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Objectives Pathology and laboratory medicine (PALM) services in low- and middle-income countries are essential to combat the increasing prevalence of cancer in addition to providing documentation of cancer types and trends for future allocation of public health resources. There are many ways PALM as a whole can engage on the global health front. This study summarizes the efforts and results of a global health educational and clinical elective for pathology residents in Quetzaltenango, Guatemala. Methods Pathology residents led and implemented the project, working alongside an in-country pathologist and project collaborator to instill project sustainability and allow for future capacity building. Results An educational elective was established between the pathology departments of the University of Virginia and Hospital Regional de Occidente in Quetzaltenango, Guatemala. Two residents at a time engaged in a month-long educational elective assisting and learning from the in-country pathologist in anatomic pathology clinical work. Conclusions The project is an example of a global health initiative centering on the enhancement of PALM services in a low-resource environment via a bidirectional, sustainable educational exchange.

Background: The adoption of electronic medical records (EMRs) in lower-income nations has progressed slowly due to the lack of adequate infrastructure, funding, and training. However, EMRs have been successfully implemented previously in resource-limited health systems in South Africa, Haiti, Cameroon, Kenya, and Peru. Detailed, organized, and easily accessible medical records are particularly important in emergency departments due to the volume and acuity of the patient population. Methods: In order to further study the plausibility of an EMR in a resource-limited emergency department, a web-based, Spanish-language EMR known as SABER was developed for use in Hospital Nacional Jose Felipe Flores in Totonicapan, Guatemala. The software collects patient data including demographics, triage, initial evaluation, review of systems, physical exam, and evaluation and plan. It then generates a .pdf file consistent with information requirements of the Guatemalan Ministry of Health. Local physicians, medical students, and nurses were trained in the use of the software, which debuted in July 2016. To assess the effectiveness of SABER as an EMR, focus groups and Likert scale surveys were conducted with six physicians and 31 medical students working in the Hospital Nacional emergency department. Results: Thirty of 32 medical students and six of six doctors would recommend SABER to another provider. Positive aspects identified by staff include ease of use, quick data entry, and the potential for large data set research. Discussion: Remaining challenges include incorporating electronic nursing orders and lab results, troubleshooting technology problems including printer difficulties, a lack of electronic signature capability, and lack of integration with the rest of the hospital. Our study is consistent with other studies that show use of an EMR may help to reduce health disparities through improved patient records, medical data collection, and organization.

Objective. To describe the prevalence of noncommunicable disease (NCD) risk factors and assess knowledge of those risk factors in the indigenous community of Santiago Atitlán in Guatemala, a lower-middle income country. Methods. A population-based, cross-sectional study was conducted using a modified version of the World Health Organization’s STEPS protocol. Adults aged 20–65 years were surveyed regarding demographics and NCD risk factors, and the survey was followed by anthropometric and biochemical measurements. Results. Out of 501 screened individuals, 350 respondents were enrolled. The mean age was 36.7 years, and 72.3% were women. Over 90% reported earning less than US$ 65 per month. Almost 80% were stunted. Among women, 37.3% were obese and over three-quarters had central obesity. Over three-quarters of the entire group had dyslipidemia and 18.3% had hypertension, but only 3.0% had diabetes. Overall, 36.0% of participants met criteria for metabolic syndrome. There was no significant association between participants’ education and NCD risk factors except for an inverse association with obesity by percent body fat. Conclusions. Santiago Atitlán is a rural, indigenous Guatemalan community with high rates of poverty and stunting coexisting alongside high rates of obesity, particularly among women. Additionally, high rates of hypertension and dyslipidemia were found, but a low rate of diabetes mellitus. Knowledge of NCDs and their risk factors was low, suggesting that educational interventions may be a high-yield, low-cost approach to combating NCDs in this community.

Background Nutritional deficits in early life have been associated with a higher prevalence of the metabolic syndrome (MetS) in adulthood. Early childhood diarrhea contributes to undernutrition and may potentially increase the risk for adult noncommunicable diseases. Our objective was to examine associations between early childhood diarrhea burden and later development of MetS. Methods We studied individuals who participated in the Institute of Nutrition of Central America and Panama Nutritional Supplementation Longitudinal Study (1969–1977) and were followed up in 2002–2004. We used logistic regression to determine associations of diarrhea burden at ages 0 to 6, 6 to 12, and 12 to 24 months with odds of MetS and elevations in its components as adults. Results Among 389 adults age 25 to 42 years at follow-up, the prevalence of MetS was 29%. Adjusting for several confounders including adult body mass index (BMI), each absolute 1% increase in diarrhea burden at age 0 to 6 months (but not at other time periods) was associated with increased odds of MetS (odds ratio [OR], 1.03; 95% confidence interval [CI], 1.01–1.06). This was attributable primarily to associations with elevated blood pressure (OR, 1.03; 95% CI, 1.00–1.06) and waist circumference (OR, 1.03; 95% CI, 1.00–1.06). Conclusions Childhood diarrhea burden at 0 to 6 months is associated with MetS in adulthood after controlling for childhood growth parameters and adult BMI.

We tested a prototype stool enzyme-linked immunosorbent assay (ELISA) (TRI-COMBO) that is simultaneously diagnostic for Giardia lamblia, Cryptosporidium parvum, and Entamoeba histolytica in a rural pediatric clinic in Guatemala. We compared its results to those of three individual ELISAs for these parasites, assessed the prevalence of these parasites, and compared our findings to those found by stool microscopy. We tested 620 non-diarrheal stools. The TRI-COMBO diagnosed 57 positive samples and 52 (91%) had a correlating positive result in an individual assay, giving a kappa coefficient of 0.90. Giardia spp., E. histolytica, and Cryptosporidium spp. were detected in 52 (8.4%), 2 (0.3%), and 3 (0.5%) samples, respectively. Twenty-three (40%) samples positive by ELISA for Giardia spp. were identified by microscopy. This study is the first to test the TRI-COMBO in this setting and, to our knowledge, represents the first assessment of these parasites in Guatemala by stool ELISA.

Objective The implementation of chest pain centers (CPC)/units (CPU) has been shown to improve emergency care in patients with suspected cardiac ischemia. Methods In an effort to provide a systematic and specific standard of care for patients with acute chest pain, the Society of Cardiovascular Patient Care (SCPC) as well as the German Cardiac Society (GCS) introduced criteria for the accreditation of specialized units. Results To date, 825 CPCs in the United States and 194 CPUs in Germany have been successfully certified by the SCPC or GCS, respectively. Even though there are differences in the accreditation processes, the goals are quite similar, focusing on enhanced operational efficiencies in the care of the acute coronary syndrome patients, reduced time delays, improved diagnostic and therapeutic strategies using adapted standard operating procedures, and increased medical as well as community awareness by the implementation of nationwide standardized concepts. In addition to national efforts, both societies have launched international initiatives, accrediting CPCs/CPU in the Middle East and China (SCPC) and Switzerland (GCS). Conclusion Enhanced collaboration among international bodies interested in promoting high quality care might extend the opportunity for accreditation of facilities that treat cardiovascular patients, with national programs designed to meet local needs and local healthcare system requirements.

Introduction: Pre-hospital diagnosis of ST-segment Elevation Myocardial Infarction (STEMI) promotes pre-arrival decision making and minimizes reperfusion delays. However, many commercial ECG transmission systems are expensive and based on proprietary technology. Recent advances in phone and app technology suggest another approach. As an alternative, a smartphone app (STEMISend) was developed and partnered with an inexpensive cell signal booster. This combination was then tested in multiple head-to-head comparisons with commercial systems. Results showed the app + booster approach to be non-inferior or superior to commercial systems -particularly in signal-limited environments. Hypothesis: Based on preliminary testing, the STEMISend smartphone app was suggested as a cost-effective alternative to commercial systems. Extensive field testing looking at percent success, cycle reliability and comparative performance in low-signal environments was conducted. Procedure: The STEMISend/booster combo was field-tested on 2 national cell networks and compared to an existing commercial system. Success was defined as a successful transmission within 120 seconds. Data was collected at 125 individual waypoints at all signal strengths. Testing was in geographically diverse areas of central Virginia. App performance was evaluated on a success/failure basis and on the time required to transmit the image. Commercial system performance was similarly evaluated and the two approaches were compared. Results: Figure 1 shows STEMISend performance compared to an existing commercial system. In signal-rich environments (3-5 bars), both showed nearly equal performance. However, in signal-limited environments (2 bars or less), the app/booster combo displayed slightly higher success. In terms of transmission duration, the cell signal booster decreased transmission cycle time in both signal -rich and signal-limited environments, though the effect is more pronounced in signal-limited environments. Conclusion: The STEMISend iPhone app is a potential alternative to commercial ECG transmission systems--particularly when coupled with inexpensive cell signal boosters. STEMISend is easy to use, reliable and non-proprietary. Additional testing in EMS environments is in progress.

training of local caregivers to ensure the sustainability. The program will be evaluated in two different school populations in rural Kenya to determine the specific challenges and effectiveness in each community. While both schools serve students of extremely low socio-economic status with poor oral health and lack of access to dental care, students at one school are boarded while at other they live at home with their families. Outcomes & Evaluation: Data collection will be done through the novel University Health Network (UHN) system. The UHN is a consortium of leading research institutions, designed to allow for highly scaled infrastructure for the secure collection, storage or sharing of clinical data in a cost-effective manner. A global information system (GIS) will be used to capture geographic trends of the oral diseases in the rural villagers. This research project, besides evaluating the impact of a comprehensiveprogram on the level of oral diseases and of oral health perception and behaviors/practices, will also assess the role of utilization of school staff and family to deliver and reinforce health-promoting behaviors. We anticipate that the lessons learned from this study will be relevant in other communities and countries in East Africa and beyond. Going Forward: As a result of our findings we will develop an educational outreach program for dental students, and their supervising faculty, from the School of Dental Medicine of the University of Nairobi. At present, the dental students and residents do not have the opportunity to work with rural impoverished communities. The goals of this project, beyond the experiential and care-provision, are also to expose dental students to the needs of these communities, and to become advocates for improving oral health services for those in Kenya in need. Thus, this program will also include leadership and advocating skills. Funding: Do a Little Foundation, San Francisco, California. Abstract #: 01NCD023

Diabetes and heart disease are two of the leading causes of death for Hispanics living in the United States (American Heart Association [AHA] in Circulation 123:e18-e209. doi: 10.1161/CIR.0b013e3182009701 , 2010). As the Hispanic population continues to grow, the need for low-cost, non-invasive methods to detect at risk populations for such diseases becomes more important. Once at risk individuals are detected, prevention strategies can be implemented. Studies have shown that Latino community health workers (CHWs) are effective educators, patient advocates and health promotion motivators for patients with known heart disease or diabetes. This pilot study examined the accuracy with which Latino CHWs could determine migrant farmworkers at risk for diabetes or cardiovascular disease (CVD) in rural Virginia. This quasi-experimental study supports the hypothesis that Latino CHWs can use non-invasive diabetes and CVD screening tools with similar accuracy as a registered nurse. The screening tools used were the American Diabetes Association's diabetes risk calculator and a non-laboratory screening tool for CVD risk designed by Gaziano et al. (Lancet 371:923-931, 2008). The terms Latino and Hispanic will be used interchangeably.

Introduction: Optimizing interfacility transfer of STEMI patients for emergent PCI remains a challenge due to difficulty calculating a customized time goal for transfer from a single non-PCI to PCI center (known as a “STEMI conduit”). Here we propose a simple formula to calculate conduit-specific interfacility STEMI transfer goals, allowing hospitals to assess and optimize their performance. Hypothesis: Optimal time for individual STEMI transfers within a STEMI conduit can be reliably calculated with the formula (V1+ V2 + 60 minutes). In this formula, V1 is the optimal EMS response time to the transferring facility (in minutes) and V2 is the interfacility (door-to-door) transfer time (in minutes). V1 and V2 are site-specific and vary across STEMI conduits. The additional 60 minutes represents the sum of the goal Door to Decision, EMS Scene Turn-around, and PCI Door to Reperfusion times. The goal times for these individual parameters are based on established expectations or national best practices and remain constant for all STEMI conduits. Using this formula, the expected facility-to-facility transfer time [F2F(expected)] can be calculated and this value can then be compared to actual performance [F2F(observed)] with the ratio of observed/expected performance. A ratio close to 1 suggests the transfer occurred in a realistic yet optimized time frame. Methods: Interfacility STEMI transfers occurring within five STEMI conduits over four years were evaluated retrospectively. A performance goal was determined for each conduit using the formula [F2F(expected) = V1 + V2 + 60 minutes] and a ratio of F2F(observed /expected) was calculated for each transfer. Results: The figure demonstrates the F2F ratio ranges and means for transfers within each conduit. Four of five conduits exhibit mean ratios close to 1, indicating that the calculated expected performance is representative of what is optimized and sustainable within that conduit. The remaining conduit has a mean ratio of 0.81 indicating observed performance better than predicted. Conclusion: The proposed formula [F2F(expected) = V1 + V2 + 60 minutes] can be used to accurately calculate interfacility transfer goals for STEMI conduits and can thereby serve as a marker for quality improvement, enabling hospitals to monitor and optimize STEMI transfer performance.

Introduction: Field transmission of pre-hospital ECG images to facilitate advance activation of the STEMI system of care has been shown to be effective in improving patient care and prognosis. Current commercial wireless ECG transmission systems available are effective, but are expensive to implement and maintain. Head to head testing was performed using an iPhone application to mediate ECG image transmission on three national cellular carriers in comparison to one commercial ECG transmission system. This resulted in comparable performance particularly in signal rich coverage environments contained three or greater bars. Hypothesis: An iPhone application that mediates pre-hospital ECG image transmission could be a cost effective alternative to current commercial systems incorporated in the pre-hospital STEMI system of care. Using a mobile phone signal booster could enhance application performance to consistently transmit in all signal coverage environments. Testing: The application was tested on three national cellular carriers with two commercial systems. Data was collected with and without the addition of the mobile phone signal booster at 75 waypoints covering the entire signal coverage environment. Application performance was determined by the average percentage of successful transmission and duration of transmission within the total signal coverage environment. Results: As shown in Figure 1, using the mobile phone signal booster resulted in marked improvements to successful transmission. Performance of the carriers under boosted conditions was merged, as the differences were not statistically significant enough to warrant differentiation. Within the signal-limited environment, the application displayed superior reliability to the two commercial systems, while in the signal rich environment it was comparable. Minimal improvement was also seen in the duration of transmission of the application, although it was more apparent within the signal limited environment than in the signal rich environment. Conclusion: The addition of the mobile phone signal booster enhances the performance of the iPhone application, allowing it to successfully transmit ECGs more consistently than commercial systems in all signal coverage environments.

Glatiramer acetate is an approved drug for the treatment of multiple sclerosis (MS). RANTES is a β-family chemokine that manifests chemoattractant activity for T lymphocytes and monocytes/macrophages implicated in the pathogenesis of MS lesions. However, the effect of glatiramer acetate on the regulation of RANTES secretion in glial cells is unknown. In the present study, we demonstrate for the first time that treatment of human U-251 MG astrocytic cells with glatiramer acetate blocks tumor necrosis factor-α (TNF-α)-induced RANTES mRNA and protein in a dose- and time-dependent manner. This effect is attributed to inhibition of transcription and a 40% decrease in transcript stability. Furthermore, our electrophoretic mobility shift assays of nuclear extracts from TNF-α-treated cells reveal an increase in DNA-binding activity specific for the nuclear factor-kappa B (NF-κB) binding site, in the 5′-flanking promoter region of the human RANTES gene, and that this increase in NF-κB binding activity is prevented by pretreatment with glatiramer acetate or the NF-κB inhibitors. These findings suggest that glatiramer acetate may exert its therapeutic effect in MS partially through inhibiting NF-κB activation and chemokine production.

We have cloned the promoter regions of the genes for the mouse and human gamma2 subunits of the type A receptors for gamma-aminobutyric acid (GABA). For the mouse, the two major transcription start sites were at +1 (by definition) and +43, as established by rapid amplification of cDNA ends (RACE) and primer extension. This numbering places the start methionine at +297. There was no TATA or CCAAT box. Both mouse and human sequences have a candidate neuron-restrictive silencer element (NRSE) site in the first intron (+956 in mouse). We made assorted mouse-based promoter/reporter (luciferase) constructs starting from a core extending from -331 to +136, varying sizes at both ends, and including and excluding the putative NRSE and more proximal sequences. These were tested by transient transfection in several neuron-like and non-neuronal cell lines. Both proximal and distal downstream elements appeared to help direct expression to neuron-like cells, the NRSE in the intron, by repression in non-neurons, and a 24-bp portion of the 5' untranslated region starting at +113 (named GPE1) by preferentially promoting expression in neuron-like cells. Cotransfected human NRSF (transcription factor for NRSE) reduced reporter expression in neuron-like cells for constructs containing the NRSE in two locations. In gel mobility shift assays, the mouse gamma2 NRSE and a consensus NRSE both bound in vitro translated NRSF very similarly, and the NRSF gave the same major shifted band with the mouse gamma2 NRSE as was observed with nuclear extracts.

γ-Aminobutyric acid (GABA), one of the most important neurotransmitters in the brain, is also found in the periphery. GABAA receptors are chloride channels opened by GABA whose presence in the rat superior cervical ganglion has been indicated by functional and binding measurements. We describe the first molecular data on the possible subunit composition of these receptors, detecting mRNAs for 12 subunits (α1–5, β1–3, γ1–3, δ) by reverse-transcriptase polymerase chain reaction. Preliminary quantitation gave highest levels (in descending order) for the γ2 (both short and long forms), β3, γ3, and α1 subunits.

We describe a synthetic 769-bp DNA internal standard, GABARQuant 1, for measuring mRNAs of 13 GABA A receptor subunits by reverse transcriptase-polymerase chain reaction (RT-PCR). When it is transcribed into cRNA, added in known amounts to target mRNAs in extracts from rat or mouse tissue, competitively reverse transcribed into cDNA, and amplified by the polymerase chain reaction (PCR), the relative intensities of the amplified, stained target and standard DNA bands enable measurement of small amounts of mRNAs for GABA A receptor subunits α 1–6, β 1–3, γ 1–3 and δ and the three cellular markers β -actin, light neurofilament protein, and glutamine synthetase. For the subunits, most standard products (263–504 bp) differ in size from target products (398–564 bp) by 10–20%. Primer pairs span at least one intron, to prevent interference by genomic DNA, and at least one rat versus mouse restriction fragment length polymorphism (RFLP), to enable rat products to be distinguished from mouse products.

Introduction: Transmission of pre-hospital ECG images to STEMI treatment centers aids in optimizing STEMI reperfusion times by promoting activation of the STEMI treatment process prior to patient arrival. Several proprietary systems have been designed for this purpose but they are expensive and incur substantial ongoing system costs. ECG photo images may also be sent by personal cell phone but require large file sizes to maintain diagnostic clarity, which results in lower reliability and speed and greater process variability. Hypothesis: A low-cost iPhone app that optimizes speed and reliability of the ECG pre-hospital transmission process could be developed to address many of these issues and provide an acceptable alternative to commercially available systems. Methods: A faculty/student team designed an iPhone app that takes a photo of the ECG, centers the image and reduces image size to < 30 kb with minimal loss of detail. The image is then sent over cell phone networks to a secure server. Successful transmission is noted with visual and audio cues to the user. The app terminates the send cycle after 120 seconds if not successful. Average time to obtain and initiate ECG transmission is 8-11 seconds. Testing: App image transmission was compared to four different photo sizes utilizing three cell phone networks. Fifty standardized physical waypoints were utilized and multiple test cycles were completed at each waypoint, totaling over 1500 separate transmission attempts. Acceptable success was defined as transmission within 120 seconds. Results: Image transmission via the app consistently outperformed standard photo transmission of all size photos with the difference becoming more marked as photo file size increased. This effect was seen within all cell phone networks. The app was significantly faster, exhibited substantially less standard deviation and had less than a 0.5% failure rate at 120 seconds, compared to failure rates of 3%, 71.2% and 15.5% for full-sized photos on the three networks tested. Average transmission times were also documented. (See figure I). Conclusion: The iPhone app outperformed the standard photo-to-email process -even in high signal-intensity suburban environments. Phase II testing is now underway in rural areas and other signal-limited environments.

GABAA receptors link binding of GABA (gamma-aminobutyric acid) to inhibitory chloride flux in the brain. They are the site of action of several important classes of drugs, and have been implicated in animal models of epilepsy and in the actions of alcohol. We compare the sequence and expression of the beta 1, beta 2 and beta 3 subunits of GABAA receptors in two inbred strains of mice, DBA/2J and C57BL/6J, which differ markedly in seizure susceptibility and in a variety of behaviors related to alcohol. Only the beta 3 subunit had strain differences in cDNA nucleotide sequence, which did not affect amino acid sequence but which did create restriction fragment length polymorphisms (RFLPs) potentially useful in gene mapping. We have also tested mouse beta 1 and beta 2 subunits for internal alternative splicing, detecting none.

The role of the gamma 2 subunit in etomidate potentiation of GABAA receptor-gated chloride current was studied by whole cell patch clamp experiments on H293 cells expressing GABAA receptors. The GABAA receptor subunits alpha 1 beta 1 with or without the gamma 2 subunit expressed well, with an overall peak current of 157 +/- 42 pA/pF. At a clinically relevant concentration, etomidate potentiates the peak current induced by GABA equally well in receptors with or without the gamma 2 subunit. In contrast, the time course of current decay was prolonged only in receptors with the gamma 2 subunit. This gamma 2 subunit-dependent prolongation of the current time course was not blocked by the benzodiazepine receptor antagonist flumazenil. These results show that etomidate, an imidazole general anesthetic, interacts with the GABAA receptor in a gamma 2 subunit-dependent manner.

Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in brain, opens chloride channels through actions on GABAA receptors. We now report base and amino acid sequences of the α1, α2, and α3 subunits from GABAA receptors of audiogenic seizure-prone (DBA/2J) and -resistant (C57BL/6J) inbred strains of mice. Inbreeding had fixed different alleles of the α1 subunit in the two strains, giving five base differences in the cDNAs. None of these affected amino acid sequence, but one did create a NsiI restriction site potentially useful in mapping genomic DNA. No base or amino acid sequence differences between the strains were detected for the other two subunits. Northern blots revealed no apparent strain differences in message levels for these three subunits in whole brains of the mice at 3 weeks of age, the peak of seizure susceptibility in DBA/2J, but did reveal distinct regional and developmental patterns of expression among the subunits in mouse brain.

GABAA receptors are multisubunit inhibitory chloride channels in the brain which open in response to binding of gamma-aminobutyric acid (GABA) and are thought to be involved in some forms of seizures. We compare the sequence and expression of the GABAA receptor delta subunit in audiogenic seizure prone (DBA/2J) and seizure resistant (C57BL/6J) inbred strains of mice and also report this subunit's postnatal developmental profile. We did not detect any unique features in the delta subunits of DBA/2J mice which might explain their seizure susceptibility, but did detect in some clones from both DBA/2J mice and C57BL/6J mice an unusual substitution of His for a conserved Tyr in the delta subunit's first putative transmembrane region.

The gamma-aminobutyric acid type A (GABAA) receptor gamma 2-subunit is important for benzodiazepine action. In previous studies, two forms of gamma 2-subunit have been found which are generated by RNA alternative splicing (gamma 2L and gamma 2S) and differ by the presence or absence of an 8-amino acid insertion in the major intracellular loop between proposed transmembrane domains M3 and M4. We have used the RNase protection assay to study regulation of expression of the two forms of gamma 2-subunit in mouse brain, as judged from mRNA levels. We find that gamma 2L and gamma 2S are differentially expressed in brain regions and that gamma 2S is expressed at a fairly constant level during brain development while gamma 2L increases dramatically with maturation. Their differentially regulated expression suggests further that gamma 2L and gamma 2S form receptors with important functional differences.

Expression of brain mRNA or cRNAs in Xenopus oocytes was used to determine what subunits of the GABAA receptor are required for modulation by barbiturates, benzodiazepines, and ethanol. Mouse brain mRNA was hybridized with antisense oligonucleotides complementary to sequences unique to specific subunits and injected into oocytes. Antisense oligonucleotides to the alpha 1, beta 1, gamma 1, gamma 2S + 2L, gamma 2L, or gamma 3 subunits did not alter GABA action or enhancement by pentobarbital. Action of diazepam was prevented by antisense oligonucleotides to gamma 2S + 2L and reduced by antisense sequences to gamma 2L, but was not affected by the other oligonucleotides. Ethanol enhancement of GABA action was prevented only by antisense oligonucleotides to gamma 2L (which differs from gamma 2S by the addition of 8 amino acids). Expression of either the alpha 1 beta 1 gamma 2S or the alpha 1 beta 1 gamma 2L subunit cRNA combination in oocytes resulted in GABA responses that were enhanced by diazepam or pentobarbital, but only the combination containing the gamma 2L subunit was affected by ethanol.

The ECG classification of acute myocardial infarctions has had a profound influence on the treatment of patients with AMI. Deciding whether a patient has ST-segment elevations or a new left bundle branch block or neither of these findings on ECG launches the treating physician down two different treatment pathways: patients with ST-elevation MI need to be assessed for immediate re-perfusion therapy, whereas patients with non-ST-elevation MI are best treated with aggressive medical management without acute reperfusion.

GABA A (γ-aminobutyric acid type A) receptors are ligand-gated ion channels composed of five subunits, generally two αs, two βs, and a γ2. Recent research in which sets of subunits containing α1 or α6 subunits were artificially linked has revealed the importance of subunit position in determining GABA A receptor function. Sensitivity to benzodiazepines depended on juxtaposition of an α1 subunit with the γ2 subunit, whereas sensitivity to furosemide depended only on the presence of an α6 subunit and not on its specific location. The major utility of the linked subunit approach is to provide a mechanism for discovering the functional signatures of defined subunit arrangements, and thus a route to identifying such arrangements in vivo.

A number of important drugs act on GABA(A) receptors, pentameric GABA-gated chloride channels assembled from among 19 known subunits. In trying to discover the roles in the brain of the subunits and their combinations, with the goal of developing more selective drugs, one tool has been to reduce expression of the subunits and examine the functional consequences. After briefly examining the properties of GABA(A) receptors, this review surveys the means available for receptor subunit reduction, and some of the observations to which their application has led. The methods discussed include radiation-induced deletion, gene knockout, knock-in mutations, antisense, ribozymes, RNA interference, dominant negative constructs, and transcriptional regulation, e.g., via decoy oligonucleotides.

GABA(A) receptors, important sites of drug action, are chloride channels composed of 5 subunits chosen from among 19 or more. Alternative splicing for alpha 5, alpha 6, and rho 1 subunits results in truncated proteins which appear to lack function. We report a similar, relatively common (about 20%) form of alternative splicing of the alpha 4 subunit mRNA in mice and humans which, remarkably, creates a severely truncated message containing only the first two and last coding exons, with a frameshift in between. The only apparent translation product includes a short piece (39 amino acids) of the N-terminus right after the signal peptide. The splicing was developmentally and regionally regulated; the highest proportions of truncated alpha 4 mRNA, about 40%, were observed in embryonic day 18 whole brain and adult cerebellum. The truncated mRNA, when coexpresssed in human embryonic kidney (HEK) 293 cells with the complete alpha 4 subunit and beta1 and gamma 2 S subunits, reduced observed GABA currents without kinetic alterations. No such effect of truncated alpha 4 was observed with alpha1 subunit-containing receptors. Thus, the truncated alpha 4 N-terminus may play a post-translational regulatory role in intracellular folding/glycosylation/assembly of the alpha 4 subunit.

RANTES is a C-C (beta)-family chemokine that is implicated in the migration of peripheral blood leukocytes to brain lesions in multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS). Glial cells are active participants in the inflammatory response in the CNS, and they have been shown to respond to and produce a number of cytokines and chemokines in vivo and in vitro. Recently, we have shown inducibility of RANTES gene expression by TNF-alpha in human astrocytic cells. Therefore, the goal of the current study was to investigate the transcription activating factor involved in the process. We found that the induction of RANTES mRNA and protein by TNF-alpha in human astrocytic cells is associated with increased NF-kappaB DNA-binding activity. p65 and p50 were determined to be the components of the activated NF-kappaB transcription factor complex by supershift assay. In addition, the blockade of NF-kappaB activation by three known NF-kappaB inhibitors markedly reduced the TNF-alpha-induced RANTES expression at the mRNA and protein levels. Furthermore, the reduction in NF-kappaB binding activity to the promoter of the human RANTES gene caused by the NF-kappaB inhibitors parallels a decrease in RANTES expression in these cells. Our data suggest that NF-kappaB may mediate the induction of RANTES gene expression, in human glial cells, through its cognate cis-acting element.

gamma-Aminobutyric acidA (GABAA) receptors are multisubunit ligand-gated ion channels which mediate neuronal inhibition by GABA and are composed of at least four subunit types (alpha, beta, gamma, and delta). The gamma 2-subunit appears to be essential for benzodiazepine modulation of GABAA receptor function. In cloning murine gamma 2-subunits, we isolated cDNAs encoding forms of the subunit that differ by the insertion of eight amino acids. LLRMFSFK, in the major intracellular loop between proposed transmembrane domains M3 and M4. The two forms of the gamma 2-subunit are generated by alternative splicing, as demonstrated by cloning and partial sequencing of the corresponding gene. The eight-amino-acid insertion encodes a potential consensus serine phosphorylation site for protein kinase C. These results suggest a novel mechanism for the regulation of the GABAA receptor by protein phosphorylation.

Gamma-aminobutyric acid (GABA) type A receptors are multisubunit ligand-gated ion channels which mediate inhibition in the brain. The GABA(A) receptor alpha3 subunit gene exhibits extensive variation in its developmental and regional expression, but the detailed mechanisms governing the expression patterns of this gene remain unknown. We have cloned and begun to characterize the murine alpha3 subunit gene Gabra3. All but one of the 10 exons and the intron-exon boundaries have been sequenced; the first intron is in the 5' untranslated region (5'UTR) of the alpha3 mRNA. Rapid amplification of the cDNA 5'-end (5'-RACE) and RNase protection indicated many transcription start sites, with the major site (=+1) corresponding to a 5'UTR of 178 bases. Most sites were in or just downstream of a region of 55 (mouse) and 25 (human) GA repeats in the proximal promoter, as revealed by genome walking of Gabra3 and the human gene GABRA3. No canonical TATA or CAAT boxes or initiator (Inr) sites were found in either promoter, but both contained conserved consensus sites for several transcription factors. Progressive deletion of the mouse promoter produced positive or negative effects on expression of reporter (luciferase) constructs, with the highest observed activity in several types of transiently transfected cells for a construct containing bases -320 to +35. The GA repeats and a much shorter nearby series of four GC repeats, the first three of which are part of a consensus E2F site, appear to contribute significantly to mouse promoter activity. Upstream GA repeats enhanced activity of the SV40 promoter, and the GA repeat sequence bound nuclear proteins from several tissues.