Cell specificity and efficiency of the Semliki forest virus vector- and adenovirus vector-mediated gene expression in mouse cerebellum

Establishing efficient gene transfer and expression in post-mitotic neurons is important in understanding the genetic basis of neural circuits with cellular complexity. This study evaluates the properties of exogenous green fluorescent protein (GFP) expression mediated by the Semliki forest virus (SFV) and adenovirus (Ad) vectors in dissociated and slice cultures of the mouse cerebellum. Infection with SFV-GFP resulted in early-onset and high-level GFP expression in about 90% of Purkinje cells and in about 40% of granule cells in dissociated cultures at 1 day after infection. Two days after infection, GFP-positive cells showed signs of SFV-derived cytotoxicity. Ad-GFP infected almost all astrocytes and granule cells in dissociated cultures, and showed a steady increase in GFP fluorescence with a plateau at around 2 days post-infection. Ad vector-mediated GFP expression lasted for several weeks with no significant cell damage. In the slice cultures, both viral vectors mainly infected astroglial cells, but also showed a similar cell preference as that in dissociated cultures. These data indicate that the use of different viral vectors and infection conditions offers a powerful means of expressing exogenous genes in cerebellar cultures with different cell-type specificity and timing and duration of expression.     

Lumbar commissural interneurons with reticulospinal inputs in the cat: morphology and discharge patterns during fictive locomotion

The purpose of this study was 1). to characterize the morphology of lumbar commissural neurons (CNs) with reticulospinal inputs and 2). to quantitate their activity during locomotor rhythm generation. Intraaxonal recordings at the L4-7 level of the spinal cord were obtained in 67 neurons in the decerebrate, paralyzed cat. Fourteen of them were subsequently nearly fully visualized following their intraaxonal injection with the tracer neurobiotin. All 14 were CNs with axons projecting across the midline of the spinal cord. Their somata were located mainly in lamina VIII and additionally in laminae VII-VI. Most of the lamina VIII CNs were excited monosynaptically from reticulospinal pathways. They were judged to be interneuronal CNs if they had no, or a short, rostral projection. These CNs commonly gave off multiple axon collaterals in and around their somata's segmental level. They projected mainly to laminae VIII-VII and some additionally to lamina IX. Some laminae VIII and the laminae VII-VI CNs were excited polysynaptically from reticulospinal pathways or were not excited. They were judged to be long propriospinal or ascending tract CNs because they had only an ascending axon. Most lamina VIII CNs discharged rhythmically during fictive locomotion evoked by stimulation of the mesencephalic locomotor region, exhibiting one peak per locomotor cycle. The peak was in phase with neurographic activity of either a left or a right hindlimb extensor nerve. These results suggested that lamina VIII CNs are reciprocally connected bilaterally at each segmental level. Such an arrangement suggests their participation in the generation and coordination of reciprocal and bilateral locomotor activity.     

Differential expression of Homer family proteins in the developing mouse brain

Homer acts as a postsynaptic adaptor protein that links multiple targets, such as proteins involved in glutamate receptor signaling. We report the differential expression of the long form of Homer proteins produced from three distinctive genes during postnatal development of the mouse brain. Homer 1b/c and Cupidin/Homer 2a/b are widespread throughout the developing brain and are down-regulated in hindbrain-origin regions, such as the cerebellum, pons, and medulla oblongata. In contrast, Homer 3a/b is restricted to the cerebellum, hippocampus, and neonatal olfactory bulb. In the cerebellum, Homer 1b/c and Cupidin/Homer 2a/b predominate in the postsynapses of developing granule cells, whereas Homer 3a/b is concentrated in the dendritic spines of Purkinje cells and their axons. The down-regulation of Homer 1b/c and Cupidin/Homer 2a/b is in marked contrast to the up-regulation of Homer 3a/b between the first and the second postnatal weeks. In the hippocampus, Homer 1b/c and Cupidin/Homer 2a/b are largely located in the CA1 region and the CA1-CA2 region, respectively, whereas Homer 3a/b is largely distributed in the CA2-CA3 region and peaks around the third postnatal week. In hippocampal cell cultures, Homer 1b/c and Cupidin/Homer 2a/b are expressed in inhibitory and excitatory neurons, whereas Homer 3a/b is largely expressed in excitatory neurons but not in inhibitory neurons. In the developing olfactory bulb, Homer 1b/c and Cupidin/Homer 2a/b are up-regulated in the granular, external plexiform, and glomerular layers, whereas Homer 3a/b drastically decreases in these regions within the first postnatal week. Cupidin/Homer 2a/b is also expressed in olfactory sensory neurons within a distinct olfactory epithelial zone and is then widely distributed to both the axons in the olfactory nerve layer and the cilia in the olfactory epithelium. These results demonstrate that Homer family members have distinct regional, cellular, and subcellular distributions in time and space during postnatal brain development.     

Redox status of plasma coenzyme Q10 indicates elevated systemic oxidative stress in Parkinson's disease

Oxidative stress is suggested to play an important role in the pathogenesis of Parkinson's disease (PD). However, no elevation of plasma oxidative stress marker has been reported. We measured percent content of the oxidized form of coenzyme Q10 in total coenzyme Q10 (%CoQ-10) because %CoQ-10 has been shown to be a sensitive marker of oxidative stress. A slight but significant elevation in %CoQ-10 was observed in PD patients when compared with age/gender-matched normal subjects, suggesting elevated systemic oxidative stress in PD patients.     

Antisense therapy specific to mutated K-ras gene in hamster pancreatic cancer model. Can it inhibit the growth of 5-FU and MMC-resistant metastatic and remetastatic cell lines?

K-ras point mutation at codon 12 has a relationship greater than 90% with pancreatic cancer. Cancer therapy should also include the treatment of metastatic disease because it is known that the properties of metastatic cells may vary considerably from those of the primary tumor. AIM: To clarify if the same drugs, which can inhibit the tumor growth in the parental cell line, can inhibit the pancreatic metastatic and remetastatic cell lines at the same concentrations and to compare the inhibition with antisense oligonucleotides mismatched to K-ras gene, in Syrian golden hamsters. MATERIALS AND METHODS: HaP-T1, a BHP-induced hamster pancreatic cancer cell line, MS-PaS-1 (a metastatic cell line established from "return trip" metastases from the liver to the pancreas) and MS-PaS-2 named as a "remetastatic cell line", i.e., metastases from MS-PaS-1 were used. MTT and MTT-agarose assays were performed, using 5-Fluorouracil (5-FU), Mitomycin C (MMC) and antisense oligonucleotide specific to K-ras oncogene. RESULTS: The inhibitory concentration (IC50) of 5-FU, which inhibited HaP-T1, had to be increased by 50-fold to inhibit MS-PaS-1 and 100-fold to inhibit MS-PaS-2. MMC had to be increased by 10-fold to inhibit MS-PaS-1 and 50-fold to inhibit MS-PaS-2. However, IC50 was the same when antisense oligonucleotide was tried in these 3 cell lines. CONCLUSION: Antisense oligonucleotide-targeted K-ras gene may be a good choice for therapy because it could inhibit the growth in metastatic and remetastatic cells as well as in primary tumor cells.     

Distribution and disposition of benzalkonium chloride following various routes of administration in rats

Benzalkonium chloride (BZK) is a cationic surfactant used widely as a disinfectant, preservative and sanitizer in hospitals, at home and many public places. The toxicity of BZK is not well established although several human fatalities have been reported over the years. In this study, distribution and disposition of BZK following oral administration (PO) and intravascular (jugular vein (JV), femoral artery (FA), femoral vein (FV) and jugular artery (JA)) administration in rats were investigated along with pathological examinations. Toxic doses of 250 and 15 mg/kg of BZK were used for PO and intravascular administration, respectively. The fatal effects of BZK appeared soon in JV-, FV- or JA-rats, but took hours in PO or FA-rats. No rat receiving BZK via FA survived longer than 1 day. The PO-rats that aspirated BZK into their lungs had some systemic symptoms and higher blood and tissue concentrations of BZK. The blood BZK levels and kinetics were similar among the different routes of intravascular administration, but the lung and kidney levels were higher in JV-rats. Pathological examinations confirmed severe congestion and edema in the lungs and kidneys. These results suggest that (1) the toxic effects of BZK varied depending on the route of administration, (2) the degree of toxicity correlated with peak blood and tissue concentrations in orally dosed rats, (3) different toxicological progressions and manifestations were observed in FA- and JV-dosed rats even though these groups had similar blood concentration profiles, and (4) lung and kidney are reservoirs for BZK and considered to be the target organs of BZK.     

Tumor-promoting activity and mutagenicity of 5 termiticide compounds

The tumor-promoting activities of 5 commercial compounds used in termiticides were measured by a cell-transformation assay employing Bhas 42 cells. Their initiating activities were also measured by the microsuspension assay employing S. typhimurium TA98 and TA100 strains. The results of the transformation assay confirmed the tumor-promoting activities of fenitrothion, silafluofen and bifenthrin. Furthermore, the mutagenicity of S-421 and fenitrothion were also confirmed. Consideration of 2-stage carcinogenesis suggests that concurrent use of and long-term exposure to these compounds that have tumor-promoting and initiator activity, and compounds exhibiting either type of activity individually should be avoided as much as possible.     

Potential risk factors aggravating airway allergy--aspects of involvement of airtight housing and passive smoking

OBJECTIVES: The involvement of tightly insulated housing conditions and passive smoking in atopic sensitization, a major risk factor for airway allergy, was examined with nonsmoking adult women and school-age children. SUBJECTS AND METHODS: The subjects were 382 nonsmoking healthy adult women (housewives) who underwent medical examinations for prevention of adult diseases conducted in a district of Osaka from 1995 to 1997, and 214 elementary school-children 9-12 years old living in an urban district of Osaka who underwent medical examinations at a health center in April, 2000 to prevent allergic diseases. We also examined the correlation between tightly insulated housing conditions and the amount of passive smoking based on family smoking habits with 170 children under 12 years old who had been under the care of a hospital pediatrics department between December, 1993 and May, 1994. A questionnaire was administered to all subjects to survey the housing structure (concrete/wooden housing), family smoking habits and visible mold proliferation in the kitchen in relation to airtight housing conditions, passive smoking and exposure to inhalant allergens. Atopic sensitization was assessed by positivity for serum house dust mite-specific IgE, and passive smoking was defined as a urinary cotinine level of more than 6 ng/mgCr. RESULTS: 1. Among the three factors, indoor mold proliferation and family smoking habits were positively and synergistically related with atopic sensitization to house dust mites. 2. Airtight conditions of concrete housing showed a promotional effect on passive smoking for housewives, but a suppressive effect for school-age children. 3. Taking into account the above results, the promotional effects of passive smoking on atopic sensitization appeared predominantly in the concrete housing-residence group of housewives and the wooden housing-residence group of school-age children. 4. Effects of visible mold proliferation in the kitchen on atopic sensitization appeared predominantly in wooden housing-residence group of housewives. CONCLUSIONS: The results suggest that involvement of the three factors in atopic sensitization is due to increased exposure to indoor inhalant allergens or enhanced IgE-antibody production (adjuvant effects of tobacco smoke) and the extent of their inpact varies depending on the individual life styles of the housewives and school-age children.