or serum lipoproteins induced growth of mouse peritoneal macrophages in vitro. In this paper, we compared the macrophage growth-stimulating activity of cardiolipin (CL), an active phospholipid with that of CSF-1. Growth kinetics and maximal degree of growth of exudated macrophages induced by CL were similar to those of CSF-1. CL did not stimulate macrophages to release soluble macrophage growth factors. Also, the activity of CL was not blocked as much by anti-CSF-1, suggesting that most of the effect of CL was direct and not mediated by CSF-1 or other protein factors. There was no synergistic effect between CL and CSF-1. CL induced growth of both exudate and resident macrophages, whereas CSF-1 induced very little resident macrophage growth. Furthermore, although the growth-stimulating activities of both substances were inhibited by IFN-gamma and TNF, CL was more resistant to these inhibitory effects. These results suggest that the lipid has some different characters from CSF-1 and may induce the growth of resident macrophages in inflammations or tumors.