Follicular expression of a human beta-cell leukaemia/lymphoma-2 (Bcl-2) transgene does not decrease atresia or increase ovulation rate in swine.

Transgenic (TG) gilts carrying a human Bcl-2 cDNA transgene driven by mouse inhibin-alpha subunit promoter were produced and evaluated to determine if ectopic expression of Bcl-2 in the ovaries would decrease the frequency of atresia in antral follicles and increase ovulation rate. Immunohistochemical analysis showed that the Bcl-2 transgene protein was expressed in granulosa and theca cells, in 86% of healthy and 54% of atretic follicles analysed in TG prepubertal and Day 50 pregnant gilts combined (n = 24). In contrast, Bcl-2 transgene protein was expressed in only 1.4% of healthy and 0% of atretic follicles in non-TG littermates (n = 13). Real-time reverse transcription-polymerase chain reaction analysis confirmed that human Bcl-2 was expressed in follicles of TG gilts. The atresia rate for the TG and non-TG groups did not differ (P > 0.05) for prepubertal (45 v. 59%) and Day 50 pregnant gilts (53 v. 52%) respectively. The mean +/- s.e.m. ovulation rate did not differ (P > 0.5) between TG (15.9 +/- 0.8, n = 12) and non-TG (16.4 +/- 0.6, n = 7) Day 50 pregnant gilts. The molecular basis of the failure of ectopic Bcl-2 expression to increase the ratio of healthy to atretic follicles is unknown, but it is possible that the activity of the mitochondrial-dependent cell death pathway was not neutralized by ectopic expression of human Bcl-2 or that other cell death pathways compensated for the decreased mitochondrial-dependent cell death.

Ectodysplasin-A1 is sufficient to rescue both hair growth and sweat glands in Tabby mice.

Mutations in the human ectodysplasin-A (EDA) are responsible for the most common form of the ectodermal dysplasia and the defective orthologous gene in mice produces the tabby phenotype, suggesting its vital role in the development of hair, sweat glands and teeth. Among several EDA splice isoforms, the most common and the longest EDA splice isoforms, EDA-A1 and EDA-A2, differing by only two amino acids, activate NF-kappaB-promoted transcription by binding to distinct receptors, EDAR and XEDAR. The extent to which any particular isoform is sufficient for the formation of hair, sweat glands or teeth has remained unclear. Here we report that transgenic expression of the mouse EDA-A1 isoform in tabby (EDA-less) males rescued development of several skin appendages. The transgenic tabby mice showed almost complete restoration of hair growth, dermal ridges, sweat glands and molars. The number of hair follicles in the transgenic mice is the same as in wild-type; though the development of follicles and associated glands varies from indistinguishable from wild-type to smaller and/or only partially formed. These results suggest that the other EDA isoforms may not be absolutely required for skin appendage formation, but consistent with distinctive temporal and spatial expression of the EDA-A2 isoform, are likely required for appropriate timing and completeness of development. Our data provide the first direct physiological evidence that EDA-A1 is a key regulator of hair follicle and sweat gland initiation; its soluble ligand form could aid in deriving therapeutic reagents for conditions affecting hair and sweat gland formation.

A murine dopamine neuron-specific cDNA library and microarray: increased COX1 expression during methamphetamine neurotoxicity.

Due to brain tissue heterogeneity, the molecular genetic profile of any neurotransmitter-specific neuronal subtype is unknown. The purpose of this study was to purify a population of dopamine neurons, construct a cDNA library, and generate an initial gene expression profile and a microarray representative of dopamine neuron transcripts. Ventral mesencephalic dopamine neurons were purified by fluorescent-activated cell sorting from embryonic day 13.5 transgenic mice harboring a 4.5-kb rat tyrosine hydroxylase promoter-lacZ fusion. Nine-hundred sixty dopamine neuron cDNA clones were sequenced and arrayed for use in studies of gene expression changes during methamphetamine neurotoxicity. A neurotoxic dose of methamphetamine produced a greater than twofold up-regulation of the mitochondrial cytochrome c oxidase polypeptide I transcript from adult mouse substantia nigra at 12 h posttreatment. This is the first work to describe a gene expression profile for a neuronal subtype and to identify gene expression changes during methamphetamine neurotoxicity.

Application of cDNA microarrays to examine gene expression differences in schizophrenia.

Using cDNA microarrays we have investigated gene expression patterns in brain regions of patients with schizophrenia. A cDNA neuroarray, comprised of genes related to brain function, was used to screen pools of samples from the cerebellum and prefrontal cortex from a matched set of subjects, and middle temporal gyrus, from a separate subject cohort. Samples of cerebellum and prefrontal cortex from neuroleptic naive patients were also included. Genes that passed a 3% reproducibility criterion for differential expression in independent experiments included 21 genes for drug-treated patients and 5 genes for drug-naive patients. Of these 26 genes, 10 genes were increased and 16 were decreased. Many of the differentially expressed genes were related to synaptic signaling and proteolytic functions. A smaller number of these genes were also differentially expressed in the middle temporal gyrus. The five genes that were differentially expressed in two brain regions from separate cohorts are: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide; sialyltransferase; proteasome subunit, alpha type 1; ubiquitin carboxyl-terminal esterase L1; and solute carrier family 10, member 1. Identification of patterns of changes in gene expression may lead to a better understanding of the pathophysiology of schizophrenia disorders.

Region-specific transcriptional response to chronic nicotine in rat brain.

Even though nicotine has been shown to modulate mRNA expression of a variety of genes, a comprehensive high-throughput study of the effects of nicotine on the tissue-specific gene expression profiles has been lacking in the literature. In this study, cDNA microarrays containing 1117 genes and ESTs were used to assess the transcriptional response to chronic nicotine treatment in rat, based on four brain regions, i.e. prefrontal cortex (PFC), nucleus accumbens (NAs), ventral tegmental area (VTA), and amygdala (AMYG). On the basis of a non-parametric resampling method, an index (called jackknifed reliability index, JRI) was proposed, and employed to determine the inherent measurement error across multiple arrays used in this study. Upon removal of the outliers, the mean correlation coefficient between duplicate measurements increased to 0.978+/-0.0035 from 0.941+/-0.045. Results from principal component analysis and pairwise correlations suggested that brain regions studied were highly similar in terms of their absolute expression levels, but exhibited divergent transcriptional responses to chronic nicotine administration. For example, PFC and NAs were significantly more similar to each other (r=0.7; P<10(-14)) than to either VTA or AMYG. Furthermore, we confirmed our microarray results for two representative genes, i.e. the weak inward rectifier K(+) channel (TWIK-1), and phosphate and tensin homolog (PTEN) by using real-time quantitative RT-PCR technique. Finally, a number of genes, involved in MAPK, phosphatidylinositol, and EGFR signaling pathways, were identified and proposed as possible targets in response to nicotine administration.

Sources of motivation for abstinence: a replication analysis of the reasons for quitting questionnaire.

The Reasons for Quitting Questionnaire (RFQ) as modified by McBride and colleagues (C. M. McBride et al., 1994) for use with substance users other than tobacco smokers, was administered to individuals approved for public-sector addiction treatment. Four motivation dimensions, similar to those found by McBride et al., were identified: self-concept issues, health concerns, legal issues, and social influence. A forced two-component solution yielded dimensions interpretable as intrinsic and extrinsic motivation. Self-concept issues provided the highest levels of motivation for abstinence in this sample, with moderate levels provided by health concerns, and the lowest levels provided by legal and social influence components. Intrinsic motivation was higher than extrinsic motivation. Logistic regression models, with adjustment for total motivation, tested the association of successful abstinence during a follow-up period with baseline extrinsic and intrinsic motivation, and with the difference between intrinsic and extrinsic levels. All three associations were significant: intrinsic motivation (positive association), extrinsic motivation (negative association), and the difference score (positive association). The results suggest the usefulness of the 20-item modified RFQ in evaluating motivation for abstinence among treatment seekers exhibiting severe negative consequences of addiction. Testing with samples varying in severity of addiction consequences is recommended.

Gene expression profile in early stage of retinoic acid-induced differentiation of human SH-SY5Y neuroblastoma cells.

PURPOSE: The human SH-SY5Y cell line is an established model for retinoic acid (RA)-induced neural differentiation. We employed a broad human 15K microarray (15,000 genes) and focused Neuroarray (1152 genes) to examine changes in gene expression early in the process of differentiation (6 hr), before morphology or growth changes are observed. METHODS: 33 P-labeled CDNA probes prepared from RNA extracts of RA-treated and control cultures were hybridized to array membranes, and levels of expression were quantified and compared. RESULTS: In the 15K array, 19 % of the genes were decreased (0.4 % were named genes and the remainder were expressed sequence tags (ESTs) or unknowns), and 9 % were increased (4.2 % named genes). In the Neuroarray, 3 % were decreased and 8 % were increased. CONCLUSIONS: Summary gene profiles are presented, which include transcription factors, genes associated with cell cycle, cell shape, neurotransmission, intermediary filaments, and others. The prevalence of down-regulated genes in the broad 15K array and up-regulated genes in the neuro-focused array suggests a pattern shift in gene expression associated with differentiation. The predominance of ESTs among the down-regulated genes indicates a great number of as-yet-unidentified genes are repressed in early stage neural differentiation.

Esophagogastric adenocarcinoma in an E1A/E1B transgenic model involves p53 disruption.

We studied tumorigenesis and p53 immunostaining in a murine transgenic model introducing E1A/E1B under the control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR) promoter in which adenocarcinoma occurs at the squamocolumnar junction in the foregut, predominantly in males, and at no other site. Mutations of p53 are frequent in human esophageal adenocarcinoma and the E1B gene product interferes with p53-mediated apoptosis, inhibiting tumor suppression at the G(1)/S checkpoint. Transgenic animals were generated utilizing a purified linear 6.7 kb fragment of plasmid DNA containing MMTV-LTR/E1A/E1B and were confirmed by dot blot hybridization of tail DNA to (32)P-labeled E1A/E1B probe and polymerase chain reaction (PCR) amplification of E1A. Transgenic and control animals were observed for morbidity and weight changes. Eleven of 45 animals were transgenic (24% efficiency) with an estimated 5 to 57 copies of the gene per genome. Profound weight loss (>20%) led to sacrifice or death of one of five females (at 12 weeks) and four of six males (at 16 to 17 weeks). Grossly visible tumors (2 to 10 mm) were noted in the forestomach at the visible margin between the proximal (squamous-lined) stomach and the distal glandular stomach. Histologic sections confirmed adenocarcinoma arising in each case at the squamocolumnar junction with glandular formation, pleomorphism, and frequent mitotic figures. Immunostaining was positive for p53 indicating accumulation of mutated or altered p53 protein. E1A/E1B transgenic animals developed macroscopic and microscopic adenocarcinoma at the squamocolumnar junction, which corresponds to adenocarcinoma at the human esophagogastric junction. Disruption of p53 was present in the transgenic model as in the human cancer.

Mice deficient in the candidate tumor suppressor gene Hic1 exhibit developmental defects of structures affected in the Miller-Dieker syndrome.

HIC1 is a candidate tumor suppressor gene which is frequently hypermethylated in human tumors, and its location within the Miller-Dieker syndrome's critical deletion region at chromosome 17p13.3 makes it a candidate gene for involvement in this gene deletion syndrome. To study the function of murine Hic1 in development, we have created Hic1 -deficient mice. These animals die perinatally and exhibit varying combinations of gross developmental defects throughout the second half of development, including acrania, exencephaly, cleft palate, limb abnormalities and omphalocele. These findings demonstrate a role for Hic1 in the development of structures affected in the Miller-Dieker syndrome, and provide functional evidence to strengthen its candidacy as a gene involved in this disorder.

Alcohol problems in women admitted to a level I trauma center: a gender-based comparison.

BACKGROUND: Male patients constitute such a large proportion of trauma patients that most studies of alcohol problems in trauma patients have been carried out with clinical data largely or totally contributed by male patients. It may be incorrect to assume that the nature of alcoholism in women and men is identical, or that the size of the problem among women is small, eliminating the need to specifically study female patients. The purpose of this study was to perform a gender-based comparison of alcohol problems in trauma patients. METHODS: Admitted injured patients underwent routine screening, including a blood alcohol concentration, serum gamma-glutamyl transpeptidase, and the Short Michigan Alcohol Screening Test. A random sample of screen positive women and men underwent a comprehensive alcohol use and psychosocial assessment, and the results were compared by gender. RESULTS: The screen-positive rate was higher for men, 51% versus 34% (p < 0.01). However, screen-positive women and men had similar problem severity as reflected by mean blood alcohol concentration (162 mg/dL vs. 142 mg/dL, p = 0.16) and Short Michigan Alcohol Screening Test scores (4.6 vs. 5.0, p = 0.32). The Alcohol Use Disorders Identification Test, NIMH-DIS, and Severity of Alcohol Dependence Data form showed that female trauma patients with alcohol problems have the same severity of dependence symptoms as men. However, women were significantly more likely to have liver dysfunction, depression, psychological distress, and recent physical, emotional, or sexual abuse. CONCLUSION: Alcohol problems are more common in male trauma patients, but women with alcohol problems are just as severely impaired, have at least as many adverse consequences of alcohol use as their male counterparts, and have more evidence of alcohol-related physical and psychological harm.

Detection of acute alcohol intoxication and chronic alcohol dependence by trauma center staff.

BACKGROUND: Trauma patients with acute alcohol intoxication or chronic alcohol dependence are at greater risk for morbidity and mortality. We hypothesized that relying on clinical suspicion to detect acute alcohol intoxication and chronic alcohol dependence in trauma patients is inaccurate, influenced by injury factors, and biased by race, gender, age, and socioeconomic status. METHODS: Trauma patients were screened with a blood alcohol concentration and with the Short Michigan Alcohol Screening Test and CAGE questionnaire. Before screening, physicians and emergency department nurses were asked whether the patient was acutely intoxicated (blood alcohol concentration > 100 mg/ dL) or had a chronic alcohol problem. Sensitivity, specificity, positive, and negative predictive values were determined by comparing responses with blood alcohol concentration, Short Michigan Alcohol Screening Test, and CAGE questionnaire results, stratified by injury and demographic factors. RESULTS: Clinical evaluations were obtained on 462 patients. Overall, 23% of acutely intoxicated patients were not identified by physicians. The miss rate increased to one third in severely injured, chemically paralyzed, or intubated patients. Specificity was also poor. Patients with a negative blood alcohol concentration were more likely to be falsely suspected of intoxication if they were either young, male, perceived as disheveled, uninsured, or having a low income (p < 0.05). Staff identified < 50% of patients with a positive Short Michigan Alcohol Screening Test or CAGE, and falsely identified 26% of patients as alcoholic. CONCLUSIONS: Formal alcohol screening should be routine because clinical detection of acute alcohol intoxication and dependence is inaccurate. Screening should also be routine to avoid discriminatory bias attributable to patient characteristics.

Alcohol interventions in a trauma center as a means of reducing the risk of injury recurrence.

OBJECTIVE: Alcoholism is the leading risk factor for injury. The authors hypothesized that providing brief alcohol interventions as a routine component of trauma care would significantly reduce alcohol consumption and would decrease the rate of trauma recidivism. METHODS: This study was a randomized, prospective controlled trial in a level 1 trauma center. Patients were screened using a blood alcohol concentration, gamma glutamyl transpeptidase level, and short Michigan Alcoholism Screening Test (SMAST). Those with positive results were randomized to a brief intervention or control group. Reinjury was detected by a computerized search of emergency department and statewide hospital discharge records, and 6- and 12-month interviews were conducted to assess alcohol use. RESULTS: A total of 2524 patients were screened; 1153 screened positive (46%). Three hundred sixty-six were randomized to the intervention group, and 396 to controls. At 12 months, the intervention group decreased alcohol consumption by 21.8+/-3.7 drinks per week; in the control group, the decrease was 6.7+/-5.8 (p = 0.03). The reduction was most apparent in patients with mild to moderate alcohol problems (SMAST score 3 to 8); they had 21.6+/-4.2 fewer drinks per week, compared to an increase of 2.3+/-8.3 drinks per week in controls (p < 0.01). There was a 47% reduction in injuries requiring either emergency department or trauma center admission (hazard ratio 0.53, 95% confidence interval 0.26 to 1.07, p = 0.07) and a 48% reduction in injuries requiring hospital admission (3 years follow-up). CONCLUSION: Alcohol interventions are associated with a reduction in alcohol intake and a reduced risk of trauma recidivism. Given the prevalence of alcohol problems in trauma centers, screening, intervention, and counseling for alcohol problems should be routine.

Practical guidelines for performing alcohol interventions in trauma centers.

Nearly 50% of trauma patients are injured while under the influence of alcohol; however, addressing alcohol problems is not considered a routine component of trauma care. A public health approach to trauma prevention should include attention to underlying risk factors in the same way that advice regarding smoking cessation is offered in adult respiratory medicine clinics, and blood pressure, cholesterol, dietary, and exercise advice is provided in coronary care units. The Department of Health and Human Services, in its recent report to Congress, stated that efforts to reduce death and disability from injuries must be combined with efforts to reduce alcohol abuse, and called for an increase in the use of alcohol interventions in trauma patients. According to the National Academy of Sciences, the responsibility to provide counseling for patients with uncomplicated cases of mild to moderate alcohol abuse lies not with specialized alcohol treatment centers, but with physicians and other health care staff in general hospital settings trained to provide brief interventions. This paper provides practical guidelines for the administration of alcohol interventions that are suitable for trauma center use, and that have documented efficacy in reducing alcohol consumption.

Alcohol interventions in trauma centers. Current practice and future directions.

Nearly half of all trauma beds are occupied by patients who were injured while under the influence of alcohol. Alcoholism plays such a significant role in trauma that efforts to reduce injury recurrence are unlikely to be successful if it remains untreated. An injury requiring hospitalization creates a unique opportunity to intervene and to motivate patients to alter their drinking behavior, thereby making trauma centers ideal sites to implement an alcohol screening, intervention, and referral program. However, despite emphasis on injury control and prevention, little has been done to incorporate alcohol intervention programs into care of the injured patient. Effective means of intervention exist that are consistent with the time, financial, and staffing constraints of trauma centers, and they should be implemented.

How much is too much? Advising patients about safe levels of alcohol consumption.

United States physicians are increasingly encouraged to advise patients about health-related behaviors, such as smoking, but there is minimal discussion in the US medical literature about the need to advise patients about safe levels of alcohol consumption. Several factors likely contribute to this lack of focus on safe drinking practices. These include the complex relationship between drinking and health, limitations in the available epidemiologic data, misinterpretation of the disease model of alcoholism, and physician attitudes. Nevertheless, epidemiologic evidence clearly relates increasing levels of alcohol consumption to increased morbidity and mortality, and research has shown that physician advice can reduce both alcohol consumption and alcohol-related problems. We propose that physicians thoroughly assess patients' alcohol consumption and advise patients who drink about safe levels of consumption.