Synaptic modulation of excitatory synaptic transmission by nicotinic acetylcholine receptors in spinal ventral horn neurons.

Nicotinic acetylcholine receptors (nAChRs) are distributed widely in the central nervous system and play important roles in higher brain functions, including learning, memory, and recognition. However, functions of the cholinergic system in spinal motoneurons remain poorly understood. In this study, we investigated the actions of presynaptic and postsynaptic nAChRs in spinal ventral horn neurons by performing whole-cell patch-clamp recordings on lumbar slices from male rats. The application of nicotine or acetylcholine generated slow inward currents and increased the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs). Slow inward currents by acetylcholine or nicotine were not inhibited by tetrodotoxin (TTX) or glutamate receptor antagonists. In the presence of TTX, the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) were also increased by acetylcholine or nicotine. A selective α4ß2 nicotinic receptor antagonist, dihydro-ß-erythroidine hydrobromide (DhßE), significantly decreased nicotine-induced inward currents without affecting the enhancement of sEPSCs and mEPSCs. In addition, a selective α7 nicotinic receptor antagonist, methyllycaconitine, did not affect either nicotine-induced inward currents or the enhancement of sEPSCs and mEPSCs. These results suggest that α4ß2 AChRs are localized at postsynaptic sites in the spinal ventral horn, non-α4ß2 and non-α7 nAChRs are located presynaptically, and nAChRs enhance excitatory synaptic transmission in the spinal ventral horn.

Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42.

An extracellular serine alkaline protease of Bacillus clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3 days at pH 10.5 and 37 degrees C. Highest alkaline protease activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate by DEAE-cellulose chromatography followed by (NH(4))(2)SO(4) precipitation, with a yield of 58%. SDS-PAGE analysis revealed the molecular weight of the enzyme to be 26.50 kDa. The optimum temperature for enzyme activity was 60 degrees C; however, it is shifted to 70 degrees C after addition of 5 mM Ca(2+) ions. The enzyme was stable between 30 and 40 degrees C for 2 h at pH 10.5; only 14% activity loss was observed at 50 degrees C. The optimal pH of the enzyme was 11.3. The enzyme was also stable in the pH 9.0--12.2 range for 24 h at 30 degrees C; however, activity losses of 38% and 76% were observed at pH values of 12.7 and 13.0, respectively. The activation energy of Hammarsten casein hydrolysis by the purified enzyme was 10.59 kcal mol(-1) (44.30 kJ mol(-1)). The enzyme was stable in the presence of the 1% (w/v) Tween-20, Tween-40,Tween-60, Tween-80, and 0.2% (w/v) SDS for 1 h at 30 degrees C and pH 10.5. Only 10% activity loss was observed with 1% sodium perborate under the same conditions. The enzyme was not inhibited by iodoacetate, ethylacetimidate, phenylglyoxal, iodoacetimidate, n-ethylmaleimidate, n-bromosuccinimide, diethylpyrocarbonate or n-ethyl-5-phenyl-iso-xazolium-3'-sulfonate. Its complete inhibition by phenylmethanesulfonylfluoride and relatively high k (cat) value for N-Suc-Ala-Ala-Pro-Phe-pNA hydrolysis indicates that the enzyme is a chymotrypsin-like serine protease. K (m) and k (cat) values were estimated at 0.655 microM N-Suc-Ala-Ala-Pro-Phe-pNA and 4.21 x 10(3) min(-1), respectively.

Down-regulation in human cancers of DRHC, a novel helicase-like gene from 17q25.1 that inhibits cell growth.

Frequent observations of allelic loss in chromosomal band 17q25.1 in a variety of human cancers have suggested that one or more tumor suppressor genes are normally present in this region. Moreover, a locus responsible for hereditary focal non-epidermolytic palmoplantar keratoderma (tylosis oesophageal cancer; TOC), a condition associated with esophageal cancer, has been mapped to the same band. During efforts to sequence, by shot-gun methods, a 1 Mb target region that we had defined as the DNA segment harboring the putative tumor suppressor gene(s) involved in these events, we identified a novel cDNA, DRHC (down-regulated in human cancers), that showed reduced expression in 28 of 95 (29%) cell lines derived from a variety of human cancers. The full-length cDNA, 6275 bp long, was expressed predominantly in thymus and brain. The predicted 1942-amino-acid product exhibited significant sequence homology to yeast enzymes belonging to the DEAD-helicase superfamily, and appeared to be a Uvr/Rep helicase with a DEXDc consensus domain. Transfection of a DRHC expression vector inhibited growth of cancer cells in liquid medium or soft agar. The results suggest that loss of expression of DRHC may play a role in human carcinogenesis.

Gene fusion involving HMGIC is a frequent aberration in uterine leiomyomas.

HMGIC, a high-mobility-group protein gene encoding an architectural transcription factor, was recently identified as the target of gene fusion in a variety of human benign mesenchymal tumors; some of these events were chromosomal translocations involving 12q13-15. HMGIC consists of three DNA-binding domains (encoded by exons 1-3), a spacer, and an acidic carboxyl-terminal regulatory domain (exons 4-5). To determine the spectrum and nature of the aberrations in uterine myomas in Japanese patients, we systematically examined the tumors of 45 patients for all possible types of gene fusions involving HMGIC, by means of 3'-rapid amplification of cDNA ends (RACE) and reverse transcriptase-polymerase chain reaction (RT-PCR) experiments. HMGIC gene fusions were found in 16 (36%) of the tumors; aberrant splicings to five cryptic sequences located in introns of the HMGIC gene were found in 11 of these cases, and translocations causing juxtaposition to other genes, such as COX6C and RA D51B, were found in 5. In all fusion transcripts, the first two or three exons of HMGIC were fused to ectopic sequences. Our results suggest that a fusion event, resulting in the separation of the DNA-binding domains of HMGIC from the spacer and the acidic carboxyl-terminal regulatory domain, is a common tumorigenic mechanism in the development of uterine myomas.

A novel MVMp-based vector system specifically designed to reduce the risk of replication-competent virus generation by homologous recombination.

Recent work highlights the potential usefulness of MVM-based vectors as selective vehicles for cancer gene therapy (Dupont et al, Gene Therapy, 2000; 7: 790-796). To implement this strategy, however, it is necessary to develop optimized methods for producing high-titer, helper-free parvovirus stocks. Recombinants of MVMp (rMVMp) are currently generated by transiently co-transfecting permissive cell lines with a plasmid carrying the vector genome and a helper plasmid expressing the capsid genes (replaced with a foreign gene in the vector genome). The resulting stocks, however, are always heavily contaminated with replication-competent viruses (RCV), which precludes their use in vivo and particularly in gene therapy. In the present work we have developed a second-generation MVMp-based vector system specifically designed to reduce the probability of RCV generation by homologous recombination. We have constructed a new MVMp-based vector and a new helper genome with minimal sequence overlap and have used the degeneracy of the genetic code to further decrease vector-helper homology. In this system, the left homologous region was almost completely eliminated and the right sequence overlap was reduced to 74 nt with only 61% homology. We were thus able to substantially reduce ( approximately 200 x), but not completely eliminate, generation of contaminating viruses in medium-scale rMVMp preparations. Since the remaining sequence homology between the new vector and helper genomes is weak, our results suggest that contaminating viruses in this system are generated by nonhomologous recombination. It is important to note, unlike the autonomously replicating helper viruses produced from the first-generation vector/helper genomes, the contaminating viruses arising from the new packaging system cannot initiate secondary infection rounds (so they are not 'replication-competent viruses'). Our findings have important implications for the design of new MVMp-based vectors and for the construction of trans-complementing packaging cell lines.

Caspase activation in high-pressure--induced apoptosis of murine erythroleukemia cells.

Murine erythroleukemia cells were subjected to physicochemical stresses such as high pressure (60--110 MPa), heating (42--45 degrees C), and ultraviolet (UV) irradiation (5--25 kJ/cm(2)). After exposure to these stresses, the cells were cultured at 37 degrees C and atmospheric pressure. The number of the cells treated at 100 MPa, 45 degrees C, or 20 kJ/cm(2) remained constant or decreased at an early stage of culture. The nuclear morphology, agarose gel electrophoresis, and flow cytometry of these cells showed that they undergo apoptosis. The activity of caspase-3 was observed in cells subjected to each stress. In particular, caspase-3 was most readily activated by high pressure under our conditions. The caspase-3 activity and apoptosis exhibited a similar pressure dependence. It is important that both caspase-3 activation and apoptosis induced by high pressure were significantly suppressed by a caspase-3 inhibitor. These results suggest that high-pressure-induced apoptosis is also characterized by the activation of caspase-3, as seen with heat- and UV-induced apoptosis.

Identification, tissue expression, and chromosomal position of a novel gene encoding human ubiquitin-conjugating enzyme E2-230k.

Through large-scale human genome mapping and sequencing of a region at chromosome 17q25.1 that is of particular interest because genes associated with breast, ovarian, and esophageal cancer are likely to be located there, we isolated the cDNA of a novel member of the family of ubiquitin-conjugating enzymes. The predicated peptide showed 97% identity in amino-acid sequence to murine ubiquitin-conjugating enzyme E2-230k (Mus UBE2-230k). The human cDNA consisted of 4878 nucleotides with an open reading frame encoding 1138 amino acids; the approximately 5 kb transcript was expressed predominantly in skeletal muscle and heart. The predicted UBE2-230k peptide contained a motif typical of the UBC domain that has been implicated in the ubiquitin-dependent proteolytic system and related pathways.

Molecular cloning, tissue expression, and chromosomal assignment of a novel gene encoding a subunit of the human signal-recognition particle.

Human cancers derived from breast, esophageal, or ovarian tissues frequently show allelic losses on chromosome band 17q25. Moreover, a locus responsible for hereditary focal nonepidermolytic palmoplantar keratoderma, a condition associated with esophageal cancer (TOC; tylosis with oesophageal cancer), has been mapped to the same band. During efforts to sequence, by shotgun methods, a 1-Mb target region that we had defined as the DNA segment harboring the putative tumor suppressor gene(s) involved in these events, we identified a novel cDNA. The full-length cDNA is 2495bp long and is expressed predominantly in skeletal muscle, heart, kidney, and placenta. The predicted product, a 627-amino-acid protein, exhibited significant sequence homology to the canine 68-kd subunit of the signal recognition particle that has been implicated in the transport of secreted and membrane proteins to the endoplasmic reticulum for proper processing. We confirmed the location of this gene at chromosome 17q25.1 by radiation-hybrid mapping and by fluorescence in situ hybridization.

Fusion of a sequence from HEI10 (14q11) to the HMGIC gene at 12q15 in a uterine leiomyoma.

Uterine leiomyoma, a benign smooth-muscle tumor of the myometrium, is the most commonly encountered neoplasm in women of reproductive age. Band q15 of chromosome 12 is often rearranged in benign mesenchymal tumors such as uterine leiomyomas, and the HMGIC gene, encoding a protein of the high-mobility-group (HMG), is present in that region. Using 3' rapid amplification of cDNA ends (3'RACE) experiments, we isolated an ectopic sequence that was fused to HMGIC in a uterine leiomyoma. Cloning of the fusion cDNA identified a gene termed rising dbl quote, left (low)homo sapiens enhancer of invasion 10" (HEI10) as the fusion partner. Radiation hybrid mapping revealed that the normal location of HEI10 is at 14q11. In the fusion transcript the first two exons of the HMGIC gene, which encode DNA-binding domains, were fused to the 3' portion of the HEI10 gene. This rearrangement implicates HMGIC in the tumorigenesis of uterine leiomyoma, and suggests that its fusion HMGIC product may play a role in mesenchymal differentiation.

Three aberrant splicing variants of the HMGIC gene transcribed in uterine leiomyomas.

Cytogenetic aberrations involving chromosome region 12q13-15 occur frequently among benign mesenchymal tumors in humans, e.g., pleomorphic adenomas of the parotid gland, pulmonary chondroid hamartomas, lipomas, or uterine leiomyomas. HMGIC, a gene encoding a protein of the high-mobility group, has been identified as a target of those events. Using the 3' rapid amplification of cDNA ends (RACE) technique, we identified six different fusion transcripts of the HMGIC gene among 13 uterine leiomyomas; three of these variants had not been described before. Radiation-hybrid mapping located all three of the novel fusion transcripts in the same chromosomal region as the HMGIC gene. Cloning of the entire HMGIC gene in a genomic contig of P1-derived artificial chromosomes and cosmids revealed that the 3' portion of each novel fusion transcript contained cryptic exonic sequences (designated a, b, and c) present in intron 3 of the HMGIC gene. Thus, aberrant alternative splicing was responsible for abnormal HMGIC isoforms in those myomas. Identification of these novel variants suggested that aberrant splicing can join chromosomal translocation and inversion as a mechanism for producing abnormal HMGIC transcripts, and that separation of the DNA binding domains of HMGIC from its acidic carboxyl-terminal regulatory domain can lead to development of benign mesenchymal tumors.

Tumor-selective gene transduction and cell killing with an oncotropic autonomous parvovirus-based vector.

A recombinant MVMp of the fibrotropic strain of minute virus of mice (MVMp) expressing the chloramphenicol acetyltransferase reporter gene was used to infect a series of biologically relevant cultured cells, normal or tumor-derived, including normal melanocytes versus melanoma cells, normal mammary epithelial cells versus breast adenocarcinoma cells, and normal neurons or astrocytes versus glioma cells. As a reference cell system we used normal human fibroblasts versus the SV40-transformed fibroblast cell line NB324K. After infection, we observed good expression of the reporter gene in the different tumor cell types, but only poor expression if any in the corresponding normal cells. We also constructed a recombinant MVMp expressing the green fluorescent protein reporter gene and assessed by flow cytometry the efficiency of gene transduction into the different target cells. At a multiplicity of infection of 30, we observed substantial transduction of the gene into most of the tumor cell types tested, but only marginal transduction into normal cells under the same experimental conditions. Finally, we demonstrated that a recombinant MVMp expressing the herpes simplex virus thymidine kinase gene can, in vitro, cause efficient killing of most tumor cell types in the presence of ganciclovir, whilst affecting normal proliferating cells only marginally if at all. However, in the same experimental condition, breast tumor cells appeared to be resistant to GCV-mediated cytotoxicity, possibly because these cells are not susceptible to the bystander effect. Our data suggest that MVMp-based vectors could prove useful as selective vehicles for anticancer gene therapy, particularly for in vivo delivery of cytotoxic effector genes into tumor cells.

Novel gene fusion of COX6C at 8q22-23 to HMGIC at 12q15 in a uterine leiomyoma.

Cytogenetic analyses have shown that aberrations involving 12q13-15 are frequent chromosomal changes in a variety of human benign mesenchymal tumors, e.g., pleomorphic adenomas of the parotid gland, pulmonary chondroid hamartomas, lipomas, and uterine leiomyomas. Recently, the high-mobility group protein gene HMGIC was identified as the target gene affected by the 12q13-15 aberrations. Using 3' rapid amplification of cDNA ends experiments, we isolated novel ectopic sequences fused to HMGIC in a uterine leiomyoma. Cloning of the fusion cDNA identified the human cytochrome c oxidase subunit VIc (COX6C) gene on 8q22-23 as the fusion partner of HMGIC. Nucleotide sequences of the fusion transcript revealed that the first 3 exons of the HMGIC gene, encoding the 3 DNA binding domains, was fused to the exon 2 of the COX6C gene. The identification of a gene rearrangement suggests a role for HMGIC in tumorigenesis of uterine leiomyoma and suggests a possible involvement of HMGIC in mesenchymal differentiation. Genes Chromosomes Cancer 27:303-307, 2000.

Two single nucleotide polymorphisms of the hSNF5/INI1 gene.

We found two single nucleotide polymorphisms at the hSNF5/INI1 gene located on 22q11.2, encoding a member of the chromatin-remodelling SWI/SNF multiprotein complexes. A guanine/adenine polymorphism at codon 299 in exon 7, and another guanine/adenine polymorphism at 39 bp upstream of exon 9 were identified. As the gene was recently identified as a tumor suppressor gene for malignant rhabdoid tumor, this polymorphism may be useful for the genetic study of susceptibility for human malignancies of various tissue origins.

Nanomolar range docetaxel treatment sensitizes MCF-7 cells to chemotherapy induced apoptosis, induces G2M arrest and phosphorylates bcl-2.

Docetaxel (Taxotere), a member of the taxoid family of chemotherapy drugs is currently being tested in clinical trials simultaneously with other apoptosis inducing drugs like doxorubicin. We show, in vitro, in MCF-7 breast cancer cells that when it is used at doses as low as 5nM, 24 hours before either doxorubicin or etoposide, docetaxel is capable of inducing a significant increase in cell death compared to the reverse sequence or simultaneous treatment. We further show that this increase in cell death is due to an increase in apoptosis, and that this sensitization coincides with a docetaxel induced G2-M arrest and phosphorylation of the bcl-2 oncoprotein. We speculate that this phosphorylation of the apoptosis blocker bcl-2 might be responsible for the sensitization, and we suggest a clinical study comparing a 24 hour docetaxel pretreatment to the current simultaneous schedules.

Method for concentrating and purifying recombinant autonomous parvovirus vectors designed for tumour-cell-targeted gene therapy.

Recent work has highlighted the use of parvoviruses as potential vectors for tumour-cell-targeted gene therapy. The oncotropic properties of the prototype strain of minute virus of mice (MVMp) suggest that this virus might be a useful vehicle for introducing selectively therapeutic genes, e.g. lymphokine or suicide genes, into tumour cells and preferentially expressing them. But the low titre of recombinant virus stocks (10(5)-10(6) infectious units per ml) and their high level of contamination by cell proteins make it practically impossible to evaluate their efficacy in in vivo systems. A technique is described for producing cellular contaminant-free stocks of recombinant virus particles, with titres up to 5 x 10(8) IU/ml.

Identification and possible function of cathepsin G in gingival crevicular fluid from chronic adult periodontitis patients and from experimental gingivitis subjects.

The levels of cathepsin G in gingival crevicular fluid (GCF) from chronic adult periodontitis patients and experimental gingivitis subjects were determined both by activity measurement using N-benzoyl-(DL)-phenylalanine-2-naphthyl ester as a substrate and by enzyme immunoassay using anti-human cathepsin G IgG. The activity level of cathepsin G in GCF of both periodontitis and experimental gingivitis has no significant correlation with all measured clinical parameters. Western immunoblotting using antibodies specific for cathepsin G or alpha 1-proteinase inhibitor revealed that the difficulty in demonstrating the association of its activity with the severity of these diseases was due largely to formation of the enzyme-inhibitor complexes. By contrast, statistically significant positive correlation was found between cathepsin G content in GCF of periodontitis, which was determined by enzyme immunoassay, and such clinical parameters as the GCF volume, the gingival index and probing depth. The increased cathepsin G content with increasing severity of periodontal inflammation was markedly diminished by the initial treatment. Although no significant activity was detectable in GCF of experimental gingivitis, a rapid increase of the immunoreactive cathepsin G was found in GCF at 3-5 d after refraining from oral hygiene measures, which rapidly decreased by 10 d. The progressively increased cathepsin G between 10th and 21st d rapidly decreased by cleaning of the teeth. The results indicate that cathepsin G is involved in the host's defensive mechanism against the invasion of etiologic microbes and/or the development of either periodontitis or gingivitis.

A cross-sectional study on osteocalcin levels in gingival crevicular fluid from periodontal patients.

The purpose of the present study was to determine the levels of osteocalcin, a bone specific matrix protein, in gingival crevicular fluid (GCF) from periodontal disease patients and to investigate the relationship between GCF osteocalcin levels and clinical parameters. Nineteen initial visit patients, 5 patients with gingivitis and 14 patients with adult periodontitis, participated in this study. The clinical parameters including probing depth, attachment level, gingival index, and tooth mobility were recorded following careful sampling of GCF with a filter paper strip harvested for 3 minutes. Osteocalcin adsorbed on a strip was extracted in a plastic tube containing 150 microliters of 10 mM sodium phosphate buffer (pH 6.5). GCF osteocalcin was determined by a newly-developed, high sensitive enzyme immunoassay which could recognize the N-terminal 20 residue peptide. In gingivitis patients, no significant amounts of osteocalcin were detected. In periodontitis patients, on the other hand, osteocalcin levels were detected, ranging between 0 and 540 pg/tube and positively correlated with these clinical parameters (P < 0.01). Moreover, in several sites in GI = 3 group, extremely higher levels of GCF osteocalcin were detected. These results strongly suggest that in addition to the presence of GCF osteocalcin the levels of osteocalcin may reflect the degree of the periodontal inflammation at the sampled sites.