Gap Junctions Mediate Glucose Transport Between GLUT1-Positive and -Negative Cells in the Spiral Limbus of the Rat Cochlea

To elucidate the role of the spiral limbus in glucose transport in the cochlea, we analyzed the expression and localization of GLUT1, connexin26, connexin30, and occludin in the spiral limbus of the rat cochlea. GLUT1 and occludin were detected in blood vessels. GLUT1, connexin26, connexin30, and occludin were also expressed in fibrocytes just basal to the supralimbal lining cells. Connexin26 and connexin30 were present among not only these GLUT1-positive fibrocytes but also GLUT1-negative fibrocytes. In vivo glucose imaging using 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-6-deoxyglucose (6-NBDG, MW 342) together with Evans Blue Albumin (EBA, MW 68,000) showed that 6-NBDG was rapidly distributed throughout the spiral limbus, whereas EBA was localized only in the vessels. Moreover, the gap junctional uncoupler heptanol inhibited the distribution of 6-NBDG. These findings suggest that gap junctions play an important role in glucose transport in the spiral limbus, i.e., that gap junctions mediate glucose transport from GLUT1-positive fibrocytes to GLUT1-negative fibrocytes in the spiral limbus.     

Conformational study of poly(L-lysine) interacting with acidic phospholipid vesicles

Circular dichroism measurements were carried out on poly(L-lysine) in the presence of vesicles of the negatively charged phospholipids, phosphatidylserine (PS; from bovine brain), phosphatidic acid (PA; prepared from egg yolk lecithin) and dimyristoylphosphatidylglycerol (DMPG). PS vesicles induced a conformational change in poly(L-lysine) from random coil to alpha-helix structure in 5 mM Tes (pH 7.0), whereas PA vesicles gave rise to beta-structure in the same buffer. The fraction of alpha-helix, F alpha (or beta-structure, F beta), increased with increasing PS (or PA) concentration, reaching a saturation value of about 0.7 (or about 1). Mixed vesicles comprising PS and dilauroylphosphatidylcholine (DLPC) also induced alpha-helix conformation, however, the saturation value of F alpha diminished with decreasing PS content in mixed vesicles. On the other hand, the spectral patterns for poly(L-lysine) in DMPG vesicle suspensions exhibited the coexistence of alpha-helix and beta-structure. Both F alpha and F beta increased with DMPG concentration and reached saturation values of about 0.5. Mixed vesicles composed of DMPG and dimyristoylphosphatidylcholine (DMPC) led to a reduction in F beta, while F alpha remained almost constant. The diversity in ordered structure induced by different phospholipid vesicles suggests the participation of lipid head groups in determining the secondary structure of poly(L-lysine) adsorbed on the vesicular surface.     

Continuous protease production in a carbon-limited chemostat culture by salt tolerant Aspergillus oryzae

Summary A chemostat culture system was investigated in order to produce protease by Aspergillus species effectively in the presence of 10% NaCl which was added to avid bacterial contamination. A salt tolerant fungus Aspegillus oryzae NISL 1913 produced protease even in the presence of 10% NaCl. The protease production by this strain was accelerated by proteins. Isolated soy protein or defatted soybean fluor (DSF) was used as a nitrogen source and an inducer of protease production, and starch was used as a carbon source. Continuous protease production was performed in a carbon-limited chemostat culture (dilution rate = 0.02). The maximum activity reached 2200 protease units (PU)/ml of the culture broth (130 PU/mg dry weight) with DSF as a nitrogen source. The culture could be continued for more than 50 days without any bacterial contamination.     

Immunohistochemical localization of proteoglycans in interstitial elements of human pancreas and biliary system

Summary The immunohistochemical localization of large proteoglycan and small proteoglycan was observed, using antibodies 2B1 and 6B6 (Sobueet al., 1988, 1989a), in fetal and adult pancreas and biliary system as well as in tumour tissues, obtained from 11 autopsies and 74 biopsies. The distribution of chondroitin 4- and 6-sulphate side chains, type I and IV collagen and elastin were also studied. In adult pancreas and all the biliary tracts examined, periductal fibrous tissues consisted mainly of dermatan sulphate small proteoglycan with networks of fibrous elements, which were composed of large proteoglycan, elastin, type I collagen and type IV collagen. In the interstitial components of cystadenoma of pancreas and biliary duct carcinoma, similar small proteoglycan-rich components were relatively abundant, although large proteoglycan was present in much larger amounts than that in non-neoplastic adult tissues. In some cholangiomas, the extra-and intracellular hyaline globules formed by the carcinoma cells were found to contain chondroitin sulphate large proteoglycan, laminin and fibronectin.The distribution of proteoglycans was observed to be different in the arterial walls of the interlobular tissues of the adult and the fetal pancreas. The biological significance of large and small proteoglycans in the interstitial connective tissues was discussed.     

Characteristics of settling matter and its role in nutrient cycles in a deep oligotrophic lake

The settling flux of seston (dry weight, DW), chlorophyll a (Chl a), particulate organic carbon (POC), particulate organic nitrogen (PON), and particulate phosphorus (PP) was measured monthly in 1981–1983 at 10 different depths in Lake Chuzenji, Japan; an oligotrophic lake with a maximum depth of 163 m. The Ti concentration in entrapped matter was used to separate the sedimentation flux into allochthonous and autochthonous components. Inflow loads of dissolved nutrients (DN: 4.5, DP: 0.48 g m^-2a^-1) were almost sufficient to supply the autochthonous fluxes at 30 m (PON: 2.9, PP: 0.51 g m^-2a^-1 ), and this flux of POC (26.6 g m^-2a ^-1) was about one-third of primary production (84 g C M^-2a^-1). Sedimentation of particulate matter was the main path of losing nutrients from lake water, explaining more than 80% removal of inflow loads (TN, TP). Decomposition rates during sedimentation which were calculated from the vertical difference in the autochthonous flux agreed very closely with the results obtained by laboratory experiments of a 100-day incubation (content ratios from field observations were: POC 0.67, PON 0.65, PP 0.85; and from laboratory experiments they were: POC 0.68, PON 0.70, PP 0.94). These decomposition rates and those near the sediment interface were used to explain dissolved oxygen depletion and nitrate increase in the hypolimnion during stratification. The average sinking velocities were 1.82m d^-1 for seston and 1.16 m d^-1 for Chl a at 30m, they were influenced by Chl a content of seston.     

Induction of tumor growth inhibitory factor (TGIF) in human mononuclear cells by OK-432, a streptococcal preparation

Summary A tumor growth inhibitory factor (TGIF) was induced in the culture supernatant from mixed culture of human peripheral blood mononuclear cells (PBMC) and a streptococcal preparation, OK-432, in vitro. The activity generated in the supernatant increased in a time-dependent fashion and first appeared 6 h after the initiation of culture, reaching its maximum around 48 h. The TGIF was cytostatic against seven of ten human tumor targets, but not against three murine tumor targets. Tumor cell growth was inhibited by a transient contact, i.e., 1 h, with TGIF. The TGIF was produced by lymphocytes but not by monocytes, because the activity was usually enhanced by elimination of plastic-adherent cells from the original PBMC fraction. The TGIF was relatively stable against heating at 56° C for 30 min, but the activity was totally destroyed after heating at 70° C for 5 min. The molecular weight of TGIF was estimated to be about 43×10³ daltons by gel filtration. No interferon (IFN) activity was detected in the TGIF-positive fractions obtained by gel filtration, and the TGIF-positive fractions did not inhibit the growth of tumor necrosis factor (TNF)-sensitive mouse L929 cells. The TGIF activity was not significantly affected in neutralizing tests using specific antibodies against human IFN and TNF. The OK-432 was administered i.p. for management of cancer patients with malignant ascites. Ascites-derived mononuclear cells (ASMC) were obtained before and 3 to 5 days after OK-432 injection. The ASMC obtained after the injection produced TGIF in vitro in the absence of OK-432; the preinjection ASMC showed no such production. A positive correlation was found between TGIF-producing activity by ASMC and the effect of OK-432 injection on ascites volume. These results indicate that TGIF is induced in mononuclear cells by OK-432 not only in vitro but also in vivo and plays an important role in inhibition of tumor growth in cancer patients.     

Mouse lymphoma L1210 cells acquire a new cystine transport activity upon adaptation in vitro

Summary Mouse lymphoma L1210 cells maintained in vitro at a high cell density for a certain time period adapted themselves to the in vitro environment and were able to grow indefinitely. From these adapted cells, more than 30 clones were isolated. They all had much higher activity to take up cystine than the original L1210 cells, supporting a previous view that the deficiency of the cystine uptake limits the survival and growth of L1210 cells in vitro. The cystine uptake of one cloned cell line was characterized. The enhanced uptake of cystine in these cells was mainly mediated by a Na⁺-independent, saturable system and was potently inhibited by glutamate and some other anionic amino acids, but less by aspartate. Such activity of cystine uptake was not observed in the original L1210 cells. The results suggest that, upon adaptation in vitro, L1210 cells acquire a new cystine transport activity necessary for survival and growth in vitro.     

Immunological Determination of Labeling Index on Human Tumor Tissue Sections Using Monoclonal Anti-Brdurd Antibody

The use of a monoclonal antibody against the thymidine analogue bromodeoxyuridine together with an in vitro labeling technique allowed rapid determination of the labeling index in human tumors. The labeling index estimated by these relatively simple immunofluorexence or immunoenzymatic staining methods was equivalent to that obtained by autoradiography. The interpretation of the preparations is easy since there is a minimum of background staining. This immunohistochemical technique combined with in vitro labeling provides a suitable alternative for determining the labeling index of human tumors.     

Genetic analysis for insulitis in NOD mice

Non-obese diabetic (NOD) mice spontaneously develop diabetic signs akin to those of Type I diabetes in man. Insulitis, i.e., lymphocytic infiltration around and into the pancreatic islets is one of the characteristics of such mice. It is also the etiologic pathological lesion in the development of diabetes mellitus in NOD mice. Thus, we chose insulitis as a marker for genetic analysis of the development of diabetes mellitus in NOD mice and clarified the mode of its inheritance. In breeding studies between NOD and C57BL/6J mice, insulitis was not observed in the F1 and (F1 X C57BL/6J) backcross generations, but was found with incidences of 3.9% in females and 1.4% in males in the F2 generation and 23.7% in females and 12.1% in males in the (F1 X NOD) backcross generation. These incidences in the F2 and (F1 X NOD) backcross females corresponded approximately to 1/16 and 1/4 of the incidences of 89.7% in the NOD females, respectively. A similar relationship was observed between the F2 and (F1 X NOD) backcross males and the NOD males. When the gene expressivity of both sexes for a double recessive homozygote was assumed to be the incidences of insulitis in 9-week-old NOD females and males, respectively, the expected numbers of both sexes with and without insulitis in the F2 and backcross generations agreed well with the observed ones. These observations suggest that two recessive genes on independent autosomal chromosomes are necessary for the development of insulitis in NOD mice.     

Carboxyl Terminal Segment of T4 Gene 32 Protein is Dispensable in Vivo

We obtained a mutant of bacteriophage T4 which overcame thedeficiency in gene 49 endonuclease. The new mutation occurredin gene 32 and the mutant, which was viable, produced an amberfragment under non-suppressed conditions, lacking about 30 aminoacid residues at the carboxyl terminus. Its growth, recombination,and resistance to UV irradiation were affected to various degreesby the particular suppressor tRNA present. Growth was increasedby Su2⁺ to nearly that of the wild type, but growth of all otherswas reduced in the presence and absence of suppressors, suggestingthat the terminal domain of gene 32 protein is not indispensablefor the function but modulates it. We discuss the mechanismby which the mutation overcomes the defect in gene 49 endonuclease. ¹ This paper is dedicated to the memory of the late Dr. JojiAshida. (Received November 22, 1982; Accepted February 21, 1983)