Isolation and characterization of new microsatellites at the nm23-H1 and nm23-H2 gene loci and application for loss of heterozygosity (LOH) analysis.

The nm23-H1 gene has been suggested to be a metastasis suppressor gene. Studies about the events of loss of heterozygosity (LOH) at the nm23 locus and its correlation to metastasis are controversially discussed. To optimize detection of LOH at the nm23 locus, we screened two P1 clones for additional microsatellites. Tumor and normal DNA from 37 colorectal, 16 gastric, and 8 germ cancer patients were examined for LOH. We found two new CA repeats, one 5' to nm23-H1 and another 3' to nm23-H2. Using these nm23 locus-specific CA repeats and five other chromosome 17 loci (D17S1522, D17S1566, D17S855, D17S515, and TP53), allele loss was observed in 4/32 (12.5%) patients with colon cancer, 2/14 (14.3%) with gastric cancer, and 1/7 (14%) with germ cancer. No isolated LOH of the nm23 region was observed.

c-myb intron I protein binding and association with transcriptional activity in leukemic cells.

Specific binding of nuclear proteins to the region of transcriptional attenuation has been shown to modulate the expression of c-myb, a nuclear proto-oncogene preferentially expressed in lympho-hematopoietic cells. Here, it plays an important role in processes of differentiation and proliferation. The mechanism that regulates c-myb expression is not yet fully understood. The block of transcriptional elongation which has been mapped to a 1 kb region within murine intron 1 may represent one regulatory pathway. The DNA sequences containing the transcriptional pause site are well conserved between murine and human species, thus Implying similar transcription-control strategies. We compared the binding potential of nuclear extracts (from human fibroblasts and MOLT4 as well as murine NIH3T3- and 70Z/3B- cell lines) to oligonucleotide sequences previously shown to be target binding sites in the murine system. One complex containing a 70 D protein was found to be associated specifically with transcriptionally active leukemia cells. We performed transient expression studies with a CAT reporter construct containing this putative enhancer sequence and yielded significant CAT activity. We identified further a putative 20 kD repressor protein in transcriptionally silent cells and demonstrated that c-Jun is part of an ubiquitously present complex. Our results confirm the participation of intron 1 in transcriptional regulation of the c-myb gene (in mouse and human) and implicate multiple and complex regulatory mechanisms of activation during myelomonocytic differentiation and leukemic cell growth control.

Isolation and characterization of the human mismatch repair gene hMSH2 promoter region.

Hereditary nonpolyposis colorectal cancer (HN-PCC) is one of man's commonest hereditary diseases. Several studies have identified four responsible genes that are involved in a process known as DNA mismatch repair; hMSH2 is the most important of these four genes. In addition to mutational analysis of these genes, investigations of transcriptional regulatory mechanisms are important. Therefore, our purpose has been to isolate the hMSH2 promoter region. Using direct sequencing of P1 recombinant DNA we have characterized 1100 bp of the hMSH2 promoter.

A novel serine/threonine-specific protein phosphotransferase activity of Nm23/nucleoside-diphosphate kinase.

Two human nm23 genes have been identified, designated nm23-H1 and nm23-H2, which encode the 88% identical nucleoside-diphosphate kinase (NDPK) A and NDPK B polypeptides, respectively. The nm23-H1 gene product has been shown to play a functional role in the suppression of tumor metastasis. The Nm23 proteins/NDPK are highly conserved throughout evolution and are implicated in controlling cellular differentiation and development in various species, while the underlying mechanisms remain undefined. Neither the NDPK activity nor the DNA-binding activity, identified recently for NDPK B, can satisfactory explain the regulatory functions of Nm23. The present study provides evidence that purified Nm23 proteins are capable of transferring a phosphate group to other proteins when non-denaturing amounts of urea are present. This novel Nm23/NDPK activity was found to be specific for serine and threonine residues, and the transphosphorylation of substrate proteins occurred stoichiometrically. Because of the absence of a substrate turn-over, the novel function was termed protein phosphotransferase activity instead of protein kinase activity. It is demonstrated that urea stimulates the interaction of NDPK with other proteins. Identical phosphoprotein patterns were obtained using purified NDPK preparations from human, Drosophila, yeast and Dictyostelium in the presence of urea. Partially purified NDPK from human erythrocytes produced a similar phosphorylation pattern independent of urea addition and also acted stoichiometrically. In this preparation, a protein phosphotransferase activity of Nm23 species may possibly be generated and/or stabilized by the interaction with copurified proteins. Using different mutants of Dictyostelium NDPK it was shown that the protein phosphotransferase activity depends on the same active site as the NDPK activity. A phosphotransfer mechanism analogous to that of protein-histidine kinases is proposed, involving a high-energy phosphohistidine intermediate. Furthermore, the novel Nm23 function is compared with an apparently similar protein phosphotransferase activity which was observed previously with partially purified NDPK from different plant species.

Characterization of the human nm23-H2 promoter region and localization of the microsatellite D17S396.

The transcription of human nm23 genes (nm23-H1, nm23-H2) is involved in suppression of tumor metastasis or tumor progression. Therefore the characterization of transcriptional regulatory mechanisms for both nm23 genes is very important. In this study we have isolated and analyzed the 5'-flanking region of the human nm23-H2 gene and estimated the distance to 4 kb between nm23-H2 and nm23-H1 genes. We localized the known microsatellite D17S396 within this region. Furthermore the identification of possible binding sites for MYC proteins and additionally the NM23-H2 protein itself (the transcription factor PuF for c-myc gene activation) is of importance with respect to possible para- and autoregulatory interactions. A comparison of the promoter sequences of both human nm23 genes revealed no significant sequence homology.

Characterization of the genomic structure and the promoter region of the human intestinal trefoil factor.

Trefoil proteins form a specific group of stable secretory polypeptides. They are expressed in a lot of human cancers and inflammatory processes of the gastrointestinal tract. Recently a new human trefoil protein, ITF/hP1.B was isolated. This gene is expressed mainly in globet cells of intestinum and colon. The genomic structure of the murine and rat homologues genes were described previously and a high nucleotide sequence conservation (78-95%) was found in the coding as well as the regulatory regions. We have isolated the human genomic ITF-gene region from a P1 library and a detailed analysis yielded in a similar gene structure with a three basepair displacement of the second exon intron boundary. The promoter region of the human gene interestingly is markedly different from rat and mouse.

Presence of regulatory sequences within intron 4 of human and murine c-myb genes.

The molecular mechanisms that modulate c-myb mRNA transcription in hematopoietic cells appear to involve intron regulatory sequences. We have characterized the fourth of ten introns from both human and murine c-myb genes in regard to nucleotide sequence and specific protein binding. For this approach complete genomic c-myb intron 4 fragments were isolated from mouse and human DNA using PCR amplification with flanking exon-primers derived from the mouse gene. Comparison of the obtained sequences revealed strong homology between the two species. Using crude nuclear protein extracts from mouse and human myb expressing cells (70Z/3B; Molt4) and gel shift experiments we found specific protein interaction for both introns and to determine the protein binding site in detail, we performed DNase I footprinting. Our results indicate that the binding factor is absent in control cell lines without c-myb transcriptional activity, suggesting a possible positive regulatory function of the DNA-protein complex. To confirm these findings we introduced the human c-myb intron 4 DNA sequence into the EcoRI site of the pCAT-Promoter plasmid and transfected Molt4 cells with this chimeric construct. The transient expression studies revealed that intron 4 sequences possess enhancer activity. Thus, we have demonstrated that intron 4 sequences can be important for the regulation of c-myb proto-oncogene expression.

The quantitative measurement of spasticity: effect of cutaneous electrical stimulation.

The goal of this research was to determine if cutaneous electrotherapy would temporarily reduce muscle spasticity. Five traumatically brain injured (TBI) and five spinal cord injured (SCI) subjects, all with clinically evident spasticity, received surface electrical stimulation over the tibialis anterior muscle. Using the Spasticity Measurement System, stiffness around the ankle was measured before, immediately after, and 24 hours after treatment. With stimulation, ipsilateral ankle viscoelastic stiffness immediately decreased in 9 of 10 subjects and remained significantly depressed for up to 24 hours. Contralateral ankle spasticity did not significantly change. Using the same subjects under sham conditions, no significant decrements in spasticity occurred. In a subjective survey, only SCI participants reported functionally evident spasticity reductions. Also within this subgroup, efficacy of treatment was directly proportional to the severity of pre-stimulation clonus. We conclude that (1) cutaneous electrotherapy transiently decreases both TBI and SCI related spasticity and (2) pre-stimulation clonus may function as a clinical indicator of SCI patients most likely to benefit from this process.

Phosphorylation of nm23/nucleoside diphosphate kinase by casein kinase 2 in vitro.

We have investigated phosphorylation of human nucleoside diphosphate kinase (NDPK) and of homologous NDPK from different species by human casein kinase 2 (CK-2). The human NDPK isotypes A and B were phosphorylated by CK-2 in vitro both when the purified proteins and total lysate of HL-60 leukemia cells were used. The homologous NDPK's from Yeast and E. coli were also substrates for CK-2 in vitro, but not Drosophila NDPK. Phosphorylation of all NDPK types by the CK-2 holoenzyme was entirely polyamine-dependent. The CK-2 phosphorylation site in human NDPK A, that was about 2.5 times stronger phosphorylated than was the B isotype, was tentatively assigned to Ser-122. The location of the corresponding residue in the 3D-structure of the 80% homologous Drosophila NDPK suggests that its phosphorylation may directly influence substrate binding and/or catalysis.

Isolation and characterization of the human genomic locus coding for the putative metastasis control gene nm23-H1.

Nm23-H1 gene expression is inversely correlated with tumor metastatic potential in certain tumors, including melanomas, breast carcinomas, and hepatocellular carcinomas. Using nm23-H1 c-DNA primer and genomic polymerase chain reaction (PCR) amplification, we purified three PCR fragments (one of 4kb and two of 2 kb) covering the whole human genomic locus of the gene (8.460bp). We recombined the PCR products into pUC18 and produced a restriction map to perform subcloning. Complete sequencing of genomic PCR fragments, including the whole coding region of nm23-H1, revealed that the gene consists of five exons and four introns spanning 8.5kb. A sequence homology analysis between human nm23-H1 and the homolog gene of the rat (NDP-K beta) shows that exon-intron boundaries are well conserved between these two species.

High levels of nm23-H1 and nm23-H2 messenger RNA in human squamous-cell lung carcinoma are associated with poor differentiation and advanced tumor stages.

Expression of the candidate metastasis-suppressor gene nm23-H1 has been shown to correlate inversely with metastatic potential in some human tumors, but not in all. Until now, few studies have been carried out on the activity of the homologous nm23-H2 gene in human cancer. No nm23 transcription studies exist for human lung cancer so far. To determine whether the nm23 genes could have a metastasis-suppressor function in non-small-cell lung carcinoma (NSCLC), pulmonary sarcoma and carcinoids, we analysed both nm23-HI and nm23-H2 mRNA levels in 37 tumor samples obtained from patients who underwent potentially curative resection between 1986 and 1990, and in 4 metastatic tumors obtained from autopsy. As compared to corresponding healthy lung parenchyma, both nm23-HI and nm23-H2 transcript levels were elevated in 37 of 41 tumors. The increases in nm23 mRNA expression were stronger in advanced stages of squamous-cell carcinoma, large-cell carcinoma, sarcoma and carcinoids than in early stages of the respective tumor types. Within stages I and II of squamous-cell carcinoma, significantly higher nm23 mRNA levels were found in poorly differentiated tumors than in moderately differentiated ones. Moreover, an inverse correlation between nm23 expression and disease-free survival of the patients was observed. In conclusion, our results indicate that the increased nm23 expression in the analysed tumors is not consistent with the proposed metastasis-suppressor function, but the 2 nm23 genes nevertheless may be implicated in the mechanism of tumor progression.

A rapid method to determine the orientation of blunt end ligated polymerase chain reaction products.

Subcloning of polymerase chain reaction (PCR) fragments is often performed via blunt end ligation after previous Klenow fragment exonuclease treatment. Since insert-specific primers are at hand from the original amplification step, we used a simple PCR-based assay to determine the insert orientation of the recombinants. After purification of enriched plasmids, small aliquots were tested performing standard PCR reactions using a plasmid primer directed towards the cloning site and one of the insert specific primers, respectively. Only the distant insert primer yields a PCR product which can be visualized after gel electrophoresis. In this way both the insert orientation in the vector and the correct size of the cloned fragment is determined rapidly without sequencing or restriction fragment analysis.

Macaque anteroventral cochlear nucleus: developmental anatomy.

The development of the anteroventral cochlear nucleus (AVCN) in fetal and infant monkeys (Macaca nemestrina) was analyzed for gross morphologic changes together with growth-related modifications in constituent cell size and cell distribution. Rapid and extensive prenatal volumetric changes were followed by slow and limited postnatal volumetric changes. The time course of packing density and cell size changes paralleled the volumetric changes. At each age the packing density along the rostrocaudal axis of the AVCN was constant except in the youngest specimens (mid- to late-fetal), where local variations occurred. Similarly, the size of AVCN cells along the rostrocaudal axis remained approximately constant at any given age. In comparison with the human and mouse, the macaque exhibits relatively less pronounced postnatal change in overall volume and cellular growth features.

Inescapable versus escapable shock modulates long-term potentiation in the rat hippocampus.

A group of rats was trained to escape low-intensity shock in a shuttle-box test, while another group of yoked controls could not escape but was exposed to the same amount and regime of shock. After 1 week of training, long-term potentiation (LTP) was measured in vitro in hippocampal slices. Exposure to uncontrollable shock massively impaired LTP relative to exposure to the same amount and regime of controllable shock. These results provide evidence that controllability modulates plasticity at the cellular-neuronal level.