Alpha1 Levels Research Articles

Association between inflammatory cytokines and prostate cancer

IntroductionWe conducted bidirectional two-sample Mendelian Randomization (MR) analysis to investigate the causal relationship between 91 inflammatory cytokines and prostate cancer (PCa).Material and methodsThe Inverse Variance Weighted (IVW) model served as the primary two-sample MR analysis method, utilized to estimate the causal effect of exposure on the outcome. The Weighted Median (WM) and MR Egger methods were additionally employed to complement the IVW model. Sensitivity analyses were performed using Cochran's Q test for both the IVW and MR Egger methods. To assess the presence of horizontal pleiotropy, the instrumental variables (IVs) were subjected to the MR-Egger intercept test.ResultsFollowing Bonferroni correction, the IVW analysis revealed positive correlations between PCa and the levels of C-C motif chemokine 20 (CCL20), C-C motif chemokine 23 (CCL23), fibroblast growth factor 19 (FGF19), fibroblast growth factor 23 (FGF23), and interleukin-6 (IL-6). Notably, IL-6 exhibited the strongest positive association effect (odds ratio [95% confidence interval]: 1.0076 [1.0014, 1.0139]), followed by CCL-20 (1.0067 [1.0004, 1.0129]) and FGF23 ([1.0002, 1.0119]). Reverse MR analysis indicated a negative causal relationship between PCa and interleukin-22 receptor subunit alpha-1 levels (IL22RA1) (0.4852 [0.2390, 0.9847]).ConclusionsThis study proposes that there exists a positive correlation between the levels of CCL20, CCL23, FGF19, FGF23, and IL-6 and the occurrence of PCa. Furthermore, we found evidence to support the causal relationship between decreased levels of IL22RA1 and the development of PCa. These findings unveil novel biomarkers and pathways that could potentially be targeted for the prevention and clinical treatment of PCa.

Association of Inflammatory Factors with Cervical Cancer: A Bidirectional Mendelian Randomization.

Persistent human papillomavirus infection is thought to be the main cause of the cervical cancer development along with inflammation. However, the potential mechanisms of action of the inflammatory factors in cervical cancer remain unclear. Therefore, this study aimed to assess the relationship between inflammatory factor levels and cervical cancer risk using a two-sample bidirectional Mendelian randomization (MR). MR utilizes single nucleotide polymorphisms as a tool to infer potential causal relationships between exposure factors and outcomes. Datasets for 91 inflammatory factors and cervical cancer were obtained from publicly available pooled data. The inverse variance weighted method was used as the main method and MR-Egger, weighted median, simple mode, and weighted mode were used as auxiliary analyses. Results were tested for robustness using sensitivity tests. In addition, we assessed the possibility of reverse causality between cervical cancer and the derived inflammatory factors by performing a reverse MR analysis. Finally, a preliminary experimental validation was performed. We found that artemin and monocyte chemoattractant protein-4 levels were significantly correlated with elevated cervical cancer risk (β: 0.0024, P = 0.002 and β: 0.0010, P = 0.016, respectively. In contrast, interleukin-18 and interleukin-22 receptor subunit alpha-1 levels were associated with reduced risk of cervical cancer (β: -0.0010, P = 0.029 and β: -0.0021, P = 0.046, respectively). Sensitivity analyses were more robust as no significant heterogeneity or horizontal pleiotropy was observed. A significant causal relationship was found between the four inflammatory factors and the risk of cervical cancer, providing new evidence of their clinical implications in cervical cancer diagnosis and treatment.

Human placental proteomics and exon variant studies link AAT/SERPINA1 with spontaneous preterm birth

BackgroundPreterm birth is defined as live birth before 37 completed weeks of pregnancy, and it is a major problem worldwide. The molecular mechanisms that lead to onset of spontaneous preterm birth are incompletely understood. Prediction and evaluation of the risk of preterm birth is challenging as there is a lack of accurate biomarkers. In this study, our aim was to identify placental proteins that associate with spontaneous preterm birth.MethodsWe analyzed the proteomes from placentas to identify proteins that associate with both gestational age and spontaneous labor. Next, rare and potentially damaging gene variants of the identified protein candidates were sought for from our whole exome sequencing data. Further experiments we performed on placental samples and placenta-associated cells to explore the location and function of the spontaneous preterm labor-associated proteins in placentas.ResultsExome sequencing data revealed rare damaging variants in SERPINA1 in families with recurrent spontaneous preterm deliveries. Protein and mRNA levels of alpha-1 antitrypsin/SERPINA1 from the maternal side of the placenta were downregulated in spontaneous preterm births. Alpha-1 antitrypsin was expressed by villous trophoblasts in the placenta, and immunoelectron microscopy showed localization in decidual fibrinoid deposits in association with specific extracellular proteins. siRNA knockdown in trophoblast-derived HTR8/SVneo cells revealed that SERPINA1 had a marked effect on regulation of the actin cytoskeleton pathway, Slit–Robo signaling, and extracellular matrix organization.ConclusionsAlpha-1 antitrypsin is a protease inhibitor. We propose that loss of the protease inhibition effects of alpha-1 antitrypsin renders structures critical to maintaining pregnancy susceptible to proteases and inflammatory activation. This may lead to spontaneous premature birth.

Rapid Reconfiguration of the Functional Connectome after Chemogenetic Locus Coeruleus Activation

The locus coeruleus (LC) supplies norepinephrine (NE) to the entire forebrain and regulates many fundamental brain functions. Studies in humans have suggested that strong LC activation might shift network connectivity to favor salience processing. To causally test this hypothesis, we use a mouse model to study the effect of LC stimulation on large-scale functional connectivity by combining chemogenetic activation of the LC with resting-state fMRI, an approach we term "chemo-connectomics." We show that LC activation rapidly interrupts ongoing behavior and strongly increases brain-wide connectivity, with the most profound effects in the salience and amygdala networks. Functional connectivity changes strongly correlate with transcript levels of alpha-1 and beta-1 adrenergic receptors across the brain, and functional network connectivity correlates with NE turnover within select brain regions. We propose that these changes in large-scale network connectivity are critical for optimizing neural processing in the context of increased vigilance and threat detection.

Abstract P5-04-08: Timing provides context for the paradoxical effects of AMPK activation in ER+ breast cancer: Suppressing growing tumors, but promoting dormant tumor cell survival and recurrence

Abstract Precision oncology requires delivering the right drug to the right patient at the right time, but “time” is rarely studied preclinically before a drug enters a particular clinical setting. Despite showing efficacy against recurrent/metastatic solid tumors, drugs sometimes fail to prevent tumor recurrence when given (neo)adjuvantly. The biology and microenvironments of overt tumors likely differ substantially from dormant cancer cells. The development of preclinical models to investigate clinically dormant disease will increase understanding of residual tumor cell biology and enable the development of therapeutics for rational adjuvant trials. Despite treatment with adjuvant anti-estrogen therapies, ˜30% of patients with ER+ breast cancer (BC) experience recurrence. In contrast to other BC subtypes, ER+ relapses occur late, often appearing years to decades after initial diagnosis and treatment. This delay suggests that ER+ BC cells can undergo extended periods of clinical dormancy. We developed novel, clinically relevant xenograft models of dormancy in ER+ BC. Low systemic levels of estrogens in mice can be further suppressed by ovariectomy, mimicking the effects of aromatase inhibitor (AI)-induced estrogen deprivation (ED) therapy. In ovariectomized mice, luciferase-labeled MCF-7, HCC-1428, HCC-1500, and MDA-MB-415 cells, as well as the HCI-017 PDX model, formed palpable orthotopic tumors upon 17b-estradiol supplementation. ED induced rapid tumor regression and decreased bioluminescent signal. However, a small proportion of ER+ BC cells survived ED for >1 year in a clinically dormant, growth-suppressed state. This residual cell population stabilized after ˜90 days of ED, as evidenced by stabilization of bioluminescent signal. Transcriptional and immunohistochemical analyses revealed significant upregulation of AMP-activated protein kinase (AMPK)-alpha-2 levels and activity in clinically dormant tumor cells compared to estrogen-driven or acutely ED xenografts. Dormant tumor cells were dependent upon AMPK activity for survival, as short-term pharmacologic inhibition of AMPK reduced residual bioluminescent signal. Metformin is an AMPK-activating drug approved for the treatment of diabetes. Metformin is currently being tested as an anti-cancer agent in many clinical trials at various points in the disease progression of diverse cancer types. In our models of clinically dormant ER+ BC, AMPK activation via metformin slowed ED-induced tumor regression, promoted residual tumor cell survival, and caused earlier tumor regrowth. In vitro studies indicated that metformin promotes cell survival during ED by enhancing fatty acid β-oxidation. Conversely, metformin treatment slowed estrogen-driven tumor growth, in agreement with prior observations that metformin slows growth of various tumor subtypes. These findings suggest that AMPK activation may be efficacious against growing tumors, but deleterious when used in combination with drugs that suppress tumor growth and induce regression. More broadly, this work highlights the issue that the time in a disease course needs to be considered when testing potential anti-cancer agents such as AMPK modulators. Citation Format: Miller TW, Hampsch RA, McCleery CF, Wells JD, Fields JL, Dillon LM, Shee K. Timing provides context for the paradoxical effects of AMPK activation in ER+ breast cancer: Suppressing growing tumors, but promoting dormant tumor cell survival and recurrence [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-08.

Rapid Reconfiguration of the Functional Connectome after Chemogenetic Locus Coeruleus Activation

The locus coeruleus (LC) supplies norepinephrine (NE) to the entire forebrain, regulates many fundamental brain functions, and is implicated in several neuropsychiatric diseases. Although selective manipulation of the LC is not possible in humans, studies have suggested that strong LC activation might shift network connectivity to favor salience processing. To test this hypothesis, we use a mouse model to study the impact of LC stimulation on large-scale functional connectivity by combining chemogenetic activation of the LC with resting-state fMRI, an approach we term “chemo-connectomics”. LC activation rapidly interrupts ongoing behavior and strongly increases brain-wide connectivity, with the most profound effects in the salience and amygdala networks. We reveal a direct correlation between functional connectivity changes and transcript levels of alpha-1, alpha-2, and beta-1 adrenoceptors across the brain, and a positive correlation between NE turnover and functional connectivity within select brain regions. These results represent the first brain-wide functional connectivity mapping in response to LC activation, and demonstrate a causal link between receptor expression, brain states and functionally connected large-scale networks at rest. We propose that these changes in large-scale network connectivity are critical for optimizing neural processing in the context of increased vigilance and threat detection.

Cryofibrinogenemia: A Marker of Severity of Cryoglobulinemic Vasculitis

BackgroundCryofibrinogenemia is frequently associated with cryoglobulinemia. The aim of this study was to determine the characteristics associated with the presence of cryofibrinogenemia in patients with cryoglobulinemic vasculitis. MethodsThis was a single-center retrospective study that included patients with cryoglobulinemic vasculitis who were tested for cryofibrinogen at a tertiary referral center between January 1, 2011 and December 31, 2012. Twenty-nine patients fulfilled the CryoVas (cryoglobulinemic vasculitis) Survey criteria for cryoglobulinemic vasculitis. Eighteen patients had a detectable cryofibrinogen (CF-positive) and 11 had no detectable cryofibrinogen (CF-negative). Median cryoglobulin levels were 89 ± 129 mg/L in the CF-positive group and 68 ± 82 mg/L in the CF-negative group (P = .32). Median cryofibrinogen level was 70 ± 174 mg/L. Clinical manifestations were similar in both groups. Cancers and hematological disorders were more frequent among CF-positive patients (39% vs 0%, P = .026). Levels of alpha-1 and alpha-2 globulinemia were higher in the CF-positive group. Cryofibrinogenemia ≥ 100 mg/L was associated with cryoglobulinemic vasculitis (odds ratio [OR] 2.86; 95% confidence interval [CI], 1.06-7.73) in cryoglobulinemic patients. Presence of cryofibrinogenemia was associated with use of corticosteroids, immunosuppressants, or plasmapheresis in cryoglobulinemic vasculitis patients (OR 22.7; 95% CI, 2.02-256.44). ConclusionsOur results strongly suggest that presence of cryofibrinogenemia is associated with a more severe phenotype among patients with cryoglobulinemic vasculitis.

Analysis of Giardin Expression During Encystation ofGiardia lamblia

The present study analyzed giardin transcription in trophozoites and cysts during encystation of Giardia lamblia . Encystment was induced using standard methods, and the numbers of trophozoites and cysts were counted at various time points during the process. At all time points, RNA from both stages were assayed for levels of alpha2-, beta-, and delta-giardin mRNA as well as for cyst wall protein 3 (CWP3) mRNA using quantitative RT-PCR. In encystation medium, the number of G. lamblia trophozoites decreased, while the number of cysts increased between 0 and 72 hr. In trophozoites, alpha2- and beta-giardin transcription decreased over time, while delta-giardin transcription remained unchanged during the same time period. CWP3 transcription exhibited a slight increase in trophozoites at 8 hr, followed by a decrease at subsequent time points. Expression of alpha2-giardin increased at 48 hr in cysts followed by decreased expression at 72 hr, while beta- and delta-giardin expression was unchanged during encystation. CWP3 transcription gradually decreased from 24-72 hr in cysts. Consistent with previous studies, giardin proteins appeared to be disassembled into amorphous structures inside cysts during encystation. These findings represent the first analysis of giardin transcription in separate populations of trophozoites and cysts during encystation and indicate differential regulation of giardin mRNA expression by these developmental stages.

Effects of weight loss, induced by gastric bypass surgery, on HDL remodeling in obese women

Plasma lipoproteins and glucose homeostasis were evaluated after marked weight loss before and over 12 months following Roux-en-Y gastric-bypass (RYGBP) surgery in 19 morbidly obese women. Standard lipids, remnant-lipoprotein cholesterol (RLP-C); HDL-triglyceride (TG); apolipoproteins (apo) A-I, A-II, E, and A-I-containing HDL subpopulations; lecithin-cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) mass and activity; plasma glucose and insulin levels were measured before and at 1, 3, 6, and 12 months after GBP surgery. Baseline concentrations of TG, RLP-C, glucose, and insulin were significantly higher in obese than in normal-weight, age-matched women, whereas HDL cholesterol (HDL-C), apoA-I, apoA-II, alpha-1 and alpha-2 levels were significantly lower. Over 1 year, significant decreases of body mass index, glucose, insulin, TG, RLP-C, HDL-TG, and prebeta-1 levels were observed with significant increases of HDL-C and alpha-1 levels (all P < 0.05). Changes of fat mass were correlated with those of LDL cholesterol (P = 0.018) and LCAT mass (P = 0.011), but not with CETP mass (P = 0.265). Changes of fasting plasma glucose concentrations were inversely correlated with those of CETP mass (P = 0.005) and alpha-1 level (P = 0.004). Changes of fasting plasma insulin concentrations were positively correlated with those of LCAT mass (P = 0.043) and inversely with changes of alpha-1 (P = 0.03) and alpha-2 (P = 0.05) concentrations. These results demonstrate beneficial changes in HDL remodeling following substantial weight loss induced by RYGBP surgery and that these changes are associated with improvement of glucose homeostasis in these patients.

18 Analyzing functional genomics data and networks to understand disease

Chondrocytes have been shown to express both in vivo and in vitro a number of integrins of the beta1-, beta3- and beta5-subfamilies (Biorheology 37 (2000) 109). Normal and v-Src-transformed chick epiphyseal chondrocytes (CEC) display different adhesion properties. While normal CEC with time in culture tends to increase their adhesion to the substrate by organizing focal adhesions and actin stress fibers, v-Src-transformed chondrocytes display a refractile morphology and disorganization of actin cytoskeleton. We wondered whether the reduced adhesion and spreading of v-Src-transformed chondrocytes could be ascribed to changes in integrin expression and/or function. Integrin expression by normal CEC is studied and compared to v-Src-transformed chick chondrocytes, using monoclonal and polyclonal antibodies to integrins alpha- and beta-chains. We show the presence of alpha1-, alpha3-, alphav-, alpha6-, beta1- and beta3-chains on CEC, with very low levels of alpha2- and alpha5-chains. Alphav chain associates with multiple beta subunits in normal and transformed chondrocytes. With the exception of alpha1- and alpha2-chains, the levels of the integrin chains analyzed are higher in transformed chondrocytes as compared with normal chondrocytes. In spite of the increased levels of integrin expression, transformed chondrocytes exhibit loss of focal adhesion and actin stress fibers and low adhesion activity on several extracellular matrix constituents. These observations raise the possibility that, in addition to its effects on global pattern of integrin expression, v-Src can influence integrin function in chondrocytes.

α1,3 Fucosyltransferase VII plays a role in colorectal carcinoma metastases by promoting the carbohydration of glycoprotein CD24

Changes in the expression levels of alpha1,3 FucT-VII and carbohydrate product SLe X have been reported in certain types of malignant transformations. However, the specific role of FucT-VII in human colorectal carcinoma is still not clear. We evaluated the expression of FucT-VII in tumor specimens from 30 colorectal carcinoma patients by RT-PCR and immunohistochemistry. The alteration of metastatic potential of LOVO cells before and after alpha1,3 FucT-VII gene transfection was also assayed. The expression change of glycoprotein CD24 and carbohydrate antigen SLe X after stable transfection of LOVO was also examined in vitro. Higher mRNA and protein expression of FucT-VII were observed in colorectal tumors compared with adjacent normal ones. The cell chemotactic migration and invasion were enhanced after alpha1,3 FucT-VII transfection in LOVO cells. There was a positive correlation between alpha1,3 FucT-VII mRNA expression and lymph node status (p=0.009) and AJCC stage (p=0.007), similar correlation was also observed at protein level (p=0.042 and 0.022, respectively). Overexpression of alpha1,3 FucT-VII promoted the expression of CD24 and SLe X in LOVO cells. Our findings suggest that alpha1,3 FucT-VII might play a role in colorectal carcinoma metastases by promoting the carbohydration of glycoprotein CD24.

Cholinesterase Activity and mRNA Level of Nicotinic Acetylcholine Receptors (α4 and β2 Subunits) in Blood of Elderly Chinese Diagnosed as Alzheimer's Disease

The aim of the study is to investigate the cholinergic deficit in Alzheimer's disease (AD) and identify candidate blood biomarkers for the diagnosis of the disease. Twenty-nine elderly Chinese diagnosed with AD and 33 age-matched controls were selected. The activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in plasma were detected by a spectrophotometric method, and the mRNA levels of alpha4 and beta2 nicotinic acetylcholine receptor (nAChR) subunits in blood leukocytes were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that AChE activity in plasma was significantly lower in the AD group than in normal controls, while BuChE activity did not show any differences between AD and controls; mRNA levels of both alpha4 and beta2 nAChR subunits in blood leukocytes were significantly lower in the AD group than in controls. The AChE activity and the mRNA levels of alpha4 and beta2 nAChR subunits in the AD patients were also significantly correlated with cognitive test scores. No differences of AChE in plasma or alpha4 and beta2 nAChR subunits in blood leukocytes were detected between smoking and non-smoking subjects. The results indicated that the decreases in the activity of AChE and in the mRNA levels of nAChR alpha4 and beta2 subunits from the peripheral blood of patients with AD might serve as supplementary indicators for the clinical diagnosis of AD.

Comparative study between the effect of the peroxisome proliferator activated receptor-α ligands fenofibrate and n-3 polyunsaturated fatty acids on activation of 5′-AMP-activated protein kinase-α1 in high-fat fed rats

Obesity is a risk factor for type 2 diabetes mellitus. It results from an energy imbalance in which energy intake exceeds energy expenditure. The cellular fuel gauge 5'-AMP-activated protein kinase (AMPK) is a heterotrimeric protein consisting of one catalytic subunit (alpha) and two non-catalytic subunits (beta and gamma), and approximately equal levels of alpha1 and alpha2 complexes are present in the liver. AMPK regulates metabolic pathways in response to metabolic stress and in particular ATP depletion to switch on energy-producing catabolic pathways such as beta-oxidation of fatty acids and switch off energy-depleting processes such as synthesis of fatty acid and cholesterol. A high-fat diet alters AMPK-alpha1 gene expression in the liver and skeletal muscle of rats and results in body weight gain and hyperglycaemia. The aim of this study was to investigate and compare the potential effects of peroxisome proliferator-activated receptor (PPAR)-alpha agonists fenofibrate and n-3 polyunsaturated fatty acids (PUFAs) in modulation of AMPK-alpha1 activity in liver and skeletal muscle of high-fat diet fed rats. Reverse transcription-polymerase chain reaction was used for determination of AMPK-alpha1 in liver and soleus muscle and both PPAR-alpha and CPT-1 in hepatic tissues. Serum, total cholesterol, triacylglycerol, fatty acid and fasting blood glucose were determined colorimetrically. Both PPAR-alpha agonists, fenofibrate and n-3 PUFA, increased the mRNA expression of AMPK-alpha1 activity in liver and skeletal muscle of obese diabetic rats. Fenofibrate was superior in its activation of hepatic mRNA expression of AMPK-alpha 1 to exert more lipolytic effect and body weight reduction, as estimated through the decrease of triacylglycerol output and serum levels of fatty acid on the one hand and the increase in CPT-1 mRNA expression, the key enzyme in beta-oxidation of fatty acid, on the other hand. n-3 PUFA activated AMPK-alpha1 mRNA expression in skeletal muscle much more than fenofibrate to reveal more hypoglycaemic effect. The PPAR-alpha agonists fenofibrate and n-3 PUFA could efficiently activate AMPK-alpha1 mRNA expression in liver and skeletal muscle to exert body weight reduction and hypoglycaemic effect, respectively.

The Ubiquitin–Proteasome System Regulates the Stability of Neuronal Nicotinic Acetylcholine Receptors

Ubiquitination is a key event for protein degradation by the proteasome system, membrane protein internalization, and protein trafficking among cellular compartments. Few data are available on the role of the ubiquitin-proteasome system (UPS) in the trafficking of neuronal nicotinic acetylcholine receptors (nAChRs). Experiments conducted in neuron-like differentiated rat pheochromocytoma cells (PC12 cells) show that the alpha3, beta2, and beta4 nAChR subunits are ubiquitinated and that their ubiquitination is necessary for degradation. A 24-h treatment with the proteasome inhibitor PS-341 increased the total levels of alpha3 and the two beta subunits in both whole cell lysates and fractions enriched for the ER/Golgi compartment. nAChR subunit upregulation was also detected in plasma membrane-enriched fractions. Inhibition of the lysosomal degradation machinery by E-64 had a significantly smaller effect on nAChR turnover. The present data, together with previous results showing that the alpha7 nAChR subunit is a target of the UPS, point to a prominent role of the proteasome in nAChR trafficking.

Genomic organization of zebra finch alpha and beta globin genes and their expression in primitive and definitive blood in comparison with globins in chicken

How alpha and beta globin genes are organized and expressed in amniotes is of interest to researchers in a wide variety of fields. Data regarding this from avian species have been scarce. Using genomic and proteomic approaches, we present here our analysis of alpha and beta globins of zebra finch, a passerine bird. We show that finch alpha globin gene cluster has three genes (alphas 1-3), each orthologous to its chicken counterpart. Finch beta globin gene cluster has three genes (betas 1-3), with an additional pseudogene at the 3' end. Finch beta3 is orthologous to chicken betaA, but the orthology of beta1 and beta2 to chicken counterparts is less clear. All six finch globins are confirmed to encode functional proteins. Gene expression in both globin gene clusters is regulated developmentally. Adult finch blood has a globin profile similar to that of adult chicken, with high levels of beta3 and alpha3 and moderate levels of alpha2. Finch embryonic primitive blood exhibits a globin profile very different from that of equivalent stage chick embryos, with all six globins expressed at high levels. Overall, our data provide a valuable resource for future studies in avian globin gene evolution and globin switching during erythropoietic development.

Cannabinoid modulation of cortical adrenergic receptors and transporters.

We previously reported that administration of the synthetic cannabinoid agonist WIN 55,212-2 causes an increase in norepinephrine (NE) efflux in the frontal cortex (FC). The present study examined the expression levels of alpha2- and beta1-adrenergic receptors (ARs) as well as the norepinephrine transporter (NET) in the FC of rats following exposure to WIN 55,212-2. Rats received systemic injection of WIN 55,212-2 (3 mg/kg) acutely or for 7 days. Another group of rats received repeated WIN 55,212-2 treatment followed by a period of abstinence. Control rats received vehicle injections. Rats were euthanized 30 min after the last WIN 55,212-2 injection, the FC was microdissected, and protein extracts were probed for alpha2-AR, beta1-AR, and NET. Results showed that beta1-AR expression was significantly decreased following repeated WIN 55,212-2 treatment but significantly increased following a period of abstinence. alpha2-AR expression showed no significant change in all groups examined. NET expression was significantly decreased following acute WIN 55,212-2 treatment, with no changes following chronic administration or a period of abstinence. Alterations in NET may arise from modulation of cannabinoid receptors (CB1) that are localized to noradrenergic axon terminals as we demonstrate colocalization of CB1 receptor and NET in the same cortical axonal processes. The present findings support significant alterations in adrenergic receptor and NET expression in the FC after WIN 55,212 exposure that may underlie the reported changes in attention, cognition, and anxiety commonly observed after cannabinoid exposure.

Protracted Developmental Trajectories of GABA A Receptor α1 and α2 Subunit Expression in Primate Prefrontal Cortex

In schizophrenia, working memory dysfunction is associated with altered expression of gamma-aminobutyric acid (GABA)(A) receptor alpha1 and alpha2 subunits in the dorsolateral prefrontal cortex (DLPFC). In rodents, cortical alpha subunit expression shifts from low alpha1 and high alpha2 to high alpha1 and low alpha2 during early postnatal development. Because these two alpha subunits confer different functional properties to the GABA(A) receptors containing them, we determined whether this shift in alpha1 and alpha2 subunit expression continues through adolescence in the primate DLPFC, potentially contributing to the maturation of working memory during this developmental period. Levels of GABA(A) receptor alpha1 and alpha2 subunit mRNAs were determined in the DLPFC of monkeys aged 1 week, 4 weeks, 3 months, 15-17 months (prepubertal), and 43-47 months (postpubertal) and in adult monkeys using in situ hybridization, followed by the quantification of alpha1 subunit protein by western blotting. We also performed whole-cell patch clamp recording of miniature inhibitory postsynaptic potentials (mIPSPs) in DLPFC slices prepared from pre- and postpubertal monkeys. The mRNA and protein levels of alpha1 and alpha2 subunits progressively increased and decreased, respectively, throughout postnatal development including adolescence. Furthermore, as predicted by the different functional properties of alpha1-containing versus alpha2-containing GABA(A) receptors, the mIPSP duration was significantly shorter in postpubertal than in prepubertal animals. In contrast to rodents, the developmental shift in GABA(A) receptor alpha subunit expression continues through adolescence in primate DLPFC, inducing a marked change in the kinetics of GABA neurotransmission. Disturbances in this shift might underlie impaired working memory in schizophrenia.

Low Serum Alpha-1 Antitrypsin (AAT) in Family Members of Individuals with Autism Correlates with PiMZ Genotype

Deficiency of Alpha-1-antitrypsin (AAT) can be a genetic condition that increases the risk of developing liver, lung and possibly gastrointestinal disease. Since many autistic children also have gastrointestinal disorders, this study was designed to measure serum concentration of AAT and establish AAT genotypes in autistic children, age and gender matched non-autistic siblings, parents and controls. We used an indirect ELISA with monoclonal IgG to AAT to measure AAT serum concentrations in 71 members from 16 families of individuals with autism and 18 controls (no family history of autism). We used a duplex polymerase chain reaction to detect M, S and Z alleles for alpha-1 antitrypsin expression in 52 members of 12 of the above families. A significantly high number of autistic family members had lower than normal serum levels of AAT when compared to controls. Autistic children with regressive onset had significantly lower levels of AAT compared to controls, and a significant number of autistic children with low serum AAT also had hyperbilirubinemia, gastrointestinal disease and respiratory problems. We also found that a significantly high number of these individuals had the PiMZ genotype and correspondingly low levels of serum alpha-1 antitrypsin. Knowing that low levels of alpha-1 antitrypsin may be inherited, and that low levels of AAT may be associated with GI disease in autistic children, genotyping autistic children may help identify individuals susceptible to developing digestive problems.

Reduced expression of nicotinic α subunits 3, 7, 9 and 10 in lesional and nonlesional atopic dermatitis skin but enhanced expression of α subunits 3 and 5 in mast cells

The skin cholinergic signalling system is modulated in atopic dermatitis (AD). To investigate of the role of nicotinic acetylcholine receptors (nAChRs) in the pathogenesis of AD. We investigated the expression and localization of nAChR alpha subunits in AD by quantitative reverse transcription-polymerase chain reaction and immunohistochemistry of biopsies from lesional and nonlesional areas of AD skin and of skin biopsies from healthy control persons. Our data demonstrate the presence of mRNA and protein of the nAChR alpha subunits 3, 5, 7, 9 and 10 in keratinocytes and mast cells in healthy and AD skin. Expression of the alpha subunits 3, 7, 9 and 10 was generally reduced in the skin of patients with AD whereas mast cells in AD but not in healthy skin showed alpha3 and alpha5 subunit immunoreactivity. Differences in the subunit mRNA levels between lesional and nonlesional skin were obtained for the alpha subunits 3, 9 and 10 with higher levels of alpha3 but lower levels of alpha10 subunit mRNA in lesional areas. No differences in the expression of the alpha subunits was found between the groups of extrinsic, intrinsic or mixed AD types, between genders and between smokers and nonsmokers. This supports the idea that the cholinergic system is dysregulated independently from inflammation in AD and that inflammation further modulates individual nAChR subunits.

Prenatal Diazepam Exposure Alters Respiratory Control System and GABAA and Adenosine Receptor Gene Expression in Newborn Rats

In experimental animals, prenatal diazepam exposure has clearly been associated with behavioral disturbances. Its impact on newborn breathing has not been documented despite potential deleterious consequences for later brain development. We addressed this issue in neonatal rats (0-2 d) born from dams, which consumed 2 mg/kg/d diazepam via drinking fluid throughout gestation. In vivo, prenatal diazepam exposure significantly altered the normoxic-breathing pattern, lowering breathing frequency (105 vs. 125 breaths/min) and increasing tidal volume (16.2 vs. 12.7 mL/kg), and the ventilatory response to hypoxia, inducing an immediate and marked decrease in tidal volume (-30%) absent in controls. In vitro, prenatal diazepam exposure significantly increased the respiratory-like frequency produced by pontomedullary and medullary preparations (+38% and +19%, respectively) and altered the respiratory-like response to application of nonoxygenated superfusate. Both in vivo and in vitro, the recovery from oxygen deprivation challenges was delayed by prenatal diazepam exposure. Finally, real-time PCR showed that prenatal diazepam exposure affected mRNA levels of alpha1 and alpha2 GABAA receptor subunits and of A1 and A2A adenosine receptors in the brainstem. These mRNA changes, which are region-specific, suggest that prenatal diazepam exposure interferes with developmental events whose impact on the respiratory system maturation deserves further studies.