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The role of transcription in the development of electrical properties of neuronal membranes has been largely unexplored. To study the molecular events which result in the expression of these properties it is useful to describe the timing of the underlying RNA synthetic events. For example, the timing of the transcription involved in denervation-induced action potentials in frog slow muscle fibres and brain extract-induced sodium channels in chick muscle cells has been investigated. Previous studies of Xenopus laevis spinal neurones have established that the timing of the development of the neuronal action potential ionic dependence in dissociated cell cultures parallels that seen in vivo. This culture system, therefore, allows the determination of transcription-dependent periods necessary for the development of membrane properties known to have in vivo relevance. In the study described here, actinomycin D was used to examine the timing of the RNA synthetic events necessary for (1) neurite outgrowth and (2) development of the ionic dependence of the action potential. I report that inhibition of transcription at an early stage specifically blocks the appearance of the mature sodium-dependent action potential without affecting either neurite outgrowth or the development of delayed rectification.

Difluoromethylornithine (DFMO) is an investigational chemopreventive agent that inhibits ornithine decarboxylase (ODC) activity, lowers cellular polyamine concentrations, and decreases cell proliferation in vivo and in vitro. In five subjects we have compared the polyamine concentrations in rectal mucosal biopsies and in exfoliated buccal mucosal cells (EBM) before and after DFMO treatment to assess the suitability of EBM as an easily accessible marker tissue for DFMO suppression of polyamine synthesis in the rectal mucosa. One month of 3 g/m2/day of DFMO treatment caused a statistically significant decrease in putrescine and spermidine concentrations in rectal mucosa biopsy specimens but not in EBM samples. ODC activity in EBM was high (approximately 1 mumol/min/mg protein), resistant to DFMO inhibition (Ki = 4200 microM), dependent on GTP concentration (maximal at 0.1 mM), and was reduced concomitantly with bacterial concentration by antiseptic mouthwashing. Bacteria adherent to EBM were visible by electron microscopy. Forty bacterial colonies/ng protein were culturable from washed EBM samples. Oral bacteria preclude the use of EBM samples as a marker tissue of DFMO effect in the rectal mucosa, but oral DFMO therapy is effective in depleting polyamines in rectal mucosa.

The efficiency of replating of cells from primary colonies grown in semisolid medium has been used to detect and quantitate self-renewal in vitro. A positive correlation has been found by others between the replating efficiency of cells from myelogenous leukemia and patient survival. In the current study we measured primary and secondary replating efficiency of metastatic melanoma cells from subcutaneous tissues or lymph nodes of twelve patients and related these results to patient survival from time of biopsy. No relationship was found between primary and secondary plating efficiency nor for primary or secondary replating efficiency and survival. These results suggest that colony-forming melanoma cells grown under anchorage-independent conditions do not identify a stem cell population important for survival distinct from highly proliferative cells. These studies do not, however, rule out the possibility that a non-clonogenic transitional cell population exists in the tumor.

In the pathogenic bacterium Chlamydia trachomatis, a transcriptional repressor, HrcA, regulates the major heat shock operons, dnaK and groE. Cellular stress causes a transient increase in transcription of these heat shock operons through relief of HrcA-mediated repression, but the pathway leading to derepression is unclear. Elevated temperature alone is not sufficient, and it is hypothesized that additional chlamydial factors play a role. We used DNA affinity chromatography to purify proteins that interact with HrcA in vivo and identified a higher-order complex consisting of HrcA, GroEL, and GroES. This endogenous HrcA complex migrated differently than recombinant HrcA, but the complex could be disrupted, releasing native HrcA that resembled recombinant HrcA. In in vitro assays, GroEL increased the ability of HrcA to bind to the CIRCE operator and to repress transcription. Other chlamydial heat shock proteins, including the two additional GroEL paralogs present in all chlamydial species, did not modulate HrcA activity.

Irradiation-resistant natural killer (NK) cells in an F(1) recipient can reject parental bone marrow, and host NK cells can also prevent engraftment of allogeneic bone marrow. We show here that repopulating bone marrow cells in certain mouse strains expressed retinoic acid early inducible 1 proteins, which are ligands for the activating NKG2D NK cell receptor. Treatment with a neutralizing antibody to NKG2D prevented rejection of parental BALB/c bone marrow in (C57BL/6 x BALB/c) F(1) recipients and allowed engraftment of allogeneic BALB.B bone marrow in C57BL/6 recipients. Additionally, bone marrow from C57BL/6 mice transgenic for retinoic acid early inducible 1epsilon was rejected by syngeneic mice but was accepted after treatment with antibody to NKG2D. If other stem cells or tissues upregulate expression of NKG2D ligands after transplantation, NKG2D may contribute to graft rejection in immunocompetent hosts.

Nephrin is a cell surface receptor of the Ig superfamily that localizes to slit diaphragms, the specialized junctions between the interdigitating foot processes of the glomerular epithelium (podocytes) in the kidney. Mutations in the NPHS1 gene encoding nephrin lead to proteinuria and congenital nephrotic syndrome, indicating that nephrin is essential for normal glomerular development and function. To identify nephrin-binding proteins, we performed mass spectrometry on proteins obtained from pull-down assays with GST-nephrin cytoplasmic domain. Nephrin specifically pulled down six proteins from glomerular lysates, MAGI-2/S-SCAM (membrane-associated guanylate kinase inverted 2/synaptic scaffolding molecule), IQGAP1 (IQ motif-containing GTPase-activating protein 1), CASK (calcium/calmodulin-dependent serine protein kinase), a-actinin, all spectrin, and beta II spectrin. All of these scaffolding proteins are often associated with cell junctions. By immunofluorescence these proteins are expressed in glomerular epithelial cells, where they colocalize with nephrin in the foot processes. During glomerular development, IQGAP1 is expressed in the junctional complexes between the earliest identifiable podocytes, MAGI-2/S-SCAM is first detected in junctional complexes in podocytes after their migration to the base of the cells. Thus, the nephrin-slit diaphragm protein complex contains a group of scaffolding proteins that function to connect junctional membrane proteins to the actin cytoskeleton and signaling cascades. Despite their special morphology and function, there is considerable compositional similarity between the podocyte slit diaphragm and typical junctional complexes of other epithelial cells.

DAP12 is a signaling adaptor containing an immunoreceptor tyrosine-based activation motif (ITAM) that pairs with receptors on myeloid cells and natural killer cells. We examine here the responses of mice lacking DAP12 to stimulation through Toll-like receptors (TLRs). Unexpectedly, DAP12-deficient macrophages produced higher concentrations of inflammatory cytokines in response to a variety of pathogenic stimuli. Additionally, macrophages deficient in spleen tyrosine kinase (Syk), which signals downstream of DAP12, showed a phenotype identical to that of DAP12-deficient macrophages. DAP12-deficient mice were more susceptible to endotoxic shock and had enhanced resistance to infection by the intracellular bacterium Listeria monocytogenes. These data suggest that one or more DAP12-pairing receptors negatively regulate signaling through TLRs.

BACKGROUND: The primary aim of this study is to demonstrate the feasibility of utilizing doxycycline to suppress matrix metalloproteinase-9 (MMP-9) in brain arteriovenous malformations (AVMs). METHODS: Ex-vivo treatment of AVM tissues: Intact AVM tissues were treated with doxycycline for 48 hours. Active and total MMP-9 in the medium were measured. Pilot trial: AVM patients received either doxycycline (100 mg) or placebo twice a day for one week prior to AVM resection. Active and total MMP-9 in BVM tissues were measured. RESULTS: Ex-vivo treatment of AVM tissues: Doxycycline at 10 and 100 microg/ml significantly decreased MMP-9 levels in AVM tissues ex-vivo (total: control vs 10 vs 100 microg/ml = 100 +/- 6 vs 60 +/- 16 vs 61 +/- 9%; active: 100 +/- 8 vs 48 +/- 16 vs 59 +/- 10%). Pilot trial: 10 patients received doxycycline, and 4 patients received placebo. There was a trend for both MMP-9 levels to be lower in the doxycycline group than in the placebo group (total: 2.18 +/- 1.94 vs 3.26 +/- 3.58, P = .50; active: 0.48 +/- 0.48 vs 0.95 +/- 1.01 ng/100 microg protein, P = .25). CONCLUSIONS: A clinically relevant concentration of doxycycline decreased MMP-9 in ex-vivo AVM tissues. Furthermore, there was a trend that oral doxycycline for as short as one week resulted in a decrease in MMP-9 in AVM tissues. Further studies are warranted to justify a clinical trial to test effects of doxycycline on MMP-9 expression in AVM tissues.

Women who inject drugs in cities where syringe exchange programs (SEPs) are well established may have different risks for HIV infection. In 1997, we interviewed 149 female syringe exchangers in San Francisco, CA, a city with high rates of injection drug use that is home to one of the largest and oldest SEPs in the United States. In this report, we describe their sociodemographics, health, and risk behavior, and we examine factors associated with recent syringe sharing. Fifty percent of respondents were women of color and the median age was 38 years. Most (86%) injected heroin and nearly half were currently homeless or had recently been incarcerated. One-third of all women reported needle sharing in the prior month. This was higher than the rate of needle sharing reported by a mixed gender sample of San Francisco exchangers in 1993, although it resembled the rate reported by a mixed gender sample in 1992. In a multivariate analysis, syringe sharing was associated with age, housing status, and sexual partnerships. Syringe sharers were more likely to be young, homeless, or have a sexual partner who was also an injection drug user. While wide access to sterile syringes is an important strategy to reduce HIV transmission among injection drug users (IDU), syringe exchange alone cannot eradicate risky injection by female IDU. Additional efforts to reduce risky injection practices should focus on younger and homeless female IDU, as well as address selective risk taking between sexual partners.

OBJECTIVE: To analyse the development and execution of the "Creative Solutions" Benson & Hedges advertising campaign to understand its social, political, and commercial implications. METHODS: Searches of the Philip Morris documents and Legacy Tobacco Documents websites for relevant materials; Lexis/Nexis searches of major news and business publications; and denotative and connotative analyses of the advertising imagery. RESULTS: Philip Morris developed the Creative Solutions campaign in an effort to directly confront the successes of the tobacco control movement in establishing new laws and norms that promoted clean indoor air. The campaign's imagery attempted to help smokers and potential smokers overcome the physical and social downsides of smoking cigarettes by managing risk and resolving internal conflict. The slogans suggested a variety of ways for smokers to respond to restrictions on their habit. The campaign also featured information about the Accommodation Program, Philip Morris's attempt to organise opposition to clean indoor air laws. CONCLUSION: The campaign was a commercial failure, with little impact on sales of the brand. Philip Morris got some exposure for the Accommodation Program and its anti-regulatory position. The lack of commercial response to the ads suggests that they were unable to successfully resolve the contradictions that smokers were increasingly experiencing and confirms the power of changing social norms to counter tobacco industry tactics.

Twisted Gastrulation (Tsg) is a secreted molecule which regulates BMP signalling in the extracellular space as part of an evolutionarily conserved network of interacting proteins. In Xenopus, maternal xTsg mRNA can be found throughout the early embryo. After gastrulation, xTsg is expressed as part of the BMP4 synexpression group until late tadpole stages. Here we report the identification of a second Xenopus Tsg gene (xTsg-2). Xenopus Tsg-2 is highly homologousto xTsg. In particular, amino acid residues which have been shown to be required for the binding of xTsg to BMP and to Chordin are conserved. The expression of Xenopus Tsg-2 mRNA was restricted to late stages of embryonic development; it was detected at tadpole stages in lateral plate mesoderm, neural crest, branchial arches and head mesenchyme. In microinjection experiments, the activity of xTsg-2 mRNA was similar to that of xTsg. We conclude that two Tsg genes act in distinct temporal and spatial territories in the course of Xenopus embryonic development.

OBJECTIVE: To investigate the genesis and development of tobacco company Philip Morris's recent image enhancement strategies and analyse their significance. DATA SOURCES: Internal Philip Morris documents, made available by the terms of the Master Settlement Agreement between the tobacco companies and the attorneys general of 46 states, and secondary newspaper sources. STUDY SELECTION: Searches of the Philip Morris documents website (www.pmdocs.com) beginning with terms such as "image management" and "identity" and expanding as relevant new terms (consultant names, project names, and dates), were identified, using a "snowball" sampling strategy. FINDINGS AND CONCLUSIONS: In the early 1990s, Philip Morris, faced with increasing pressures generated both externally, from the non-smokers' rights and public health communities, and internally, from the conflicts among its varied operating companies, seriously considered leaving the tobacco business. Discussions of this option, which occurred at the highest levels of management, focused on the changing social climate regarding tobacco and smoking that the tobacco control movement had effected. However, this option was rejected in favour of the image enhancement strategy that culminated with the recent "Altria" name change. This analysis suggests that advocacy efforts have the potential to significantly denormalise tobacco as a corporate enterprise.

We studied prevalence and correlates of injection drug use, awareness of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), and risky behaviors among drug users serviced by a nongovernmental organization catering to drug users in three Pakistani cities (Quetta, Peshawar, and Rawalpindi). Logistic regression analysis was used to identify correlates of injection drug use. Of 608 drug users, 99.8% were male; median age was 32 years, and 44% were married. Most (79.8%) were Pakistani; 15.3% were Afghani. The majority used heroin (98.7%), mostly by inhalation; 15.2% injected drugs. Only 41% had heard of HIV/AIDS, and 30% had been paid for donating blood. Injection drug use and needle sharing were highest in Quetta. Injecting drug users (IDUs) were nearly twice as likely to have donated blood and to have heard about HIV/AIDS compared to other drug users. Interventions to discourage transitions to injection, increase HIV testing, and safeguard the blood supply in Pakistan are urgently needed.

In activated mouse natural killer (NK) cells, the NKG2D receptor associates with two intracellular adaptors, DAP10 and DAP12, which trigger phosphatidyl inositol 3 kinase (PI3K) and Syk family protein tyrosine kinases, respectively. Here we show that cytotoxicity, but not cytokine production, is triggered by NKG2D in activated NK cells lacking either DAP12 or the Syk family members Syk and ZAP70. Inhibition of PI3K blocks this cytotoxicity, suggesting that the DAP10-PI3K pathway is sufficient to initiate NKG2D-mediated killing of target cells. Our results highlight signaling divergence in the effector functions of NKG2D and indicate that alternative associations between a receptor and its adaptors may provide a single receptor with a dual 'on-switch', giving mouse NK cells more choices through which to trigger cytotoxicity.

Objetivo: Comparar la calidad y la fuente de financiamiento de los estudios cuyas conclusiones señalan una repercusión económica negativa de las políticas de ambientes libres de humo de tabaco en el sector de la hostelería con las de los estudios que no llegan a esas conclusiones.Fuentes de datos: Los investigadores buscaron todos los estudios realizados antes del 31 de agosto de 2002. Se encontraron artículos publicados en revistas científicas utilizando mecanismos de búsqueda como Medline, Science Citation Index, Social Sciences Citation Index, Current Contents, PsychInfo, Econlit y Healthstar. Se encontraron asimismo estudios inéditos en sitios web de empresas tabacaleras y en internet.Selección de los estudios: Se incluyeron 97 estudios que contenían afirmaciones sobre las repercusiones económicas. El 93% de los estudios encontrados cumplían los criterios de selección determinados por consenso entre los diferentes investigadores encargados de la revisión.Extracción de datos: Dos investigadores clasificaron por separado los resultados y las características de los estudios (además de la fuente de financiamiento). Participó también en la clasificación un tercer evaluador que desconocía el objetivo de este trabajo y la fuente de financiamiento de los estudios.Síntesis de los datos: En los estudios cuyas conclusiones señalaban repercusiones negativas, las probabilidades de que utilizara una medida subjetiva de los resultados fueron 4,0 veces más altas (intervalo de confianza de 95% (IC) 1,4 a 9,6; p = 0,007) que las de los estudios que no señalaban en sus conclusiones un impacto negativo y las probabilidades de que el trabajo no fuese sometido a una revisión científica por pares fueron 20 veces más altas (IC de 95%, 2,6 a 166,7; p = 0,004). Todos los estudios que indicaban repercusiones negativas fueron financiados por la industria tabacalera. El 94% de los estudios realizados con financiamiento de empresas tabacaleras señaló repercusiones negativas. En contraste, ninguno de los estudios que no contaban con ese respaldo mostró repercusiones negativas.Conclusiones: Los estudios mejor diseñados, en todos los casos, señalaban que las leyes que creaban ambientes libres de humo de tabaco en restaurantes y bares no tuvieron repercusiones, o que si las tuvieron fueron positivas, en las ventas o el empleo. Las autoridades pueden tomar medidas para proteger a trabajadores y clientes de los productos tóxicos por la exposición al humo del tabaco ajeno, rechazando con toda confianza los reclamos de la industria de que habrá efectos económicos adversos.OBJECTIVE: To compare the quality and funding source of studies concluding a negative economic impact of smoke-free policies in the hospitality industry to studies concluding no such negative impact. DATA SOURCES: Researchers sought all studies produced before 31 August 2002. Articles published in scientific journals were located with Medline, Science Citation Index, Social Sciences Citation Index, Current Contents, PsychInfo, Econlit, and Healthstar. Unpublished studies were located from tobacco company websites and through internet searches. STUDY SELECTION: 97 studies that made statements about economic impact were included. 93% of the studies located met the selection criteria as determined by consensus between multiple reviewers. DATA EXTRACTION: Findings and characteristics of studies (apart from funding source) were classified independently by two researchers. A third assessor blind to both the objective of the present study and to funding source also classified each study. DATA SYNTHESIS: In studies concluding a negative impact, the odds of using a subjective outcome measure was 4.0 times (95% confidence interval (CI) 1.4 to 9.6; p = 0.007) and the odds of not being peer reviewed was 20 times (95% CI 2.6 to 166.7; p = 0.004) that of studies concluding no such negative impact. All of the studies concluding a negative impact were supported by the tobacco industry. 94% of the tobacco industry supported studies concluded a negative economic impact compared to none of the non-industry supported studies. CONCLUSION: All of the best designed studies report no impact or a positive impact of smoke-free restaurant and bar laws on sales or employment. Policymakers can act to protect workers and patrons from the toxins in secondhand smoke confident in rejecting industry claims that there will be an adverse economic impact.

Nonobese diabetic (NOD) mice, a model of insulin-dependent diabetes mellitus, have a defect in natural killer (NK) cell-mediated functions. Here we show impairment in an activating receptor, NKG2D, in NOD NK cells. While resting NK cells from C57BL/6 and NOD mice expressed equivalent levels of NKG2D, upon activation NOD NK cells but not C57BL/6 NK cells expressed NKG2D ligands, which resulted in downmodulation of the receptor. NKG2D-dependent cytotoxicity and cytokine production were decreased because of receptor modulation, accounting for the dysfunction. Modulation of NKG2D was mostly dependent on the YxxM motif of DAP10, the NKG2D-associated adaptor that activates phosphoinositide 3 kinase. These results suggest that NK cells may be desensitized by exposure to NKG2D ligands.

A recent study by Ingram et al. [2000b: Teratology 62:393-405] suggests a (His)73(Arg) polymorphism (A:G) in HOXA1 contributes substantially to a liability for autism. Using 68 individuals diagnosed with Autism Spectrum Disorders, they found a significant dearth of G homozygotes and biased transmission of G alleles from parents to affected offspring, especially from mothers. Because the connection between HOXA1 and liability to autism is compelling, we attempted to replicate their finding using a larger, independent sample from the Collaborative Programs of Excellence in Autism (CPEA) network. In our data, genotype frequencies conform to Hardy-Weinberg equilibrium; allele transmissions meet Mendelian expectations; and there is no obvious sex-biased allele transmission. Based on our sample size, calculations suggest that we would have at least 95% power to detect linkage and association even if the A:G polymorphism were to account for only 1% of the heritability of autism. Therefore, although we cannot exclude the possibility that the samples in the two studies are intrinsically different, our data from our sample argue against a major role for HOXA1 (His)73(Arg) in liability to autism.

In this paper, we present a review of semantic priming experiments in schizophrenia. Semantic priming paradigms show utility in assessing the role of deficits in semantic memory network access in the pathology of schizophrenia. The studies are placed in the context of current models of information processing. In this review we include all English-language reports (from peer-reviewed journals) of single-word semantic priming studies involving participants with schizophrenia. The studies to date show schizophrenic patients to exhibit variable semantic priming effects under automatic processing conditions, and consistent impairments under controlled/attentional conditions. We also describe associations with other neurocognitive dysfunction, neurochemical and electrophysiological disturbances, and clinical manifestations (such as thought disorder).

Metastatic melanomas are typically resistant to radiation and chemotherapy. The underlying basis for this phenomenon may result in part from defects in apoptotic pathways. Nuclear factor kappa B (NFκB) has been shown to control apoptosis in many cell types and normally functions as an immediate stress response mechanism that is rigorously controlled by multiple inhibitory complexes. We have previously shown that NFκB binding is elevated in metastatic melanoma cells relative to normal melanocytes. In the current study, Western blot analysis showed that, compared with normal melanocytes, melanoma cell lines have higher nuclear levels of the NFκB subunits p50 (7-fold) and RelA (5-10-fold). In response to tumor necrosis factor-alpha (TNFα), both melanocytes and melanoma cells showed increased nuclear p50 and RelA levels, but levels in melanoma cells remained higher than in melanocytes. We also found that melanoma cells expressed higher cytoplasmic levels of RelA, p105/p50 and the inhibitory protein, inhibitor of kappa B alpha (IκBα than melanocytes. To directly test whether RelA expression has an impact on melanoma cell survival, we used antisense RelA phosphorothioate oligonucleotides and found that melanoma cell viability was significantly decreased compared with untreated or control cultures. The constitutive activation of NFκB in metastatic melanoma cell cultures may, therefore, support an inappropriate cell survival pathway that can be therapeutically manipulated.

Previous studies in rat, showing a transient pattern of expression of the alpha 7 nicotinic acetylcholine receptor in the ventrobasal thalamus and barrel cortex during the first 2 postnatal weeks, suggest that these receptors may play a role in development of the thalamocortical system. In the present study, in situ hybridization and radiolabeled ligand binding were employed to examine the spatiotemporal distribution of alpha 7 mRNA and alpha-bungarotoxin binding sites in the thalamocortical pathway of mouse during early postnatal development. As in the rat, high levels of alpha 7 mRNA and alpha-bungarotoxin binding sites are present in the barrel cortex of mouse during the first postnatal week. Both alpha 7 mRNA and its receptor protein are observed in all cortical laminae, with the highest levels seen in the compact cortical plate, layer IV, and layer VI. When viewed in a tangential plane, alpha 7 mRNA and alpha-bungarotoxin binding sites delineate a whisker-related barrel pattern in layer IV by P3-5. Quantitative analysis reveals a dramatic decrease in the levels of expression of alpha 7 mRNA and alpha-bungarotoxin binding sites in the cortex by the end of the second postnatal week. Unlike in the rat, only low levels of alpha 7 mRNA or alpha-bungarotoxin binding sites are present in the ventrobasal complex of the mouse thalamus. The broad similarities between the thalamocortical development of rat and mouse taken together with the present results suggest that alpha 7 receptors located on cortical neurons, rather than on thalamic neurons, play a role in mediating aspects of thalamocortical development.

Alternative splicing results in production of four agrin proteins (agrin0, agrin8, agrin11, and agrin19) with different AChR aggregating activities. However, the cellular origin of mRNAs encoding each agrin isoform remains unknown. Using single-cell PCR, we demonstrate that in the chick ciliary ganglion, nonneuronal cells express only mRNA encoding agrin0, whereas neurons express one or any combination of agrin mRNAs. Moreover, significant differences were observed between the agrin mRNA profiles of ciliary and choroid neurons in the ganglion. The abundance of each agrin mRNA, the fraction of neurons expressing each transcript, and the combinations of transcripts expressed by neurons also change during development. Our results demonstrate that transcripts encoding agrin proteins with high AChR aggregating activity are expressed exclusively by neurons in the ciliary ganglion and that alternative splicing of agrin mRNA is regulated during development and in a cell-specific manner.

The most convincing evidence for a preventive role for any modality is obviously demonstration of incidence reduction produced by that modality. However, cancer prevention trials with cancer incidence as an endpoint have logistic problems rendering them essentially impossible to conduct for most malignancies. Hence a workable strategy often involves analysis of other, indirect lines of evidence to reach conclusions. For oral cancer, dietary epidemiologic evidence points to a protective role for foods rich in carotenoids. Other anti-oxidants, such as vitamin C, are also implicated. Similarly, laboratory evidence points to a carcinogenesis inhibitory role for both retinoids and carotenoids. Clinical studies have targeted premalignant lesions, i.e., oral leukoplakia. For over two decades the efficacy of retinoids, natural and synthetic, has been known. Nevertheless, it has been difficult to translate this into a recommendation for prevention because of the toxicity of retinoids. The synthetic retinoid most often used in these trials is 13-cis-retinoic acid. This compound is toxic even at very low doses (0.1 mg/kg/day), particularly when given over several weeks to months. Hence, although effective, it cannot be advocated for prevention or oral cavity cancer. Studies with nontoxic antioxidants, such as beta-carotene, are much more recent. Early results are promising in that beta-carotene, alone or in combination with other nutrients, can reverse oral leukoplakia without toxicity in short-term trials. Studies currently under way will demonstrate whether durable remissions can be obtained using this strategy. It should be emphasized that such long-term trials are problematic to conduct with the toxic retinoids because the risks of prolonged exposure to them outweighs the chance of cancer development in the usual leukoplakia lesion.

A role for neutral endopeptidase 24.11 (NEP) in growth and development is supported by the demonstration that NEP hydrolyses and inactivates a number of peptide growth factors including atrial natriuretic peptide, endothelins, bombesin-like peptides, and opioid peptides, including the enkephalins. In the present study, suspensions of cells obtained from the ventral mesencephalon or cortex of rat embryos (ED14) were implanted into the striatum of the adult rat brain. Three to 15 weeks after transplantation the relative distribution of NEP-positive cellular elements was visualized histochemically. NEP staining in the transplants consistently appeared before NEP staining in the surrounding host striatum supporting a relative increase in NEP activity in the transplants. The NEP staining richly visualized cells of varying size and morphology which lacked the normal organization of the host striatum. The histochemical staining in the transplants and the surrounding host tissue was completely blocked by a 100 nM concentration of the selective NEP inhibitors phosphoramidon or JHF-26, supporting the exclusive localization of NEP by this method. NEP localization in the embryonic (ED14) cortex and ventral mesencephalon was also confirmed, suggesting one possible origin for the NEP-positive cells visualized in the transplants. Fluorescent double-labeling studies for NEP and glial fibrillary acidic protein (GFAP) or transforming growth factor alpha precursor (TGF alpha p) revealed the presence of rich glial labeling within the transplants for both GFAP and TGFap. NEP-labeled cells in the transplants were closely associated with glial elements, however, only occasional glial elements in the transplants stained for NEP; supporting a non-astrocytic localization for the NEP in the transplants. The marked enhancement of NEP staining in the transplants may have significance for controlling the rate or pattern of growth of the transplanted cells through inactivation of peptide growth factors produced by, or in response to, the transplants.

Normal human melanocytes require 12-O-tetradecanoylphorbol 13-acetate (TPA) for prolonged growth in vitro. In contrast, the growth of human malignant melanoma cells is often inhibited by TPA. In this study, we have confirmed and extended these observations. Since protein kinase C (PKC) is an important mediator of the effects of TPA, we have investigated the nature of this differential growth response by examining PKC expression and activity in primary cultures of human neonatal melanocytes and metastatic melanoma cell strains. PKC, when measured by immunoreactivity or a functional assay, was found to be more abundant in melanoma cells than in melanocytes. When specific isotypes were examined by Northern analysis, PKC-alpha and -epsilon were expressed in both melanocytes and melanoma. PKC-beta was expressed in melanocytes, but was undetectable by Northern analysis in 10 out of 11 melanoma cell strains. Southern analysis revealed that no gross deletions or rearrangements of the PKC-beta gene had occurred. These data suggest that down-regulation of the PKC-beta gene occurs frequently during the process of transformation of melanocytes. Furthermore, differential expression of PKC isotypes may explain the different effects of TPA on melanocyte and melanoma cell growth.

Recent studies of the p53 tumor suppressor locus (designated TP53) in primary hepatocellular carcinoma (PHC) have identified a high frequency of codon 249 mutations. Due to the geographic location from which the samples were obtained and the substitution observed, the mutation was suggested to be attributable to aflatoxin B1 (AFB1) exposure. To determine the generality of this phenomenon, we have examined PHC tissues from 107 geographically and ethnically diverse sources. The frequency of p53 gene mutations was evaluated by using PCR/restriction-digest methods, GC-clamp (G+C-rich sequence) denaturing gradient gel electrophoresis, and DNA sequencing. The mutation rate observed in tumors from high-AFB1-exposure regions (25%) was more than double the rate observed in low-exposure regions (12%) but lower than the 50% frequency previously reported. Codon 249 mutations occurred at a much lower frequency than previously reported (2 of 107 samples examined). These results suggest that changes in DNA encoding p53 may not represent primary oncogenic effects but instead represent genetic changes related to tumor progression. High AFB1 levels may facilitate the generation of these progressional changes, but not by inducing a specific p53 gene mutation at codon 249 as previously reported.

Cultivation of human melanocytes requires several growth factors for cell proliferation. For example, basic fibroblast growth factor (bFGF) is an essential growth agent for melanocyte proliferation in vitro and has been proposed to be an autocrine growth factor in human melanoma cells. Studies using either anti-bFGF antibodies or antisense oligonucleotides partially inhibited the proliferation of human melanoma cells. However, one group was unable to detect bFGF RNA transcripts in human melanoma cells using a human complementary DNA probe. These contradictory results prompted us to investigate the bFGF gene expression in human primary melanocytes and metastatic melanoma cells using Southern, Northern, and Western blot analyses. No gross rearrangements in the bFGF gene were detected in the genomic DNA. Although high levels of bFGF RNA transcripts were detected in melanocytes, no bFGF protein was detected using Western blot analysis. In contrast, melanoma cells expressed much lower levels of bFGF RNA transcripts, and cells from three of four cell strains synthesized the multiple isoforms of bFGF protein. In one of the melanoma cell strains, no bFGF protein was detected using Western blot analysis. Although three of four melanoma cell strains expressed bFGF protein, this molecule does not appear to function as an autocrine growth factor, and expression of the bFGF protein was not a consistent alteration in all melanoma cell strains.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder of unknown aetiology that affects numerous body systems including skin, brain and kidneys. Some TSC has been linked to chromosome 9, additional TSC genes on chromosomes 11 and 12 have been proposed, but the majority of TSC families remain unlinked. Using TSC families in which data had excluded linkage to chromosome 9, we failed to detect linkage with loci on chromosomes 11, 12 and others. One marker examined was D16S283, the closest locus on the proximal side of the polycystic kidney disease type 1 (PKD1) gene. Linkage between TSC and D16S283 demonstrated a lod score of 9.50 at theta = 0.02 with one family independently presenting a lod score of 4.44 at theta = 0.05. These data reveal an important TSC locus near the region of PKD1 on chromosome 16p13.

Autism is a behavior disorder with genetic influences indicated from twin and family studies and from the co-occurrence of autism with known genetic disorders. Tuberous sclerosis complex (TSC) is a known genetic disorder with behavioral manifestations including autism. A literature review of these two disorders substantiates a significant association of autism and TSC with 17-58% of TSC subjects manifesting autism and 0.4-3% of autistic subjects having TSC. In initial data collected on 13 TSC probands and 14 autistic probands in our family study of autism and TSC, we identified 7 TSC subjects with autism. The seven TSC autistic probands are similar to non-TSC autistic probands on the Social and Communication domains of the Autism Diagnostic Inventory (ADI) (Le Couteur et al., 1989), but show fewer Repetitive Rituals. There are more male TSC probands with autism than female, despite an equal sex ratio among TSC probands. The TSC probands with autism have significantly more seizures and mental retardation than those without autism; however, the extent and etiology of associations require further study. Our preliminary findings suggest that a fruitful approach for delineating genetic influences in autism may come from further investigation of possible mechanisms underlying the association of autism and TSC.

Adherence interventions were implemented in a 1-year community-based colon cancer prevention clinical trial (n = 110) using wheat bran fiber and calcium dietary supplements. The adherence promotion strategy was guided by a theoretical model.The adherence intervention contains both a generalized portion given to all participants and an individualized portion given to marginal (50-74% intake) and low (under 50% intake) adherers. A regression model was employed to assess the effectiveness of the interventions both at the first intervention and at subsequent times.The Health Behavior in Cancer Prevention Model-based adherence promotion intervention was associated with retention of participants, both during the run-in period and after randomization (P = 0.05); and maximization of the percentage of the 13.5-g recommended fiber supplement consumed during the trial (92.5%). The positive effects of the adherence intervention were greater with first-time nonadherers and the control group than with the experimental group. The high-fiber group had notably more biological GI effects from the increased fiber intake, more preexisting comorbidities, and lower perceived cognitive and physical health status.Randomized participants had excellent adherence overall. Retention rates in the trial were better than would be expected without the adherence intervention, especially among those participants who may have been at higher risk for dropping out of the study. This suggests that a systematic, theoretically based adherence strategy should be further tested in clinical trial settings in which lower adherence is a problem.

The hereditary human disease ataxia-telangiectasia (AT) is characterized by phenotypic complexity at the cellular level. We show that multiple mutant phenotypes of immortalized AT cells from genetic complementation group D (AT-D) are corrected after the introduction of a single human chromosome from a human-mouse hybrid line by microcell-mediated chromosome transfer. This chromosome is cytogenetically abnormal. It consists primarily of human chromosome 18, but it carries translocated material from the region 11q22-23, where one or more AT genes have been previously mapped by linkage analysis. A cytogenetically normal human chromosome 18 does not complement AT-D cells after microcell-mediated transfer, whereas a normal human chromosome 11 does. We conclude that the AT-D gene is located on chromosome 11q22-23.

Retinoids, the natural and synthetic analogs of vitamin A, are growth-inhibiting and differentiation-inducing agents and show clinical promise as chemopreventive and antineoplastic agents. Fenretinide, a new synthetic retinoid, has antitumor activity in certain in vitro and in vivo model systems and was relatively nontoxic in phase I trials. Based on these data, we designed a phase II study of Fenretinide involving 31 patients with advanced breast cancer [15] and melanoma [16], two cancers shown to be responsive to this agent in preclinical models. Fenretinide was inactive in patients with advanced disease. Toxicity was mild, and reversible. Mucocutaneous side effects occurred in 16 (52%) patients. Nyctalopia developed in three patients one of whom developed decreased B-wave amplitude of the scotopic electroretinogram. The minimal toxicity and significant activity in preclinical studies make this an attractive agent for future breast cancer chemoprevention studies.

The value of chromosomal analysis is well established in human hematologic neoplasms. In contrast, the relation between chromosomal abnormalities and clinical outcome in solid tumors in humans has received little study. We undertook this study to determine whether chromosomal abnormalities could provide information on the survival of patients with malignant melanoma. Chromosome-banding analysis was performed on tumor-biopsy samples from 62 patients with metastatic melanoma, and recurring cytogenetic abnormalities were correlated with survival. Patients with structural abnormalities of chromosome 7 or 11 had significantly shorter survival than patients without these abnormalities. We conclude that cytogenetic analysis may provide useful prognostic information about patients with metastatic melanoma.

Tuberous sclerosis (TSC) is a dominantly inherited disorder characterized by hamartomas and hamartias in one or more organs, most often in skin, brain, and kidneys. Analysis of the basic genetic defect in tuberous sclerosis would be greatly expedited by definitive determination of the chromosomal location of the TSC gene or genes. We have carried out genetic linkage studies in 15 TSC families, using 34 polymorphic markers including protein markers and DNA markers. Pairwise lod scores were calculated using LIPED, and multipoint analyses were carried out using MENDEL. In the pairwise linkage analysis, using a penetrance value of 90%, a significant positive lod score was obtained with MCT128.1 (D11S144), 11q22-11q23, Zmax 3.26 at theta = 0.08. The tyrosinase probe TYR (11q14-11q22) gave a maximum lod score of 2.88 at theta = 0. In the multipoint analyses the most likely order is (TYR,TSC)-MCT128.1-HHH172. Homogeneity analysis was carried out using the USERM9 subprogram of MENDEL, which conducts the admixture test of C. Smith (1963, Ann. Hum. Genet. 27: 175-182). This test provided no evidence for genetic heterogeneity (that is, non-11-linked families) in this data set.

ABSTRACT 6-[18F]-Fluoro- l-dopa (FDOPA) has been widely used as a biomarker for catecholamine synthesis, storage, and metabolism-its intense uptake in the striatum, and fainter uptake in other brain regions, is correlated with the symptoms and pathophysiology of Parkinson's disease (PD). 6-[18F]fluoro- m-tyrosine (FMT), which also targets l-amino acid decarboxylase, has potential advantages over FDOPA as a radiotracer because it does not form catechol- O-methyltransferase (COMT) metabolites. The purpose of the present study was to compare the regional distribution of these radiotracers in the brains of PD patients. Fifteen Parkinson's patients were studied with FMT and FDOPA positron emission tomography (PET) as well as high-resolution structural magnetic resonance imaging (MRI). MRI's were automatically parcellated into neuroanatomical regions of interest (ROIs) in Freesurfer (); region-specific uptake rate constants (Kocc) were generated from coregistered PET using a Patlak graphical approach. The essential findings were as follows: (1) regional Kocc were highly correlated between the radiotracers and in agreement with a previous FDOPA studies that used different ROI selection techniques; (2) FMT Kocc were higher in extrastriatal regions of relatively large uptake such as amygdala, pallidum, brainstem, hippocampus, entorhinal cortex, and thalamus, whereas cortical Kocc were similar between radiotracers; (3) while subcortical uptake of both radiotracers was related to disease duration and severity, cortical uptake was not. These results suggest that FMT may have advantages for examining pathologic changes within allocortical loop structures, which may contribute to cognitive and emotional symptoms of PD. Synapse 68:325-331, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Human alcohol dehydrogenase (ADH) consists of a complex group of isozymes encoded by at least five non-identical genes, two of which have previously been shown through enzymatic analysis to possess polymorphic variants. Using a cDNA probe the ADH2 gene encoding the beta subunit of human ADH was mapped to human chromosome 4. The cDNA probe for ADH2 was also used to detect a restriction fragment length polymorphism present in human populations. This polymorphism may help establish whether certain ADH allelic variants are linked with certain types of altered alcohol tolerance observed in various individuals. The restriction fragment length polymorphism may also be of use in genetic linkage studies of other genes located near ADH on human chromosome 4.

New RFLPs are described for INP10 and interleukin 2. The 55 pairwise genetic linkage relationships for these two loci and nine additional loci on the long arm of chromosome 4 (4q) are reported. Fifteen new linkages are established, and new data are added to the four previously reported linkages on 4q. Tight linkage of interleukin 2 (T-cell growth factor), epidermal growth factor, and alcohol dehydrogenase is described. Significant differences were observed between male and female recombination rates. The female rate was estimated to be 1.27 times the male rate. On the basis of these pairwise results, the order for the 11 loci is D4S35-GC-(ALB/AFP)-MT2P1-D4S1-INP10-ADH3-( EGF/IL2)-(FBB/FBA/FBG)-MNS. This preliminary order can serve as a starting point for more detailed multipoint analysis.

Human alcohol dehydrogenase (ADH) exists as a heterogeneous group of isozymes capable of oxidizing a wide variety of aliphatic and aromatic alcohols. The five distinct human ADH subunits, each encoded by a separate gene, are differentially expressed during development and are subject to tissue-specific regulation. To analyze the organization and regulation of human ADH genes we first isolated a cDNA clone (pADH12) encoding the 3' portion of the beta ADH gene. In the current study pADH12 was used to screen a human genomic library, and several overlapping and nonoverlapping clones were selected. Hybridization and partial nucleotide sequence analyses of the clones indicated that three full-length human ADH genes encoding the alpha, beta, and gamma subunits were isolated. Human genomic DNA hybridization results indicate that the alpha, beta, and gamma ADH genes form a closely related gene family and suggest that the other known human ADH genes (i.e. those encoding the pi and chi subunits) share a more distant evolutionary relationship. Nucleotide sequence analysis of the beta ADH gene reveals that the coding region is interrupted by eight introns and spans approximately 15 kilobases. A presumptive transcription initiation site for the beta ADH gene was located by S1 nuclease mapping at a position 70 base pairs upstream of the start codon. The 5' flanking region possesses a TATA box promoter element as well as two tandem DNA sequences which display homology to previously examined glucocorticoid-responsive elements.

We have used three different methods to map the human liver/bone/kidney alkaline phosphatase (ALPL) locus: (1) Southern blot analysis of DNA derived from a panel of human-rodent somatic cell hybrids; (2) in situ hybridization to human chromosomes; and (3) genetic linkage analysis. Our results indicate that the ALPL locus maps to human chromosome bands 1p36.1-p34 and is genetically linked to the Rh (maximum lod score of 15.66 at a recombination value of 0.10) and fucosidase A (maximum lod score of 8.24 at a recombination value of 0.02) loci. These results, combined with restriction fragment length polymorphisms identified by ALPL DNA probes, provide a useful marker for gene mapping studies involving the short arm of chromosome 1. In addition, our results help to elucidate further the structure and evolution of the human alkaline phosphatase multigene enzyme family.

An acid-sensitive growth factor for human melanoma has been partially purified from human platelets. TSK gel filtration HPLC provides a molecular weight estimation of 60,000 daltons. This factor is not only mitogenic for human melanoma, but also stimulates 3H-thymidine uptake and increases the number of bovine aortic endothelial cells, while fibroblasts are nonresponsive. Radioiodination of active HPLC fraction has been accomplished. The human melanoma cell line, M1RW5 demonstrates specific, time-dependent binding of the labeled protein. These studies suggest that the growth of human melanoma may in part be regulated by a newly described growth factor present in human platelets.

PGA1 and PGE1 reduced the plating efficiency and inhibited proliferation of Cloudman S-91 murine melanoma cells in a dose dependent manner, as assessed by their effects on colony formation in soft agar. PGF2α did not reduce plating efficiency but was as effective as PGA1 in raising cAMP and cGMP levels. This data suggests that the inhibition of Cloudman S-91 murine melanoma cell growth occurs via a non-cyclic nucleotide mechanism. © 1982.

The radiation response of murine and human melanoma cells assayed in bilayer soft agar and monolayer was examined. Cells from the murine melanoma Cloudman S91 CCL 53.1 cell line and three human melanoma cell strains (C8146C, C8161, and R83-4) developed in our laboratory were irradiated by single dose X-rays and plated either in agar or on plastic. D0 values were the same within 95% confidence intervals for cells from the human melanoma cell strains C8146C, C8161, and R83-4 but were dissimilar for the murine cell line CCL 53.1 Dq values were different for all cells studied. The shape of the survival curve for all four melanomas was not identical for cells assayed in soft agar versus cells grown on plastic. This would indicate that apparent radiosensitivity was influenced by the method of assay although there were no apparent consistent differences between the curves generated by monolayer or bilayer soft agar assays.