Vascular endothelial growth factor and its receptor Flk-1 are expressed in the hippocampus following entorhinal deafferentation.

Vascular endothelial growth factor (VEGF) has been thought of as a mitogen that promotes proliferation of endothelial cells and as a neurotrophic factor that stimulates neurogenesis and axonal growth in both peripheral and central nervous systems. To investigate the potential involvement of VEGF in the lesion-induced reorganization in the brain, the expression changes of VEGF and its receptor Flk-1 were analyzed in the mouse hippocampus after transections of the entorhinal afferents. In situ hybridization and immunohistochemistry showed the time-dependent expression upregulation of VEGF mRNA and protein in the entorhinally denervated hippocampal stratum lacunosum-moleculare and dentate outer molecular layer, which initiated by 3 days postlesion, reached its maximum at 7-15 days postlesion, still persisted by 30 days postlesion for protein, and recovered to the normal levels at 30 days postlesion for mRNA and at 60 days postlesion for protein. Double labeling of VEGF and glial fibrillary acidic protein revealed that VEGF-expressing cells in the denervated areas were reactive astrocytes. Semi-quantitative RT-PCR analysis showed that VEGF receptor Flk-1 mRNA was also time-dependently upregulated in the deafferented hippocampus with its maximal elevation at 7-15 days postlesion while the Flt-1 mRNA levels remained unchanged at any time point we examined. Immunohistochemistry analysis also displayed the upregulation of Flk-1 protein in the denervated stratum lacunosum-moleculare and outer molecular layer with a time course similar to that of VEGF mRNA upregulation. Flk-1 receptors were found to be expressed not only by reactive astrocytes but also by neurites, which most likely belong to sprouting axons by 7 days postlesion and regrowing dendrites by 15-30 days postlesion. From these data we suggest that the spatiotemporal upregulation of VEGF and Flk-1 in the hippocampus is induced by entorhinal deafferentation and that VEGF may be involved in the structural reorganization in the deafferented hippocampus via directly or indirectly promoting neurite growth.

Kinetic studies on the mechanism of atomization in electrothermal atomic absorption spectrometry with and without chemical modifiers.

Over the past decade a few methods for determining kinetic data for atom formation from the absorption signal in electrothermal atomic absorption spectrometry (ETAAS) have been developed in the author's laboratory. These approaches include improvement to the Smets method, and development of new methods for simultaneous determination of kinetic order and activation energy for atom formation at increasing or constant temperatures. The steady-state approximation and first-order kinetic assumption for atom formation have been avoided during their derivation. One of the most distinct features of these new methods is their suitability for quantitative determination of the kinetic order for atom formation from absorption signals under normal analytical conditions, even for atomization processes with fractional reaction orders and/or with multiple mechanisms. The application of the developed methods to the study of the mechanism of atomization in the graphite-furnace atomizer, with and without chemical modifiers, is reviewed with emphasis on research work in the authors' laboratory.

[Recent developments of the atomization kinetics in electrothermal atomic absorption spectrometry].

Recent advances of the atomization kinetics in electrothermal atomic absorption spectrometry are critically reviewed with 31 references during 1994-2000. These include new methods for the determination of kinetic parameters, a two-parameters, a two-precursor model for the kinetics of analyte atomization, a kinetic model of atomization with redeposition, and the influences of matrices, chemical modifiers and atomizer surfaces on analyte atomization in an electrothermal atomizer.

Determination of eight synthetic food colorants in drinks by high-performance ion chromatography.

Eight synthetic food colorants (Amaranth, Brilliant Blue, Indigo Carmine, New Red, Ponceau 4R, Sunset Yellow, Tartrazine, Allura Red) were determined by high-performance ion chromatography on an anion-exchange analytical column with very low hydrophobicity and visible absorbance detection. Gradient elution with hydrochloric acid-acetonitrile effected both the chromatographic separation of these colorants and the on-line clean-up of the analytical column, which was very advantageous for routine analysis. High-performance ion chromatography may be a solution to the chromatographic analysis for some water-soluble, organic analytes with strong hydrophobicity. The method has been applied to the determination of colorants in drinks and in instant drink powder. No time-consuming pretreatment, as used in conventional liquid chromatography, was needed.

Human chromosome pellicle antibody recognizing centromere protein-C (CENP-C), the main component of the kinetochore.

Recently the antichromosome antisera from several scleroderma patients have been found to recognize the pellicle of metaphase and anaphase chromosomes. In order to identify the pellicle components, we used these antichromosome antisera to screen a human embryonic cDNA library. The sequences of the positive clones are identical to the cDNA gene sequence of CENP-C (centromere protein C), a human centromere autoantigen. This result suggests that CENP-C is a component of the pellicle of human metaphase and anaphase chromosomes.

[Expression of foreign tyrosine hydroxylase gene in the rat model of Parkinson's disease].

A total of 75 adult rats were used to produce hemiparkinson's disease (PD) model by unilateral injection of 6-hydroxydopamine (6-OHDA) into the right substantial nigra. Four weeks following the lesions, rats were tested for apomorphine (APO) -induced asymmetric rotation. Only rats that showed more than seven turns per minute were selected as PD models for transplantation. A tyrosine hydroxylase expression plasmid (pSVK3-TH) mixed with lipofectin was transplanted into the right striatum of 15 PD rats. Another 20 PD rats transplanted either with lipofectin or with pSVK3-TH served as controls. Every three days following transplantation, the performance of asymmetric rotation were tested and stained with the TH-immunohistochemistry. Only the animals grafted with pSVK3-TH mixed with lipofectin were found to show the decreased rotational behavior and a few TH-positive cells in the right striatum at the postgraft day 3-12, but not day 18. The results indicate that there exists a correlation between amelioration of asymmetric rotation and the TH gene expression in the denervated striatum. It is suggested that the lipofectin may mediate foreign TH gene expression in the PD rat brain.

[The molecular cloning of mouse centromeric DNA (SFA-DNA)].

The centromeric DNA (SFA-DNA) isolated from enriched mouse centromeric preparation has been cloned. The vector adopted for the cloning was ELBL3. About two thousand clones were obtained. The average size of the SFA-DNA inserts was estimated by measuring the inserts obtained from a mixture of 20 radomly picked clones. It was found that the average size of the SFA-DNA inserts is approximately 14 Kb.

[Fate of human fetal dopamine neurons transplanted into rhesus monkey model of Parkinson's disease: a tyrosine hydroxylase immunocytochemical study].

To predicate the value of human fetal substantia nigra transplantation in clinical treatment of Parkinson's disease (PD), dissociated cells of substantia nigra from 8-12 week old abortive human fetus were grafted into the neostriatum of 5 adult rhesus monkeys with hemiparkinsonism induced by unilateral injection of MPTP. At 2, 5 and 12 months after transplanting the monkeys were sacrificed for tyrosine hydroxylase (TH) immunocytochemistry to examine the survival and possible synaptic contact of transplanted dopamine (DA) neurons. Transplanted TH immunoreactive cells took a pattern of patches scattered in the neostriatum. Each of the cell patches consisted of 3-10 cells. The TH immunoreactive fiber network was seen in the neostriatum. Electron microscopic survey revealed that TH+ buttons arising from grafted DA neurons formed symmetric or asymmetric synapses with TH- dendritic shafts/spines, and TH+ dendrites were seen to form synapses with TH- axons of the host. Additionally, there were a few synapses formed by TH+ axonal terminals with negative buttons. The results suggest that DA neurons from 8-12 week old abortive human fetus are able to survive grafting into the neostriatum of monkey, a species phylogenetically very close to human, and to establish reciprocal synaptic connectivity with the host even at 2 months post-transplanting. It is, therefore, inferable that embryonic human DA neurons transplanted into human neostriatum may have the same fate as in monkeys.

[Studies on the centrosome antiserum from a scleroderma patient].

Using ascites cells as screening system and by means of indirect immunofluorescence microscopy, several antisera against centrosome from scleroderma patients were discovered. Since centrosome is chemically complex cellular structure and the autoimmune antiserum is polyclonal, further investigation was made using one of the antisera against centrosome. L929 cultured cells were also employed for the antigen localisation. It was found that the antiserum decorated microtubules, mitotic spindle, centrosome as well as some nuclear structure. Immunoblots of the cell lysate with the antiserum revealed that in addition to the main bands of tubulin there were several less distinct bands. This result confirmed the indirect immunofluorescence observation.

[The study of metaphasic chromosome pellicle].

Among 12 antichromosome antisera from scleroderma patients, four were found to be the antisera against the pellicle of metaphase chromosomes. Western blotting with these sera were achieved on the protein bands resolved by SDS-PAGE of the whole cell lysate and nuclear lysate of the ascites cells. The result showed 11 bands of the nuclear lysate reacted with antisera. Moreover, additional 8 were obtained in the whole cell lysate.

[Fluorescent in situ hybridization of enriched SFA-DNA on pachytene chromosomes in mouse].

In the present study, the primary constrictions of mouse pachytene chromosomes stained with Hoechst 33258, and those stained by indirect immunofluorescence method with CREST antiserum were compared with those hybridized by the probe DNA isolated from the enriched mouse spindle fiber attachments. It was found that the DNA probe hybridized not only with the centromeric regions on synaptonema complexes but also with the DNA of pericentromeric heterochromatin of all autosomes. Moreover, the DNA probe hybridized also with the centromeric regions of both X and Y chromosomes. It was concluded that the DNA isolated from the enriched mouse spindle fiber attachments contains a complete set of the centromeric DNAs of all autosomes, X and Y chromosomes.

Kinetics of indium atomization from different atomizer surfaces in electrothermal atomic absorption spectrometry (ETAAS).

The kinetic parameters of indium atomization in electrothermal atomic absorption spectrometry (ETAAS) have been determined by a newly proposed method. Effect of the atomizer surface and the palladium modifier on the kinetics of indium atomization has been investigated. The mechanisms of indium atomization seem to be identical for the pyrolytically coated graphite and the uncoated graphite tubes, i.e. the rate-limiting step for the atomization changes from a first order kinetics at lower temperatures into a nearly 1/3 order kinetics at higher temperatures, which may suggest that the analyte moves from a dispersed state to agglomates with increasing temperature. However, for the zirconium coated graphite tube, the atomization of indium is controlled by a single mechanism with the kinetic order of near 2/3 and the activation energy of 186 +/- 13 kJ/mol. Relatively weak indium-zirconium carbide interactions and the release of indium from the sphere of molten indium metal on the zirconium coated surface are suggested. In the presence of palladium, a simple mechanism, i.e. the release of indium from the solid solution of the In and the Pd on the pyrolytically coated graphite surface, is proposed to account for the observed first order kinetics and the activation energy of 421 +/- 27 kJ/mol.

[The characteristic of the spindle fiber attachment (SFA) enriched fraction from mouse nuclei].

The characteristics of the particulate mouse centromere enriched fraction from isolated nuclei obtained in our laboratory were investigated by indirect immunofluorescence, test of the activity of microtubule organizing center (MTOC), SDS-PAGE, and fluorescence in situ hybridization. Most of the particles of the fraction are complexes of DNA and kinetochore proteins and show MTOC activity. The DNA isolated from the fraction hybridizes the DNA in the regions of the primary constrictions of all chromosomes of ascite cells. The kinetochore proteins of the proteins isolated from the fraction are mainly those with molecular weight of 55 and 59 KD. It is evident that the fraction obtained is a centromere enriched nuclear fraction as indicated in our previous report.

[Types of anticellular structure antisera in scleroderma patients].

The sera of 338 scleroderma patients were cytologically examined with indirect immunofluorescence method. The sera were classified according to the cellular structure, to which they were against respectively. Among these sera, six main types of cellular structure antiserum were found: antikinetochore, antinucleolus, antichromatin, antiheterochromatin, antichromosome and anticentrosome. Some of these main types can further be subtyped with respect to the substructure or the chemical components (antigens) of a particular cellular structure. These types of antiserum may possibly be correlated with the clinical signs of scleroderma patients. For exploring the possibility to use for diagnosis.

[A study of transpial migration of implanted serotonergic neurons in rat spinal cord].

Transpial migration of implanted 5-HT neurons from the subarachnoid space into the spinal cord was studied in adult Wistar rats. Embryonic raphe tissue or cell suspension containing 5-HT cells was used as grafts. The implanted 5-HT cells were monitored by 5-HT immunohistochemical method. The results are as follows: (1) 10 d after cutting the spinal cord at lower thoracic level, 5-HT fibers disappeared in the transected spinal cord. (2) Raphe tissue was implanted into the subarachnoid space of the thoracic lumbar segment after the spinal cord was cut. One month later, 5-HT positive cells could be found in the transected spinal cord with fibers extending into both the grey and the white matters. (3) If the raphe cell suspension instead was implanted, a number of 5-HT positive cells appeared in the grey matter near the implanted region and the distribution of these cells in the grey matter was quite consistent with the implanted range of the cell suspension in the subarachnoid space. The 5-HT positive cells which had entered into the spinal cord sent out fibers and reestablished a new fiber network in the grey matter. (4) After implantation, the number of the 5-HT positive fibers in the transected grey matter became more and more sparsely distributed with increasing distance from the cell bodies and the 5-HT positive fibers reappeared in the white matter were much less than that in the grey matter. Present results show that the implanted 5-HT neurons are able to migrate transpially from the subarachnoid space into the spinal cord.

[Guiding effect of embryonic fimbria graft on cholinergic fiber growth in hippocampus of adult rats].

In view of the fact that in embryonic and neonatal central nervous system (CNS), the pathway of developing fiber tracts is capable of guiding the axonal growth, it would be interesting to know whether a similar effect exists on the axonal growth in adult CNS. Embryonic fimbria was grafted into the hippocampus of the adult rat. Two weeks later, the grafts were examined for cholinergic fibers with AChE histochemical method. It was found that a lot of cholinergic fibers appeared in the embryonic graft, but none of them in the adult fimbria graft as control. If the fimbria-fornix was transected at the time of grafting, no cholinergic fibers could subsequently be detected in both the embryonic graft and the host hippocampus. If a suspension of embryonic fimbria was used as a graft, only a few of long cholinergic fibers could be found in the grafted area. However, if tissue fragments of embryonic fimbria adhered to a strip of nitrocellulose filter were grafted as previously, numerous cholinergic fibers from the host hippocampus were found to be attracted around the strip and grow along the surface of the filter. The results seem to indicate that grafted embryonic fimbria or its tissue fragments are able to guide cholinergic fiber growth in adult hippocampus. It is possible that embryonic fimbria and other pathways of developing CNS fiber tracts provide a natural substrate for guiding axonal growth in adult CNS.

Enrichment of the spindle fiber attachments from mouse liver cell nuclei.

Spindle fiber attachments (SFAs) were enriched in a fraction of nuclei isolated from mouse livers. The enrichment method combines sonication, treatment with 2 mol/L NaCl and high speed centrifugation. SFA was enriched 27-fold on the average (n = 4) when measured by radioimmunoassay. The basic method offers opportunities for further increases of yield and for the enrichment of SFA of other vertebrates.

Involvement of a nucleolar component, perichromonucleolin, in the condensation and decondensation of chromosomes.

A component of the nucleolar material, perichromonucleolin (PCN), was identified by a specific antiserum against nucleoli obtained from a scleroderma patient. The distribution and changes of PCN during the mitotic cycle were followed using this antiserum and indirect immunofluorescence microscopy. Based upon the behavior of PCN during mitosis, it could be differentiated into chromosomal and nonchromosomal portions. During prophase the former gradually associates with the surface of the condensing chromosomes and forms the coat or pellicle around the metaphase and anaphase chromosomes. This pellicle PCN is carried by the anaphase chromosomes to the daughter nuclei. During the time from telophase to interphase, the pellicle PCN dissociates from the decondensing chromosomes and is incorporated into the new nucleoli. The nonchromosomal PCN, after the breakdown of the nuclear membrane, distributes in the cytoplasmic region around the condensed chromosomes and diffuses into the cytoplasm during anaphase. Preliminary biochemical analysis by immunoblotting showed that the PCN consists of two main proteins with molecular masses of 36 kDa and 30 kDa.

The demonstration of the spindle fiber attachments in the amphibian liver and sperm nuclei by the sera from scleroderma patients.

The isolated liver nuclei and the mature sperm nuclei from Rana nigromaculata and Bufo bufo asiaticus had been investigated by indirect immunofluorescence method with anti serum specific to the spindle fiber attachments (SFAs) from the scleroderma patients with CREST syndrome. It was found that in the isolated liver nuclei and the mature sperm nuclei of these animals there are discrete speckles which bind the antibody specific to the SFAs. The maximum number of these speckles counted in a single sperm nuclei of the frog corresponds with the haploid chromosome number of this animal (n = 13). The haploid chromosome complement of the toad is 11 and the maximum number of the speckles counted in a single sperm nucleus of this species is also 11. These findings suggest that the scleroderma serum can be used to identify the SFAs of the amphibias and the antigens of SFAs are conserved in the mature sperms of these animals. The implication of these conclusions is discussed.