There is evidence that nitric oxide (NO), a reactive free radical produced in large quantities by resident synoviocytes, chondrocytes and infiltrating macrophages, is elevated in the synovial fluid and serum of rheumatoid arthritis (RA) patients. Observations within our lab suggest that NO may be capable of causing mutations of significant size, including large-scale deletion (LSD) mutations. Since NO has been shown to cause DNA damage and since genetic changes also occur in the inflamed joints, we hypothesize that NO may participate in the genotoxic environment of RA joints. To determine if LSD mutations contribute to the increase in T cell hprt MF observed earlier, hprt mutant ( hprt-) T cells were isolated from RA patients and screened by multiplex-PCR (MP-PCR) for deletion of hprt exons. To determine if NO is capable of inducing LSD mutations such as those found in RA and OA T cells, two human B lymphoblastoid cell lines (TK6 and WIL2-NS) were exposed to NO-donating drugs and the resulting hprt - clones were examined for hprt LSD mutations by MP-PCR. (Abstract shortened by UMI.)

The process of Excitation-Contraction (E-C) coupling in cardiac muscle is dependent on calcium. Many proteins that regulate calcium homoeostasis in the cardiocyte are also calcium binding proteins. Two cDNAs were previously isolated by screening a rabbit heart cDNA expression library with an antiserum that recognized two calcium binding polypeptides of the sarcolemma. These cDNAs and other related cDNAs, which were isolated from cardiac cDNA libraries, were shown to encode a novel family of sarcolemmal associated proteins (SLAPs). The SLAP cDNAs had divergent $5\sp\prime$ termini and could potentially encode polypeptides of 37, 46, and 74 kDa molecular designated SLAP 1, SLAP2 and SLAP3 respectively. Three SLAP transcripts of 5.9, 4.6 and 3.5 kb were found in heart. The 5.9 kb transcript was ubiquitously expressed while the 3.5 kb and 4.6 kb transcripts were predominantly expressed in cardiac, soleus and smooth muscle. SLAP was encoded by a single gene that mapped to chromosome 3p14.3-21.2 and thus the various transcripts were likely generated by alternative splicing or alternative promoter usage. The primary structure of SLAP predicted that it would have large regions of coiled-coil structure, which included an eleven heptad acidic amphipathic a-helical segment containing two leucine zippers, and a carboxyl terminal transmembrane domain. Three SLAPs of 81, 45 and 35 kDa were shown to be associated with the cardiac sarcolemmal membranes. SLAPs could only be solubilized from cardiac membrane with detergents suggesting that they were integral membrane proteins. Immunohistochemical localization of SLAP in cardiac muscle revealed that SLAP associated with the sarcolemma and displayed a reticular pattern of staining that resembled the transverse-tubules and/or the sarcoplasmic reticulum. Expression studies showed that MYC-epitope tagged SLAP localized to regions of juxtaposition between neighbouring cell membranes although an intracellular pool of the protein was also present in cells undergoing apparent cleavage. SLAPs appeared to be the mammalian orthologues of $\rm TOP\sb{AP},$ a topographically graded protein expressed in the developing chick retina and tectum (Savitt et al., 1995): Immunofluorescence studies with specific antibodies raised against SLAP fusion proteins showed that SLAP expression was developmentally regulated in the rat retina where it is enriched in Mueller glial cells. However, no evidence of a topographical gradient of SLAP distribution was found in either the mammalian retina, superior colliculus or the lateral geniculate nucleus. In the neonatal rat brain SLAP was selectively expressed in neurons of the hippocampus as well as in the support cells of the nerve fiber layer of the olfactory bulb. SLAP also shared 44% homology with USO1, a yeast protein involved in the fusion of vesicles between the endoplasmic reticulum and the Golgi (Sapperstein et al., 1996). SLAP expression was found to inhibit the fusion of myoblast fusion without effecting their differentiation. In view of the role of tail-anchored-membrane proteins in membrane fusion and the similarity of SLAPs to USO1, we propose that SLAPs may define a novel family of proteins involved in mediating membrane-membrane interactions.

The spinal muscular atrophies (SMAs), characterized by the degeneration of spinal cord motor neurons resulting in muscular atrophy, are among the most common autosomal recessive disorders. All forms of SMA map to chromosome 5 at 5q13. The interval containing the SMA gene was defined by linkage analysis and the identification of recombination events to an approximately 1.1 Mb region flanked centromerically by the DNA marker D5S629 and telomerically by the DNA marker D5S557. In the first part of this study I describe a recombination event on an SMA chromosome from a type I SMA family, occurring between different subloci of two complex microsatellite repeats (MSR), CMS-1 and 7613. This new proximal boundary reduced the minimal region harboring the SMA locus from ∼1.1 Mb to approximately 600 kb. It was within this newly defined SMA interval that an SMA associated gene, designated NAIP (Neuronal Apoptosis Inhibitory Protein), was cloned in 1995. NAIP exists in multiple copies in the SMA region at 5q13. Deletions in the first two coding exons of the only intact copy of NAIP are associated with approximately 66% of type I SMA cases and a lower percentage of the milder cases. The NAIP protein is homologous to the baculoviral inhibitor of apoptosis proteins, and is present in motor neurons and other neuronal populations affected in type I SMA. In the second part of this study I report the cloning and characterization of multiple copies of the mouse homologue of NAIP, all of which map to mouse chromosome 13, region D1-D3, which is syntenic to the human chromosome 5q11-q23. The analysis of the genomic organization of Naip indicated the existence of a minimum of six distinct Naip loci in the mouse. The original reason for the cloning of the mouse homologues of NAIP was to generate a knockout mouse model for SMA. Thus, as a first step toward this goal, I describe the targeted disruption of the CNS locus, Naip1, in ES cells. A report on the mapping of the multiple Naip loci to the critical region of Lgn1, a locus modulating the intracellular replication of Legionella in macrophages and therefore its pathogenicity, made Naip a candidate gene for murine Legionella resistance. Here I report further characterization of Naip2, as the locus most suited for this candidacy. Taken together, my data reveal that in contrast to the human case where there is one intact NAIP gene and multiple unprocessed truncated and deleted pseudogenes, the mouse possesses multiple potentially functional Naip loci representing a gene family. (Abstract shortened by UMI.)

The adaptive response, a phenomenon sensitive to low doses of ionizing radiation, was used to assess the relative biological effectiveness (RBE) of low doses of tritium. Normal human fibroblasts (AG1522) were grown in culture to confluence and exposed to tritium beta-particles or 60Co gamma-ray adapting doses ranging from 0.10 cGy to 50 cGy. These cells, and other unexposed cells, were allowed to adapt during a 3 h, 37°C incubation prior to a challenge dose of 4 Gy of 60Co gamma-rays. Cells were replated in the presence of cytochalasin B and adaption was assessed by measuring micronucleus frequency in binucleated cells. Fluorescence in situ hybridization using whole chromosome paints (WCP) was then employed to probe the chromosome content of radiation induced micronuclei in radiation-adapted and non-adapted fibroblasts. (Abstract shortened by UMI.)

Alpha-amylase is a digestive enzyme involved in the metabolism of starch. It is found in a wide variety of organisms, and its expression is often regulated by carbon source. The D. melanogaster $\alpha$-amylase gene was used here to show the evolutionary conservation of both a structural gene as well as its glucose repression regulatory system. The repression machinery was shown to be functionally conserved between yeast and flies, as the $\alpha$-amylase promoter was used to express luciferase-based reporter constructs in a glucose repressible manner in Saccharomyces. The repression motifs of the $\alpha$-amylase promoter were mapped to a 126 bp fragment which contains two putative binding sites for MIG1, the yeast transcription factor involved in glucose repression. Further 5$\sp\prime$ deletions or removal of the 3$\sp\prime$ MIG 1 binding site eliminated glucose repression. Additional evidence for conservation of this machinery was provided by the cloning of Drosophila genomic and cDNA copies for SNF4, a subunit of the yeast derepression complex. This genes codes for a 684 aa protein which features extended carboxy and amino terminal sequences relative to the known yeast and mammalian homologues. Southern analysis suggested the presence of two gene copies in Drosophila, which would agree with the multiple cDNA isoforms seen in mammals. Inferred phylogenies indicated that the short isoform seen in yeasts and most mammalian sequences group separately from the long isoforms seen in Drosophila, C. elegans and a second human sequence--indicating the possible organization of orthologous gene copies. Aside from low level similarity to a handful of IMP dehydrogenases and IMPDH-like proteins, SNF4 is not significantly similar to other known proteins. Last, the evolution of the $\alpha$-amylase gene itself was examined with regards to nucleotide and amino acid biases. Nucleotide bias was observed at all three codon positions, primarily as a pyrimidine preference in the coding strand. Due to a nucleotide bias at nonsynonymous sites, there were significant content differences in GC-rich and AT-rich amino acids depending on the GC content of the gene. Although an amino acid bias might be expected to affect phylogenetic determination, with $\alpha$-amylase this bias was shown to not have a strong influence on inferred gene trees.

Sarcolemmal-associated proteins (SLAPs) are a family of acidic amphipathic alpha-helical proteins associated with the membrane. Different size SLAP transcripts were identified and shown to encode polypeptides of 37, 46 and 74 kDa designated by SLAP1, SLAP2 and SLAP3, respectively. The SLAP1 and SLAP2 transcripts were expressed in cardiac, soleus and smooth muscle, whereas SLAP3 was expressed ubiquitously. The subcellular distribution studies localized SLAPs to cell membrane, as well as sarco/endoplasmic reticulum membrane and transverse-tubules. The SLAP family is encoded by a single gene which was mapped to a human chromosome 3p14.3-21.2 and the various transcripts are perhaps generated by alternative promoter and/or alternative splicing. In order to understand the molecular basis underlying the SLAP isoform diversity studies were carried out to elucidate the genomic organization of the SLAP gene. The 3$\sp\prime$ end of SLAP gene is composed of 11 exons spanning over 35 kb, that range in size from 60 to 321 bp. SLAP introns on the other hand, range in size from 0.2 to 5.5 kb and all conform to a canonical GT-AG rule. Results suggest that SLAP primary transcript is alternatively spliced and that the expression of alternative variants is regulated in a developmental and tissue specific manner. Moreover, RT-PCR and cDNA library screening isolated alternatively spliced human, rabbit and rat orthologues that have been evolutionarily conserved which is indicative of a functional role of the various variants. Although the function of various SLAP variants remains unknown the alternative splicing is predicted to introduce putative phosphorylation sites, influence secondary structure and perhaps alter subcellular localization. In summary, heterogeneity due to alternative usage of the N-terminal of SLAP combined with the diversity generated by alternative splicing is predicted to give rise to 36 distinct SLAP isoforms, that may perhaps play distinct roles in the membrane function.

Retinoids are known to strongly inhibit the growth of estrogen (E2) dependent human breast cancer cells. This growth inhibition was found to be associated with an accumulation of cells in G1 phase of the cell cycle. Since the molecular mechanisms underlying these antiproliferative effects were unknown, we investigated the effects of retinoic acid (RA) on cell cycle progression and the expression and activity of cell cycle regulatory genes in the E2 dependent breast carcinoma cell line, MCF-7. We have found that RA induced accumulation of cells in G1 was correlated with decreased phosphorylation of the retinoblastoma protein (pRb). It also resulted in a down regulation of cyclin dependent kinase 2 (cdk2) and cyclin D1 gene expression and a profound down regulation of cdk2 activity. In contrast, no changes were seen in cdk4 expression or activity. We have further demonstrated that RA did not cause a change in the overall levels of expression of the known cyclin-cdk inhibitors p21 and p27. As well, no change in the activity of the cdk activating kinase, CAK, was detected. These results suggest that the growth inhibition of MCF-7 cells is associated with a down regulation of cdk2 activity. While this reduced activity occurred in the absence of RA-mediated increases in the levels of the cdk inhibitors p21 and p27, an increased association between p27 and cdk2 was observed in RA treated lysates. Furthermore, assays of cdk2 in pooled lysates taken from RA treated and control cells showed that RA treated cells contain a p27-like transferable cdk2 inhibitory activity. Pretreatment of cells with antisense but not mismatch p27 oligonucleotides attenuated the inhibitory effects of RA on cdk2 associated activity. These findings demonstrate that RA is capable of regulating cdk2 activity through a mechanism involving p27. RA also caused an increase in p53, we therefore investigated the role of p53 in growth arrest induced by exposure to RA. MCF-7/E6 cells expressing the human papillomavirus HPV16 E6 protein contained very low levels of p53 and exhibited partial abrogation of both cdk2 inactivation and G1 arrest in response to RA. To further elucidate the role of p53 in RA mediated inhibition of cdk2, MCF-7 cells were transfected with various p53 mutant expression constructs. However, none of the stable clones generated were impaired in their ability to down regulate cdk2 or undergo G1 arrest in response to RA. These results suggest that no functional domain of p53 alone contributes to the RA induced regulation of cdk2, but rather the cooperation of several domains of p53 may be required. We also observed that RA was able to profoundly down regulate cdk2 gene expression. However, following exposure to RA, no decrease in cdk2 gene transcription was observed in MCF-7 cells which had been stably transfected with a cdk2 promoter firefly luciferase reporter gene construct. These results were confirmed by nuclear run off assays which demonstrated that RA did not cause a decrease in the rate of cdk2 gene expression. Instead we found that RA decreased cdk2 expression by triggering posttranscriptional destabilization of the mRNA. Taken together, these findings suggest that RA inhibits cell cycle progression in MCF-7 cells through the inactivation of cdk2. Several targets have been identified that participate in the RA mediated down regulation of cdk2 activity.

The dlx homeobox genes of vertebrates are transcriptional regulators involved in the development of the ventral forebrain, otic vesicle and inner ear, visceral arches, olfactory placodes and fins/limbs. The zebrafish genorne contains eight dlx genes whose expression patterns during development are partially overlapping. Experiments in cultured cells and transgenic zebrafish embryos were used to understand the functional specificity and the biochemical properties of Dlx proteins. Previous work by us and others showed that Dlx proteins can act as transcriptional activators. In order to determine how the various Dlx proteins differ from each other, transfection experiments in cultured cells were done to identify the Dlx transcriptional activation domains. I found that an activation domain was located in the N-terminal region of Dlx1, Dlx2 and Dlx4. In addition, to test the hypothesis that the partially overlapping expression of dlx genes during development is the result, in part, of specific cross-regulatory interactions between the genes, overexpression of mutant versions of the dlx genes into transgenic zebrafish embryos were carried out. Injection into one cell embryo of synthetic mRNA coding for a chimeric protein including the amino-terminal region of Dlx3 and the carboxy-terminal half of Dlx2, resulted in a loss or decrease of endogenous dlx4 expression in the visceral arches, the otic vesicles and the forebrain of 27 hours embryos. Furthermore, I demonstrated that overexpression of that chimera, which disturbed the endogenous dlx4 expression, results in malformations of the craniofacial cartilages. It suggests that Dlx3 is normally a positive regulator of dlx4 expression and that the chimera Dlx3-Dlx2 protein interferes with the function of the endogenous Dlx3. These results suggest that the combinatorial patterns of dlx expression are due at least in part to cross-regulatory interactions between the different dlx genes.

Neural signals modulate acetylcholinesterase (AChE) expression in skeletal muscle cells. However, the cellular and molecular events mediating these effects are poorly understood. The mechanisms by which nerve-evoked electrical activity and nerve-derived trophic factors regulate the expression of AChE along muscle fibers was therefore elucidated. The contribution of nerve-evoked activity versus intrinsic properties of muscle fibers was assessed for their role in dictating the distinct patterns of AChE expression displayed by fast and slow muscles. We demonstrate that nerve-evoked electrical impulses play a key role in regulating AChE synthesis in these distinct muscle types. We also demonstrate that myogenic precursor cells from fast and slow muscle fibers generate myotubes that display similar patterns of AChE expression. Together, these findings suggest that nerve-evoked electrical activity rather than intrinsic properties of muscle cells is the primary regulator of AChE expression in fast and slow muscles. The importance of transcriptional versus post-transcriptional mechanisms in mediating the activity-dependent regulation of AChE was also determined. We demonstrate that higher levels of AChE mRNA observed in fast versus slow muscles is due to post-transcriptional events. We also show that reductions of AChE mRNA seen in mature denervated muscles cannot be accounted for by a decrease in the rate of AChE gene transcription. Denervation-induced reductions in the levels of AChE mRNA were less pronounced in muscles of developing animals. This attenuated decrease in AChE transcript abundance may be due to a transient enhancement in the transcriptional activity of the AChE gene observed in denervated muscles of developing rats. Transcriptional as well as post-transcriptional mechanisms are therefore important in mediating the activity-dependent regulation of AChE in developing muscles whereas post-transcriptional events are the primary mediators controlling AChE expression in mature muscles. Finally, the activity of several rat AChE promoter fragments in synaptic versus extrasynaptic regions of muscle fibers was assessed to determined whether enhanced transcription of the AChE gene in synaptic nuclei contributes to the accumulation of AChE transcripts at the neuromuscular synapse. Our findings indicate that synapse-specific activity of the AChE gene contributes to localized expression of the enzyme and its mRNA at the neuromuscular junction. Furthermore, we observed that calcitonin gene-related peptide (CGRP) and ciliary neurotrophic factor (CNTF) downregulate AChE expression. The suppressive effects of CGRP and CNTF on AChE expression suggest that a combination of activating and inhibiting signalling cascades may function in concert to regulate the abundance of AChE transcripts at synaptic sites.

Necrotizing enterocolitis (NEC) is an acute, self-limiting, inflammatory bowel disease of unclear pathogenesis affecting premature infants. It is the most common acquired surgical emergency in the neonatal period. NEC is characterized by necrosis of the bowel wall and may lead to peritonitis, stricture formation with bowel obstruction and lower gastrointestinal hemorrhage. Approximately 10% of premature babies develop NEC with up to 94% of cases seen in infants born at less than 36 weeks gestation. As more and more premature infants survive the neonatal period, it is expected that the incidence of this disease will increase. Briefly, the protocol involves laparotomy of anesthetized animals, creation of closed loops from terminal ileum to proximal colon which are injected with acidified casein test solution separated by saline injected control loops. Because NO is involved in neural control of intestinal motility and mucosal protection, we sought to evaluate the effects of manipulation of the nitrergic system on experimentally induced NEC in this model. Developmental changes in piglet intestinal motility were then documented in vivo, as well as those modifications induced by systemic stimulation or inhibition of NO availability. Our results have shown the presence of an ontogenically determined pattern of fasting intestinal motor activity, which is more susceptible to nitrergic manipulation in earlier developmental stages as documented through motility responses of the terminal gut. NO synthase activity was then measured in different regions of the developing piglet gut as well as in various layers of the piglet gut wall and compared to the activity found in NEC-induced gut specimens (test loops) at different stages of development and in different gut regions. Total NOS, iNOS and cNOS activity was assayed using the conversion of 14C L-arginine to 14C citrulline in full thickness gut, and in muscular and mucosal layers separated under the dissecting microscope. The potential therapeutic applications of these findings in preventing or attenuating NEC-induced intestinal damage in premature infants led to the development of a, now ongoing, clinical trial evaluating the effects of intravenous L-arginine administration in premature infants. It is expected that, based on preliminary data, a multi-centre clinical trial may lead to the prophylactic use of L-arginine as a treatment modality for NEC. (Abstract shortened by UMI.)