Cellular targets and trophic functions of neurotrophin-3 in the developing rat hippocampus.

The expression of the neurotrophins and trk receptors in the hippocampus has directed attention toward their roles in the development and maintenance of this region. We have examined the effects of the neurotrophins NT-3, BDNF, and NGF in cultures of developing rat hippocampal cells by two criteria: rapid induction of c-fos and neurotrophic responses. The selective induction of c-fos mRNA suggests the presence of functional receptors for NT-3 and BDNF, but not NGF, in embryonic hippocampal cultures. The NT-3-responsive cells were localized in pyramidal neurons of areas CA1 through CA3 and dentate granular and hilar cells of postnatal organotypic slices, as detected by c-Fos immunocytochemistry. In addition to immediate early responses, NT-3 caused a 10-fold increase in the number of cells expressing the neuronal antigen calbindin-D28k. This increase was dose dependent, with maximal stimulation at 10 ng/ml. In contrast, BDNF elicited small but significant calbindin responses. These results indicate biological responses to NT-3 in the CNS and suggest roles for for this neurotrophin during hippocampal neurogenesis.     

Biotransformation of germacrane-type sesquiterpenoids by Aspergillus niger

Three germacrane-type sesquiterpenoids, (+)-germacrone-4,5-epoxide, germacrone and (+)-curdione were biotransformed by Aspergillus niger to give hydroxylated guaiane-type sesquiterpenoids together with allylic alcohols and spirolactone.     

Distribution of 3H in rat conceptus cultured in vitro following brief administration of [3H]thymidine

Rat embryos with intact visceral yolk sacs, explanted at 12 1/2 days of gestation, were cultured in vitro for up to 60 min in medium consisting of fetal calf serum, Eagle's MEM, and [3H]thymidine (1.2 kBq ml-1), using the roller bottle method. The total amount of 3H incorporated into the conceptus during the 60-min incubation was 79.2 Bq, and approximately 33, 23, and 44% of this activity was distributed to the embryo, the yolk sac, and the fluid in the exocoelom and amniotic cavity, respectively. The rate of 3H accumulation in conceptuses decreased with time in culture. It appeared that the decrease in the viability of the conceptus was not responsible for this phenomenon. The concentration of 3H in the yolk sac, i.e., 3H activity per gram wet weight, was 2.1 times that in the medium at the end of culture. In contrast, the 3H concentration in the embryo was significantly lower than that in the medium. These findings suggest that the visceral yolk sac of rat conceptuses may act as a barrier to the transport of tritiated thymidine between the medium and embryo.     

Biosensor system for continuous flow determination of enzyme activities. II. Simultaneous determination of plural enzyme activities.

A biosensor system for continuous flow determination of plural enzyme activities was prepared from the combination of two pyruvate sensors, a prereactor and a flow cell. This system was applied to the simultaneous determination of lactic dehydrogenase (LDH) and glutamic-pyruvic transaminase (GPT) activities in the same sample. These enzyme activities can be determined by measuring pyruvate produced by the enzyme reactions as follows. The amount of pyruvic acid can also be determined from the amount of oxygen consumed upon oxidation of pyruvic acid by pyruvate oxidase. (Formula: see text). Therefore, both of the detectors for the determination of lactic dehydrogenase and glutamic-pyruvic transaminase activities were prepared from the combination of a pyruvate oxidase membrane and an oxygen electrode. Pyruvate oxidase was covalently immobilized on a membrane prepared from cellulose triacetate. A linear relation was obtained between the output current and LDH or GPT activities in the range of 50 to 3,600 IU l-1 or 6 to 1,000 IU l-1, respectively. Each assay of these enzyme activities was completed within 15 min. The results obtained had a precision of ca. 4%. The sensor was stable for more than 25 days at 5 degrees C.     

Presence of immunoreactive endothelin in human saliva and rat parotid gland.

The presence of immunoreactive endothelin (IR-ET) in human saliva and rat parotid gland was investigated by radioimmunoassay. The IR-ET concentration (mean +/- SEM) in saliva taken from normal volunteers was 2.0 +/- 0.2 pmol/l (n = 15). The IR-ET concentration in rat parotid gland was 19.2 +/- 2.2 fmol/g wet weight (n = 10). Fast protein liquid chromatography (FPLC) of human saliva extract revealed 6 peaks; one peak eluting in the void volume, one in a position between ET-1 and -3, and the other four in the positions of synthetic ET-1, -2, -3 and big ET(1-38), respectively. A similar pattern of rat parotid gland extract was noted with FPLC, except that there was no peak after the void volume. Presence of endothelin, a potent growth factor, in saliva and salivary gland points to a role in maintaining the integrity of the oral and gastrointestinal tract mucosa.     

Epithelio--mesenchymal interactions are critical for Quox 7 expression and membrane bone differentiation in the neural crest derived mandibular mesenchyme.

In higher vertebrates, branchial arch mesenchyme (ectomesenchyme) is derived from the cephalic neural crest. The ectomesenchyme of the mandibular arch yields the Meckel's cartilage and several membrane bones. We previously reported the isolation of a quail homeobox gene, Quox 7. In common with its mouse counterpart Hox 7, Quox 7 is highly expressed in the medioventral part of the mandibular arch and later in the precursor cells of the membrane bones. Since bone differentiation from ectomesenchyme is strictly dependent upon a signal provided by the mandibular epithelium, we decided to see whether the regulation of Quox 7 gene activity might be correlated with epithelio--mesenchymal interactions. Quox 7 expression was studied in E3 mandibular ectomesenchyme cultured in vitro or grafted on the chick chorioallantoic membrane either alone or recombined with the homotopic and heterotopic epithelia. We found that Quox 7 mRNA was undetectable after 48 h in cultures of mesenchyme alone while it remained abundant in non-cartilaginous tissue of the mandibular arch ectomesenchyme recombined with its own epithelium. The signal provided by the mandibular epithelium for Quox 7 expression can also arise from various heterotopic epithelia, e.g. of dorsal or ventral body wall and of limb bud. Thus the effect of the epithelium on Quox 7 expression in mesenchymal cells strictly parallels that on bone formation. These results strongly suggest that the epithelio-mesenchymal interactions have an essential role on the regulation of Quox 7 gene, the product of which seems to be, in turn, necessary for the execution of the skeletal developmental program in the facial area.     

Mapping of the MYC gene to band 8q24.12----q24.13 by R-banding and distal to fra(8)(q24.11), FRA8E, by fluorescence in situ hybridization

The MYC gene was mapped to R-banded human prometaphase chromosomes and to chromosomes expressing fra(8)(q24.11) by fluorescence in situ hybridization. By high-resolution banding analysis, the fluorescent signals were localized to R-positive band q24.12----q24.13 of the long arm of chromosome 8. Furthermore, the signals were localized near the middle part, q24.12----q24.13, of the distal portion of fra(8)(q24.11) expression. Thus, the precise localization of MYC was to the subband 8q24.12----q24.13.     

Morphology of the granular hemocytes of the Japanese horseshoe crabTachypleus tridentatus and immunocytochemical localization of clotting factors and antimicrobial substances

Summary The structure of hemocytes in the normal state and during blood coagulation, and the intracellular localization of three clotting factors and two antimicrobial factors were examined in the Japanese horseshoe crabTachypleus tridentatus. Two types of hemocytes were found in the circulating blood: non-granular and granular hemocytes. The latter contained numerous dense granules classed into two major types: L- and D-granules. The L-granules were larger (up to 1.5 m in diameter) and less electron-dense than the D-granules (less than 0.6 m in diameter). The L-granules contained three clotting factors and one antimicrobial factor, whereas the D-granules exclusively contained the other antimicrobial factor. After treatment with endotoxin, the L-granules were released more rapidly than the D-granules, although almost all granules were finally exocytosed. The granular hemocyte possessed a single Golgi complex; possible precursor granules of L-granules and D-granules contained tubular and condensed dense material, respectively. These data are discussed in relation to the self-defense mechanisms of the horseshoe crab.     

Polyol pathway in tissues of spontaneously diabetic Chinese hamsters (Cricetulus griseus) and the effect of an aldose reductase inhibitor, ONO-2235

1. Sorbitol and fructose levels were significantly elevated in the lens, the sciatic nerve, the retina and the kidney of diabetic Chinese hamsters and inositol level was significantly decreased in the lens and sciatic nerve of diabetics. 2. The activity of an aldose reductase in the kidney was not different between normal and diabetic Chinese hamsters. 3. An aldose reductase inhibitor (ONO-2235) had no effect in sorbitol, fructose and inositol contents of all these tissues from diabetic Chinese hamsters. 4. These results suggest that diabetic Chinese hamsters produce polyol accumulation in tissues but that there is a clear species-specific difference to inhibition of aldose reductase.     

Isolation and regional localization of a large collection (2,000) of single-copy DNA fragments on human chromosome 3 for mapping and cloning tumor suppressor genes

Summary A collection of 2,000 lambda phage-carrying human single-copy inserts (> 700 bp) were isolated from two chromosome-3 flow-sorted libraries. The single-copy DNA fragments were first sorted into 3p and 3q locations and about 700 3p fragments were regionally mapped using a deletion mapping panel comprised of two humanhamster and two-human-mouse cell hybrids, each containing a chromosome 3 with different deletions in the short arm. The hybrids were extensively mapped with a set of standard 3p markers physically localized or ordered by linkage. The deletion mapping panel divided the short arm into five distinct subregions (A-E). The 3p fragments were distributed on 3p regions as follows: region A, 26%; B, 31%; C, 4%; D, 4% and E, 35%. We screened 300 single-copy DNA fragments from the distal part of 3p (regions A and B) with ten restriction endonucleases for their ability to detect restriction fragment length polymorphisms (RFLPs). Of these fragments 110 (36%) were found to detect useful RFLPs: 35% detected polymorphisms with frequency of heterozygosity of 40% or higher, and 25% with frequency of 30% or higher. All polymorphisms originated from single loci and most of them were of the base pair substitution type. These RFLP markers make it possible to construct a fine linkage map that will span the distal part of chromosome 3p and encompasses the von Hippel-Lindau disease locus. The large number of single-copy fragments (2,000) spaced every 100–150 kb on chromosome 3 will make a significant contribution to mapping and sequencing the entire chromosome 3. The 300 conserved chromosome 3 probes will increase the existing knowledge of man-mouse homologies.