Protein glycosylation.

Glycan structures can modulate the biological properties and functions of glycoproteins. This has been shown by investigation of the biological activities and glycan structures of several recombinant glycoproteins. Glycan structures of glycoproteins differ according to the species and tissue producing them, and selection of an appropriate host-cell type can generate recombinant glycoproteins with new characteristics.     

The fine structure of the mesonephros of the lamprey,Entosphenus japonicus Martens

Summary The fine structure of the mesonephric kidney of the lamprey, Entosphenus japonicus Martens, has been investigated with the electron microscope and discussed from the viewpoint of comparative morphology of the mesonephros.The structure of the capillary wall of the glomerulus essentially coincides with that of higher vertebrates, though its basement membrane is remarkably thick (300–400 m) because of a dense accumulation of fibrillar material between the endothelium and the basal lamina of epithelial cell. No obvious fenestration of the endothelial cell has been observed in the glomerulus or capillaries in any part of this organ.The kidney tubule is divided into three segments: 1. neck segment composed of ciliated cells with numerous mitochondria and glycogen particles, 2. proximal tubule composed of brush bordered cells provided with extensive pinocytotic vesicles and lysosomal granules in the apical cytoplasm and with lamellar membranes in the basal, and 3. distal tubule characterized by cells which, with their abundant mitochondria and branched tubular endoplasmic reticulum (about 500 Å diameter) with a central core, closely resemble the chloride cells in the gill filament of some teleosts. The possibility that the lamellar membranes in the proximal tubule cells correspond to basal infoldings is discussed.The extensive development of the tubular reticulum and of the mitochondria in the distal tubule cells is believed to reflect the active absorption of urine chloride in the urinary tubule of lamprey mesonephric kidney evidenced by physiologists. The proximal tubule is suggested to take a part also in the urinary transport of water and ions, as the lamellar membranes found in the cells of this portion likely correspond to the basal infoldings in more advanced forms of the kidney.The epithelial cells of the ureteric duct are characterized by granules suggesting a mucous secretion. No fine structure implying an absorptive activity in this duct has been observed.     

Induction by mycoplasmas of tumor necrosis factor-alpha from macrophages

Several species of mycoplasmas including M. pneumoniae, the causative agent of human respiratory infection, were investigated for tumor necrosis factor-alpha (TNF-alpha) induction. The cytotoxic activity to Meth A cells of peritoneal macrophages purified from BALB/c mice was enhanced markedly when cultured with either viable or nonviable mycoplasmas. The supernatant of macrophage culture mixed with mycoplasmas, M. pneumoniae or A. laidlawii, showed a potent cytotoxic activity to TNF-alpha-sensitive but not to TNA-alpha-insensitive L cells. Addition of anti-TNA-alpha antiserum inhibited completely the cytotoxic activity of the supernatant, indicating that the cytotoxic activity is due mostly to TNF-alpha. These results strongly suggest that mycoplasmas possess an activity to induce TNF-alpha, which enhances the cytotoxic activity of macrophages and prevent infection with mycoplasmas in vivo.     

Heptapeptide toxin production during the batch culture of two Microcystis species (Cyanobacteria)

Changes in the content of cyclic heptapeptide hepatotoxins called microcystins were investigated during batch culture of two Microcystis species using high performance liquid chromatography. After adsorption to ODS-silica gel cartridges and elution with methanol, the toxins were analyzed and quantified by HPLC. 35 μg per 100 mg dry cells of microcystin-RR, 34 μg of -YR and 43 μg of -LR were present at the beginning of the exponential growth phase of M. viridis. Microcystin-RR increased markedly towards the end of the exponential phase with the maximum content of 112 μg per 100 mg cells was measured at the late stage of the exponential phase. A remarkable increase of microcystin-YR from 130 μg per 100 mg cells to 1020 μg was observed during the exponential phase of a highly toxic strain of M. aeruginosa. However no clear differences were found in the pattern of change among the three toxins during the growth course.     

Glycosphingolipids are not pivotal receptors for Subtilase cytotoxin in vivo: sensitivity analysis with glycosylation-defective mutant mice

Certain glycosphingolipids play important roles as cellular receptor for bacterial toxins with high specificity and strong affinity. In particular AB(5) toxins exhibit typical modes of cell attachment with B5 and invasion and biological effects in cells with A subunit. Subtilase cytotoxin (SubAB) is the prototype of a recently discovered AB(5) cytotoxin family produced by certain strains of Shiga toxigenic Escherichia coli, and shows highly specific serine protease activity toward endoplasmic reticulum chaperone Bip. Since this toxin bound to a mimic of ganglioside GM2, GM2 has been considered to be possible receptor for SubAB. Using six kinds of glycosylation-defective knockout mice lacking certain group of glycosphingolipids, sensitivity to SubAB in vivo was analyzed. Consequently, all mutant mice died at around 70h after intraperitoneal injection of 10 microg (or 7.5 microg) of SubAB as well as wild type mice. These results indicated none of glycolipids are not pivotal receptor for SubAB in the body.     

The Identification of a Novel Gene,MAPO2, That Is Involved in the Induction of Apoptosis Triggered by O6-Methylguanine

O⁶-Methylguanine, one of alkylated DNA bases, is especially mutagenic. Cells containing this lesion are eliminated by induction of apoptosis, associated with the function of mismatch repair (MMR) proteins. A retrovirus-mediated gene-trap mutagenesis was used to isolate new genes related to the induction of apoptosis, triggered by the treatment with an alkylating agent, N-methyl-N-nitrosourea (MNU). This report describes the identification of a novel gene, MAPO2 (O⁶-methylguanine-induced apoptosis 2), which is originally annotated as C1orf201. The MAPO2 gene is conserved among a wide variety of multicellular organisms and encodes a protein containing characteristic PxPxxY repeats. To elucidate the function of the gene product in the apoptosis pathway, a human cell line derived from HeLa MR cells, in which the MAPO2 gene was stably knocked down by expressing specific miRNA, was constructed. The knockdown cells grew at the same rate as HeLa MR, thus indicating that MAPO2 played no role in the cellular growth. After exposure to MNU, HeLa MR cells and the knockdown cells underwent cell cycle arrest at G₂/M phase, however, the production of the sub-G₁ population in the knockdown cells was significantly suppressed in comparison to that in HeLa MR cells. Moreover, the activation of BAK and caspase-3, and depolarization of mitochondrial membrane, hallmarks for the induction of apoptosis, were also suppressed in the knockdown cells. These results suggest that the MAPO2 gene product might positively contribute to the induction of apoptosis triggered by O⁶-methylguanine.     

Systematic single base-pair substitution analysis of DNA binding by the cAMP receptor protein in cyanobacterium Synechocystis sp. PCC 6803

The cAMP receptor protein SYCRP1 in cyanobacterium Synechocystis sp. PCC 6803 is a regulatory protein that binds to the consensus DNA sequence (5'-AAATGTGATCTAGATCACATTT-3') for the cAMP receptor protein CRP in Escherichia coli. Here we examined the effects of systematic single base-pair substitutions at positions 4-8 (TGTGA) of the consensus sequence on the specific binding of SYCRP1. The consensus sequence exhibited the highest affinity, and the effects of base-pair substitutions at positions 5 and 7 were the most deleterious. The result is similar to that previously reported for CRP, whereas there were differences between SYCRP1 and CRP in the rank order of affinity for each substitution.     

Identification of aquaporin-5 and lipid rafts in human resting saliva and their release into cevimeline-stimulated saliva

BACKGROUND: It is unknown whether AQP5 and lipid rafts are released into human unstimulated (resting) saliva and saliva in response to secretagogues. METHODS: In order to quantitate the salivary concentration of AQP5, we produced a polyclonal antibody for human AQP5 and developed an enzyme-like immunosorbent assay (ELISA). RESULTS: AQP5 and lipid rafts were identified in human resting saliva. The amount of AQP5 in resting saliva showed a diurnal variation with high levels during waking hours, and an age-related decrease in AQP5 was coincident with the volume of resting saliva. Cevimeline, a muscarinic acetylcholine receptor (mAChR) agonist, induced the release of AQP5 with lipid rafts, amylase, mucin, and lysozyme. Changes in saliva AQP5 levels after cevimeline administration occurred simultaneously with changes in saliva flow rates. Confocal microscopy revealed that AQP5 was located in the apical plasma membrane and showed a diffuse pattern in parotid glands under resting conditions. Following cevimeline administration, AQP5 was predominantly associated with the APM and was localized in the lumen. GENERAL SIGNIFICANCE: AQP5 and lipid rafts were released with salivary proteins from human salivary glands by the stimulation of M3 mAChRs, and that changes in saliva AQP5 levels can be used as an indicator of salivary flow rate and also as a useful index of M3 mAChR agonist's action on human salivary glands.     

Mutants of Lactobacillus plantarum ML11-11 deficient in co-aggregation with yeast exhibited reduced activities of mixed-species biofilm formation

Lactic acid bacteria (LAB) mutants deficient in inter-species co-aggregation with yeast were spontaneously derived from Lactobacillus plantarum ML11-11, a significant mixed-species biofilm former in static co-cultures with budding yeasts. These non-co-aggregative mutants also showed significant decreases in mixed-species biofilm formation. These results suggest the important role of co-aggregation between LAB and yeast in mixed-species biofilm formation. Cell surface proteins obtained by 5 M LiCl extraction from the wild-type cells and non-co-aggregative mutant cells were analyzed by SDS-PAGE. There was an obvious difference in protein profiles. The protein band at 30 kDa was present abundantly in the wild-type cell surface fraction but was significantly decreased in the mutant cells. This band assuredly corresponded to the LAB surface factors that contribute to co-aggregation with yeasts.