Migration Behavior of Granule Cell Neurons in Cerebellar Cultures. II. An Electron Microscopic Study

We examined the fine structure of migrating granule cell neurons in cerebellar microexplant cultures. Radially migrating bipolar cells extended microspikes or small filopodia from their soma and processes and frequently made contact with neighboring cells. These microspikes contained microfilaments but no microtubules. At the later phase of the migration, in which they had symmetrical bipolar long processes, filopodia extending from perikarial region of cells contained microtubules, suggesting that they are precursors of the future thick perpendicular processes. When cell bodies changed orientation from radial to perpendicular, microtubules that were nucleated from perinuclear centrioles frequently extended into both thick radial and perpendicular processes from the perikarial region. Bundles of 10nm intermediate filaments also appeared in these processes. During migration by the perpendicular contact guidance, many filopodia extending from both the thick leading processes and thin trailing processes made close contacts with the radial parallel neurite. These findings suggest that; 1) The direct contact of the filopodia from both the growth cones and their processes of the granule cells to the neurite bundle plays roles in both the parallel and perpendicular contact guidances. 2) The spacial and temporal changes of cytoskeletons and the association of microtubules with perinuclear centrioles are important for the formation of perpendicular processes and initiation of the perpendicular contact guidance.     

Conformation of (2→1)-β-d-fructan in aqueous solution

The conformation and dilute solution properties of (2→1)-β-d-fructan in aqueous solution were studied by gel permeation chromatography, low-angle laser light-scattering photometry, viscometry, small-angle X-ray scattering and electron microscopy. Fractions covering a broad range of weight-average molecular weights (M_w) from 1.49 × 10⁴ to 5.29 × 10⁶ were obtained from a native sample by ultrasonic degradation and fractional precipitation. For M_w < 4 × 10⁴, the intrinsic viscosity [η] varies with M_w^0.71, indicating that the fructan chain behaves as a random coil expanded by an excluded-volume effect in this molecular weight region. For M_w > 10⁵, [η] exhibits an unusually weak dependence on M_w and finally becomes almost independent of molecular weight. This behaviour is interpreted in terms of a globular conformation of the high-molecular-weight fructan molecules. Small-angle X-ray-scattering measurements and electron microscopic observations support this interpretation of the values of [η] observed.     

Meristem-specific gene expression directed by the promoter of the S-phase-specific gene,cyc07, in transgenic Arabidopsis

A genomic clone for the cyc07 gene, which is expressed specifically at the S phase during the cell cycle in synchronous cultures of periwinkle (Catharanthus roseus) cells, was isolated. Determination of the nucleotide sequence of the clone revealed that the cyc07 gene consists of seven exons separated by six introns. Genomic Southern analysis indicated that the cyc07 gene is present as a single copy per haploid genome in periwinkle. Expression of related genes was detected in a wide range of other plants. Transgenic Arabidopsis plants were generated that expressed the gene for -glucuronidase (GUS) under the control of the promoter of the cyc07 gene. The tissue-specific pattern of expression directed by the promoter was investigated by analysis of GUS activity. Histochemical tests demonstrated that 589 bp of the 5-upstream sequence of the cyc07 gene could direct specifical expression of the GUS reporter gene in meristematic tissues in transgenic plants. The spatial pattern of expression directed by the promoter was closely correlated with meristematic activity and cell proliferation, suggesting an association between the function of the cyc07 gene and cell proliferation.     

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.     

Immunocytochemical study of tyrosine hydroxylase and dopamine β-hydroxylase immunoreactivities in the rat pancreas

An immunohistochemical and immunoelectron microscopic study was used to demonstrate tyrosine hydroxylase (TH) and dopamine -hydroxylase (DBH) immunoreactivities in the rat pancreas. Small TH immunoreactive cells were found in close contact with large TH immunonegative ganglion cells among the exocrine glands and were occasionally found in some islets. Some of these TH immunoreactive cells were also DBH immunopositive. The immunoreaction product was seen diffusely in the cytoplasm and in the granule cores of TH immunoreactive cells. All intra-pancreatic ganglion cells were immunoreactive for DBH, but not for TH. The TH immunoreactive cells were identified as small intensely fluorescent (SIF) cells due to their localization and morphological characteristics and showed no insulin, glucagon, somatostatin or pancreatic polypeptide immunoreactivities. These results indicate that SIF cells may release dopamine or noradrenaline to adequate stimuli while the intra-pancreatic ganglion cells with only DBH may not synthesize catecholamines in a normal biosynthetic pathway. TH immunoreactive nerve bundles without varicosities and fibers with varicosities, associated or unassociated with blood vessels, were found in both the exocrine and endocrine pancreas. Close apposition of TH immunoreactive nerve fibers to the smooth muscle and endothelial cells of the blood vessels was observed. A close apposition between TH immunoreactive nerve fibers and exocrine acinar cells and islet endocrine cells was sometimes found in the pancreas. The immunoreaction product was seen diffusely in the axoplasm and in the granular vesicles of the immunoreactive nerve fibers. Since no TH immunoreactive ganglion cells were present in the rat pancreas, the present study suggests that noradrenergic nerve fibers in the pancreas may be extrinsic in origin, and may exert an effect on the regulation of blood flow and on the secretory acitivity of the acinar cells, duct cells and endocrine cells.     

A novel complex mutation in the LDL receptor gene probably caused by the simultaneous occurrence of deletion and insertion in the same region

A novel complex mutation with the presence of both deletion and insertion in very close proximity in the same region was detected in exon 8 of the LDL receptor gene from two apparently unrelated Japanese families with familial hypercholesterolemia (FH). In this mutant LDL receptor gene, the nine bases from nucleotide (nt) 1115 to nt 1123 (AGGGTGGCT) were replaced by six different bases (CACTGA), and consequently the four amino acids from codon 351 to 354, Glu-Gly-Gly-Tyr, were replaced by three amino acids, Ala-Leu-Asn, in the conserved amino acid region of the growth factor repeat B of the LDL receptor. The nature of the amino acid substitution and data on the families suggest that this mutation is very likely to affect the LDL receptor function and cause FH. The generation of this complex mutation can be explained by the simultaneous occurrence of deletion and insertion through the formation of a hairpin-loop structure mediated by inverted repeat sequences. Thus this mutation supports the hypothesis that inverted repeat sequences influence the stability of a given gene and promote human gene mutations.     

Restriction Fragment Length Polymorphism (RFLP) of Genomic DNA of Moraxella (Branhamella) catarrhalis Isolates in a Hospital

Epidemiological typing, based on restriction fragment length polymorphism (RFLP) by pulsed-field gel electrophoresis (PFGE), was attempted for the 38 clinical isolates of Moraxella catarrhalis obtained at Shinshu University Hospital during the years 1987 and 1993. Digestion with SmaI or NotI generated well separable, 12 to 5 genomic DNA fragments ranging from 1,000 kb to 30 kb and the strains could be classified into 14 or 13 types, respectively. The electrophoretic profile differed with the strain in most of them and was hence useful to distinguish the each strain. Investigation for their RFLP have, however, suggested that majority of them, including the type strain ATCC25238, may have derived from a common ancestor.     

Seasonal change in the food habits of the Iriomote catFelis iriomotensis

The food habits of the Iriomote catFelis iriomotensis were studied by analyzing 177 feces collected monthly from 1987 to 1988. A total of 26 food items were identified. The frequency of lizards and frogs in the feces was higher than those of mammals and birds. The food habits changed greatly seasonally.Eumeces skinks were fed on most frequently, and found in the feces with a similar high frequency occurrence in March–April and July–September, while their proportion to the total number of food items was larger in March–April than in July–September. The cats fed on larg-sized skinks, adultEumeces kishinouyei, more in March–April than in other seasons. The number of skinks sighted in the course of a road census was greater from March to August, and large-sized skinks were sighted more in March–April than in July–August. The cats fed selectively on large-sized skinks in every season. Therefore, changes in the food habits depended on the food availability. Characteristics of food habits in the Iriomote cat are discussed in comparison with the food habits of other felids in temperate and tropical regions.     

Axillary bud flowering after apical decapitation in Pharbitis in relation to photoinduction

The flowering response of axillary buds of seedlings of Pharbitis nil Choisy, cv. Violet, was examined in relation to the timing of apical bud removal (plumule including the first leaf or second leaf) before or after a flower-inductive 16-h dark period. When the apical bud was removed well before the dark period, flower buds formed on the axillary shoots that subsequently developed, but when removed just before, or after, the dark period, different results were observed depending on the timing of the apical bud removal and plant age. In the case of 8-day-old seedlings, fewer flower buds formed on the axillary shoots developing from the cotyledonary node when plumules were removed 20 to 0 h before the dark period. When the apical bud was removed after the dark period, no flower buds formed. Using 14-day-old seedlings a similar reduction of flowering response was observed on the axillary shoots developing from the first leaf node when the apical bud was removed just after the dark period. To further elucidate the relationship between apical dominance and flowering, kinetin or IAA was applied to axillary buds or the cut site where the apical bud was located. Both chemicals influenced flowering, probably by modulating apical dominance which normally forces axillary buds to be dormant.