A metabotropic glutamate receptor antagonist, alpha-methyl-4-carboxyphenylglycine, attenuates immediate early gene mRNA expression following traumatic injury in cultured rat cortical glial cells.

The effects of three glutamate receptor antagonists, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate (MK-801) for the N-methyl-D-aspartate receptor, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f] quinoxaline-7-sulfonamide (NBQX) for the alpha-amino-3-hydroxy-5methyl-4-isoxazole propionate /kinate receptor and (S)-alpha-methyl-4-carboxyphenylglycine (MCPG) for the metabotropic receptor, on c-fos and c-jun mRNA expression were investigated in cultured cortical glial cells following traumatic scratch injury. Expression of the two genes along the edges of wounds detected by in situ hybridization was not affected by MK-801 and NBQX. However, 100 and 500 microM of MCPG remarkably reduced the hybridization signals for both c-fos and c-jun mRNAs. The present results suggest that group I metabotropic glutamate receptors might have some association with immediate early gene induction after in vitro traumatic injury in glial cells.

TRF1 is a critical trans-acting factor required for de novo telomere formation in human cells.

The duplex telomere repeat (TTAGGG)(n) is an essential cis-acting element of the mammalian telomere, and an exogenous telomere repeat can induce chromosome breakage and de novo telomere formation at the site of a break (telomere seeding). Telomere seeding requires the telomere repeat (TTAGGG)(n) more stringently than does an in vitro telomerase assay, suggesting that it reflects the activity of a critical trans-acting element of the functional telomere, in addition to telomerase. Furthermore, telomere seeding is induced at a frequency fluctuating widely among human cell lines, suggesting variation in the activity of this hypothetical factor among cells. In this study, we investigated the cellular factor(s) required for telomere formation using the frequency of telomere seeding as an index and identified TRF1, one of the telomere repeat binding proteins, as an essential trans-acting factor. The exogenous telomere repeat induces telomere formation at a frequency determined by the availability of TRF1, even in telomerase-negative cells. Our study shows clearly that TRF1 has a novel physiological significance distinct from its role as a regulator of telomere length in the endogenous chromosome. The possible role of TRF1 in cell aging and immortalization is discussed.

p53-independent transient p21(WAF1/CIP1) mRNA induction in the rat brain following experimental traumatic injury.

The expression of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) mRNA after traumatic brain injury in rats was investigated using an in situ hybridization technique, along with regulating gene p53 and stress response gene hsp70 mRNA levels. At 3 h postinjury, p21(WAF1/CIP1) mRNA was markedly increased in the cortex, white matter, thalamus, CA2, a part of CA1,3 and dentate gyrus of the injured side. Hybridization signals remained elevated at 6 h in injured cortex and hippocampus and returned to the baseline by 24 h post-insult. On the other hand, p53 mRNA induction was not observed in any brain sections throughout the post-injury time course. Slight expression of hsp70 mRNA was detected in the injured cortex 3-6 h following injury and this was similar to the temporary pattern of p21(WAF1/CIP1) mRNA expression. This study showed p21(WAF1/CIP1) mRNA to be transiently induced after traumatic brain injury, independent of p53, this possibly being an early stress response to protect cells by arresting them in the cycle and allow DNA repair.

Hypothermia modulates induction of hsp70 and c-jun mRNA in the rat brain after subarachnoid hemorrhage.

We investigated expression of hsp70 and c-jun mRNA with in situ hybridization for evaluating hypothermia effect on the brain exposed to subarachnoid hemorrhage (SAH). SAH was induced in Wistar rats with endovascular perforation. Animals were divided arbitrarily into normothermic and hypothermic groups, and they were sacrificed at 3 h or 12 h after SAH. The SAH induced hsp70 and c-jun mRNAs in the cerebral cortex, hippocampus, thalamus, hypothalamus, and caudoputamen. Mild hypothermia depressed hsp70 mRNA expression in the cortex, thalamus, and hippocampus. The c-jun mRNA expression was reduced by hypothermia in the cortex, thalamus, and CA1 of the hippocampus. Based on these findings, we speculate that hypothermia protects the brain exposed to SAH by reducing this stress response. Although it is yet difficult to employ hypothermia in the clinical settings, this study suggests its utility to those patients sustaining severe subarachnoid hemorrhage.

Identification and characterization of cell lines with a defect in a post-adsorption stage of Sendai virus-mediated membrane fusion.

In the early stage of infection, Sendai virus delivers its genome into the cytoplasm by fusing the viral envelope with the cell membrane. Although the adsorption of virus particles to cell surface receptors has been characterized in detail, the ensuing complex process that leads to the fusion between the lipid bilayers remains mostly obscure. In the present study, we identified and characterized cell lines with a defect in the Sendai virus-mediated membrane fusion, using fusion-mediated delivery of fragment A of diphtheria toxin as an index. These cells, persistently infected with the temperature-sensitive variant Sendai virus, had primary viral receptors indistinguishable in number and affinity from those of parental susceptible cells. However, they proved to be thoroughly defective in the Sendai virus-mediated membrane fusion. We also found that viral HN protein expressed in the defective cells was responsible for the interference with membrane fusion. These results suggested the presence of a previously uncharacterized, HN-dependent intermediate stage in the Sendai virus-mediated membrane fusion.

Experimental models of subarachnoid hemorrhage in the rat: a refinement of the endovascular filament model.

The rat endovascular filament model has been utilized to study subarachnoid hemorrhage (SAH). Because the severity of the hemorrhage with this model has proven difficult to modulate, we attempted to vary the hemorrhage by modifying filament size, and compared this model to the blood injection method with regards to acute physiological responses and hemorrhage size. SAH was achieved using either a 3-0 or 4-0 filament, or by injecting 0.3 cc of autologous blood into the cisterna magna. Peak ICP elevations were lowest in the 4-0 filament group. CBF decreased acutely and rose from its nadir in all three models with the injection model demonstrating the earliest recovery. In the injection group, mean arterial blood pressure rose acutely and remained elevated, whereas in the 3-0 group, MABP rose transiently and in the 4-0 group it did not rise significantly. Histologically, there was less subarachnoid blood in the 4-0 group vs. the injection or 3-0 groups and a different distribution of blood in the two experimental models. Varying filament size provides a method to modulate the severity of SAH in the filament model. In addition, the rat endovascular filament and blood injection models produce different distribution of blood and physiological responses.

Traumatic injury in vitro induces IEG mRNAs in cultured glial cells, suppressed by co-culture with neurons.

Glial changes following traumatic injury to glial monolayers as well as to neuronal-glial co-culture systems in vitro were examined with a focus on the expression of mRNAs coding for the immediate early genes (IEG) c-fos, c-jun and zif/268, demonstrated using in situ hybridization. Glial cells along scratch wound lines extended cytoplasmic processes as early as 10 min post-injury and the whole wound was covered with gliosis by 24 h. For complete restoration in the case of glial cells co-cultured with neurons, this required 48 h. Induction of the three IEG mRNAs was eminent along the edges of scratch wound, peaking at 30-60 min post-injury and subsiding by 3 h. The peak expression of IEG mRNAs was delayed to 1-3 h post-injury and became undetectable at 6 h in neuronal-glial co-cultures. The data suggest that mechanical injury to glial cells causes gliosis and the expression of IEG mRNAs, which are suppressed by co-culture with neurons, indicating some influence of neuronal-glial interactions.

A single nucleotide insertion in the 5'-untranslated region of hepatitis C virus leads to enhanced cap-independent translation.

The 5'-untranslated region (5'-UTR) of hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) that directs translation of the viral open reading frame (ORF). The 5'-UTR consists of 341 nucleotides (nt) in most strains, and multiple segments within this region are important for its IRES activity. Sequencing analysis of a full-length HCV cDNA clone derived from a Japanese HCV1b-positive patient showed the 5'-UTR was 342 nt long due to a nucleotide T insertion at position 207. The influence of this T insertion on the IRES activity in directing cap-independent translation was investigated. The IRES of the 5'-UTR342 was approximately five- and two- to sevenfold more active in directing luciferase expression in monocistronic and bicistronic expression systems, respectively, when compared with the IRES of the 5'-UTR341 of a previously reported HCV1b strain. In addition to the T insertion, another point mutation involving an A to C transition at position 119 was also present in the 5'-UTR342. Simultaneous comparison of the IRES activities in engineered constructs that contained each of the two mutations indicated that the insertion at position 207 is responsible for the enhanced IRES activity of the 5'-UTR342. Further determination of the abilities of the engineered 5'-UTRs harbouring A, G, or C insertions at the same position to initiate translation indicated that both T and non-T nucleotide insertions lead to enhanced cap-independent translation.

Gene delivery systems using the Sendai virus.

Fusogenic liposome (FL) is a delivery system that can transfer encapsulated materials into living cells directly through membrane fusion. FL is a promising approach for gene therapy because it can deliver various genetic materials much more efficiently than other non-viral vectors without damaging the cell. FL-mediated gene transfer consists of two independent membrane fusion phenomena; generation of a FL by fusing a Sendai virus (SV) particle with a simple liposome encapsulating DNA, and successive fusion of the FL with cell membrane. The former requires viral F protein but no other special molecule on the liposomal membrane, whereas the latter may require the receptor (sialic acid) and unidentified assistant molecule(s) on the cell membrane. Further analysis suggests that these assistant molecule(s), not the receptor, may control the fusion and govern the cell specificity of FL-mediated delivery. This review has described a detailed analysis of these fusion phenomena and discussed possible applications of FL-mediated gene delivery to human gene therapy.

Expression of immediate early gene c-fos in rat brain following increased intracranial pressure.

No attention has been given to an influence of the intracranial pressure (ICP) elevation on the brain at the level of the gene. In the present study, we originally attempted to evaluate the molecular biological changes of the brain, especially the expression of c-fos mRNA as a marker of cellular response, caused by increased ICP. Our results confirm that the neurons and non-neuronal cells are well able to tolerate the stress of increased ICP at the level of the gene, under the condition that cerebral blood flow (CBF) is maintained. A severe increase in ICP, which reduces CBF, enhances the c-fos mRNA expression in a similar fashion as in a forebrain ischemia model, except in the choroid plexus.

Gene transfer vectors based on Sendai virus.

A gene delivery system is a fundamental technology used in human gene therapy. In order to treat patients suffering from incurable metabolic diseases, we must be able to deliver genes efficiently in situ and induce stable gene expression in non-dividing tissue cells. However, none of the current gene transfer systems (both viral and non-viral) satisfies this goal. In order to develop a novel gene delivery system that is free from the defects of existing gene transfer vectors, we analyzed natural biological phenomena that involve gene transfer and expression, and made artificial components that mimic the functioning of these systems. Our recent results shed light on three major aspects of gene transfer and expression: (1) the direct delivery of DNA into cytoplasm using fusogenic liposomes, (2) the transfer of DNA from cytoplasm to nucleus with a nuclear localization signal, and (3) the stabilization of DNA in the nucleus as an independent replicon. The possible development of a hybrid vector by combining these components is discussed.

Differential induction of immediate early gene mRNAs following cryogenic and impact trauma with/without craniotomy in rats.

Expression of immediate early gene (IEG) mRNAs following traumatic brain injury in 3 different models-cryogenic injury, impact injury with craniotomy and impact injury without craniotomy-was investigated using in situ hybridization. Cryogenic brain injury resulted in c-fos and c-jun mRNA expression throughout the ipsilateral cortex, piriform cortex and dentate gyrus on the injured side, with peak at 30 min to 1 h post-injury. Impact injury with craniotomy was associated with hybridization signals in the same areas and also in the subcortical white matter or ependyma underlying the impact site at 30 min post-injury. The expression was rather more prolonged than with cryogenic injury. Impact injury without craniotomy induced the expression of both mRNAs throughout the ipsilateral cortex, piriform cortex and dentate gyrus at 30 min post-injury, but this was promptly attenuated by 1 h post-injury, except for bilateral elevation in the dentate gyrus. The present study, thus, demonstrated that regional and temporal expression of IEG mRNAs is influenced by the intensity, quality and manner of application of the insult. Differences in the expression of IEGs may alter the late response gene expression and affect the succeeding events.

Clinical-pathological varieties of gliosarcoma: report of two cases.

The authors present two tumour cases, in both of which the diagnosis of gliosarcoma was made. The two cases differ histologically, one being a tumour clearly consisting of two different parts, the other being a tumour seemingly composed of cells with multipotential differentiation. Pathological and radiological studies are shown as well as the clinical course of the cases.

Angiotensin-converting enzyme inhibitor cilazapril suppresses expression of basic fibroblast growth factor messenger ribonucleic acid and protein in endothelial and intimal smooth muscle cells in a vascular injury model of spontaneous hypertensive rats.

The relationship between the expression of basic fibroblast growth factor (bFGF) messenger ribonucleic acid (mRNA) and protein, a potent mitogen for vascular smooth muscle cells in vivo, and administration of the angiotensin-converting enzyme inhibitor cilazapril, which suppresses smooth muscle cells proliferation in denuded arteries, was studied in spontaneously hypertensive rats using the in situ hybridization technique and immunohistochemical study. The effect of cilazapril on neointimal formation through modification of bFGF expression was evaluated using the increased tissue expression of the renin-angiotensin system in spontaneously hypertensive rats. Arterial injury was produced by using balloon catheter denudation in the left carotid artery of rats. The effects were evaluated 2 weeks later. bFGF mRNA and protein were observed only in the endothelial cells of sham-operated rats. bFGF mRNA and protein were observed in both endothelial cells and intimal smooth muscle cells in operated rats receiving only vehicle. Expression of bFGF mRNA and protein was suppressed in both endothelial cells and intimal smooth muscle cells of operated rats receiving cilazapril. These data suggest that cilazapril suppresses smooth muscle cell proliferation through modification of the expression of bFGF mRNA and bFGF protein in addition to other genes.

BALB/c 3T3 cells co-expressing FGF-2 and soluble FGF receptor acquire tumorigenicity.

The physiological significance of the soluble fibroblast growth factor (FGF) receptors is not clear yet although they are present in blood, vitreous fluid and in the extracellular matrix of vascular endothelial cells. A hypothesis that they might help FGF-2 release from cells is very interesting because FGF-2 does not have clear secretion signal and the mechanism of the secretion of FGF-2 is still unclear. Single overexpression of FGF-2 is related neither to the secretion potential of the molecule nor to the tumorigenicity of the cells. In this report, BALB/c 3T3 cells transformed with the full length of human FGF-2 cDNA are further transformed with the cDNA coding the extracellular domain of human FGF receptor 1. The obtained transformants co-expressing FGF-2 and soluble FGF receptor are highly tumorigenic in nude mice, while the parental cells do not show any tumorigenicity. In the conditioned medium of the double-transformants, FGF-2 is immunologically detected. These results suggest that naturally produced soluble form of FGF receptor supports the release of FGF-2 from the cells and that over-expression of these two molecules leads to induce the malignant tumours in vivo.

Subarachnoid hemorrhage induces c-fos, c-jun and hsp70 mRNA expression in rat brain.

To detect stress responses of the brain to subarachnoid hemorrhage (SAH), we investigated the expression of immediate early genes (IEGs) and hsp70 mRNA by in situ hybridization. Experimental SAH was produced in 49 rats by endovascular penetration. We also monitored the intracranial pressure (ICP) changes. The genes c-fos and c-jun were induced in the cerebral cortex, hippocampus and dentate gyrus in the penetrated side. mRNA coding for hsp70 was induced in the cerebral cortex, hippocampus, thalamus, hypothalamus and caudoputamen in the penetrated side and extended to the contralateral hemisphere. IEGs in the cerebral cortex were completely blocked by MK-801 pretreatment, but hsp70 mRNA was not. This suggests that the expression of IEGs correlates with spreading depression. The IEGs and hsp70 expression may reflect the severity of SAH impact and relate to the mechanisms of symptomatic vasospasm.

Pure amnesia caused by bilateral temporal lobe astrocytoma--case report.

A 32-year-old male presented with progressive pure amnesia caused by astrocytoma invading the bilateral medial temporal lobes. Methionine positron emission tomography demonstrated the extent of tumor invasion well. His memory impairment was partially improved by treatment for the astrocytoma. Lesion of the bilateral hippocampus causes memory impairment, but pure memory loss without other associated neurological sign or deterioration of consciousness is rare in a case of cerebral neoplasm.

Expression of basic fibroblast growth factor mRNA after transient focal ischemia: comparison with expression of c-fos, c-jun, and hsp 70 mRNA.

We have reported that basic fibroblast growth factor (bFGF) prevents retrograde degeneration of thalamic neurons after middle cerebral artery (MCA) occlusion. To identify the protective mechanism of bFGF, we examined bFGF mRNA expression in a model of transient focal ischemia with in situ hybridization. Compared to c-fos, c-jun, and hsp 70 mRNA expression, upregulation of the bFGF mRNA expression was delayed until 6 h after reperfusion. By 12 h, bFGF mRNA was markedly induced in the peri-infarcted cortex, cingulate cortex, and peri-infarcted white matter. At 24 h and 2 days the induction of bFGF mRNA in these regions persisted, and disappeared by 5 day. The quantitative assessment of bFGF mRNA expression revealed that optical density ratios of the cingulate gyrus and the caudoputamen were significantly higher at 12 h, 24 h, and 2 d after reperfusion than those in sham controls. Microscopic observation indicated bFGF mRNA signals were present in several types of cortical cells, including neurons and nonneuronal cells. Since intrinsic bFGF, released from the damaged tissue, can influence the healing response through receptors upregulated by injury, it is reasonable that this pattern of bFGF mRNA expression parallels the bFGFR mRNA expression previously reported.

Marked alteration of c-fos and c-jun but not hsp70 messenger RNA expression in rat brain after cold-induced trauma: An in situ hybridization study.

Immediate early gene (IEG) mRNA induction by cryogenic injury was examined using an in situ hybridization approach and the results compared with the heat shock protein mRNA expression. Hybridization signals for c-fos and c-jun mRNA were found after 30 min in the ipsilateral cortex, the hippocampal dentate granule cells and the piriform cortex, c-jun mRNA was also detected in the contralateral dentate gyrus and the piriform cortex, but was less extensive. Return to baseline values was observed at the 24 h time point. Peak induction, with silver grains observed mainly over the neurons on emulsion autoradiograms, was demonstrated in all cases 30 min to 1 h post-injury. In contrast, only slight hsp70 mRNA expression by the neurons surrounding the cold-injured site could be detected by microautoradiography, at 6 h following the trauma. The results indicate that cryogenic brain injury induces IEGs in a similar way to mechanical modes of injury such as lateral fluid percussion, but that hsp70 mRNA is hardly expressed, implying the possible existence of differences in stress response pathways.

GLAST mRNA expression in the periventricular area of experimental hydrocephalus.

Glutamate transporters play an important role in maintaining the extracellular glutamate concentration below the neurotoxic level. We investigated the expression of glutamate/aspartate transporter (GLAST) mRNA in the periventricular region of rats with kaolin-induced hydrocephalus by in situ hybridization (ISH). The density of GLAST mRNA-positive cells and the level of hybridization signals per positive cell significantly increased in the acute stage of hydrocephalus. We also demonstrated co-localization of GLAST mRNA and GFAP immunoreactivity in a single cell using the combined methods of ISH and immunohistochemistry. These findings suggest that GLAST is expressed in the reactive astrocytes of the periventricular area and regulates extracellular glutamate concentration after hydrocephalic brain injury.