RNase Protection Assay Analysis Research Articles

Alternative Splicing and Polyadenylation Contribute to the Generation of hERG1 C-terminal Isoforms

The human ether-a-go-go-related gene 1 (hERG1) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel. Several hERG1 isoforms with different N- and C-terminal ends have been identified. The hERG1a, hERG1b, and hERG1-3.1 isoforms contain the full-length C terminus, whereas the hERG1(USO) isoforms, hERG1a(USO) and hERG1b(USO), lack most of the C-terminal domain and contain a unique C-terminal end. The mechanisms underlying the generation of hERG1(USO) isoforms are not understood. We show that hERG1 isoforms with different C-terminal ends are generated by alternative splicing and polyadenylation of hERG1 pre-mRNA. We identified an intrinsically weak, noncanonical poly(A) signal, AGUAAA, within intron 9 of hERG1 that modulates the expression of hERG1a and hERG1a(USO). Replacing AGUAAA with the strong, canonical poly(A) signal AAUAAA resulted in the predominant production of hERG1a(USO) and a marked decrease in hERG1 current. In contrast, eliminating the intron 9 poly(A) signal or increasing the strength of 5' splice site led to the predominant production of hERG1a and a significant increase in hERG1 current. We found significant variation in the relative abundance of hERG1 C-terminal isoforms in different human tissues. Taken together, these findings suggest that post-transcriptional regulation of hERG1 pre-mRNA may represent a novel mechanism to modulate the expression and function of hERG1 channels.

Molecular correlates of age-specific responses to traumatic brain injury in mice

Aged traumatic brain injury (TBI) patients suffer higher rates of mortality and disability than younger patients. Cognitive problems common to TBI patients are associated with damage to the hippocampus, a central locus of learning and memory. To investigate the molecular mechanisms of age-related vulnerability to brain injury in a mouse model of TBI, we studied the effects of TBI on hippocampal gene expression in young and aged mice. Young and aged male C57Bl/6 mice were subjected to sham injury or TBI and sacrificed 24 h post-injury. We used laser capture microdissection to obtain pure populations of neurons from the CA1, CA3, and dentate gyrus subfields of the hippocampus. We compared injury-induced gene expression in hippocampal neurons of young and aged mice using quantitative ribonuclease protection assay analysis of linearly amplified mRNA from laser captured neurons. Both increased age and TBI were associated with increased expression of neuroprotective (brain-derived neurotrophic factor), pro-inflammatory (interleukin-1β), and proapoptotic (caspase-3) genes in mouse hippocampal neurons. Our data support previous reports that suggested the CA3 subregion is highly susceptible to fluid percussion TBI and that age-related changes in gene expression are one potential mechanism of increased vulnerability of the aged brain to TBI.

This Month in Anesthesiology

This Month in Anesthesiology

Suppression of experimental crescentic glomerulonephritis by interleukin-10 gene transfer

Investigated were effects of overexpression of interleukin-10 (IL-10) on the outcome and progression of crescentic glomerulonephritis in Wistar-Kyoto (WKY) rats. Rats were singly or simultaneously injected with antiglomerular basement membrane (a-GBM) antibody and adenoviral vector encoding rat IL-10 (Ad-rIL-10) or LacZ (Ad-LacZ) (3 x 1010 pfu/rat) intravenously, and were sacrificed at day 7. Their kidneys and other organs were isolated and examined by histology and immunohistochemistry. The In vivo expression of IL-10 mRNA in the liver of Ad-rIL-10-injected rats was confirmed by both reverse transcription-polymerase chain reaction (RT-PCR) and ribonuclease protection assay analysis and its translated protein was measured in the serum by enzyme-linked immunosorbent assay (ELISA). The exogenous IL-10 mRNA was strongly expressed in the liver in a dose-dependent manner and was intense at days 4 and 7 but was less intense at day 14. Ad-rIL-10 treatment significantly reduced the incidence of glomerular crescent formation from 67%+/- 1.9% in a-GBM antibody-treated group or 69.8%+/- 1.9% in a-GBM antibody + Ad-LacZ-treated group to 21.6%+/- 1.8% (P < 0.001), the glomerular infiltration of macrophages from 35.7 +/- 6.3 cell s/gcs (a-GBM antibody) or 37.6 +/- 8.6 cells/gcs (both a-GBM antibody + Ad-LacZ) to 17.9 +/- 5.5 cells/gcs (P < 0.001), that of major histocompatibility complex (MHC) class II-positive cells from 14.4 +/- 5.3 cells/gcs (a-GBM antibody) or 15 +/- 4.6 cells/gcs (a-GBM antibody + Ad-LacZ) to 5.7 +/- 2.3 cells/gcs (P < 0.0001) at day 7, the glomerular and immune tissue expression of IL-1beta mRNA, as well as the proteinuria from 159.0 +/- 22.7 mg/24 hours (a-GBM antibody) or 166 +/- 28 mg/24 hours (a-GBM antibody + Ad-LacZ) to 42.2 +/- 35.2 mg/24 hours (P < 0.01) at day 7. The serum creatinine and blood urea nitrogen levels were also reduced from 2.8 +/- 0.1 mg/dL (a-GBM antibody) or 2.8 +/- 0.1 mg/dL (a-GBM antibody + Ad-LacZ) to 1.0 +/- 0.1 mg/dL (P < 0.001) and from 63.2 +/- 8.9 mg/dL (a-GBM antibody) or 61.3 +/- 5.2 mg/dL (a-GBM antibody + Ad-LacZ) to 27.0 +/- 4.5 mg/dL (P < 0.001), respectively. However, the glomerular accumulation of CD8+ T cells was unaffected: 5.4 +/- 1.1 cells/gcs (a-GBM antibody + Ad-rIL-10), 5.9 +/- 1.5 cells/gcs (a-GBM antibody), and 5.8 +/- 1.1 cells/gcs (a-GBM antibody + Ad-LacZ) (P= NS). IL-10 gene transfer significantly attenuated the glomerular lesions and injury in the anti-GBM crescentic glomerulonephritis of WKY rats.

Predominantly neuronal expression of cytochrome P450 isoforms cyp3a11 and cyp3a13 in mouse brain

Despite the very small amounts of cytochrome P450 enzymes expressed in different areas and cell populations of the brain as compared with the liver, there is significant evidence for their specific involvement in brain development, function, and plasticity. Nevertheless, the current discussion about occurrence and importance of cerebral cytochrome P450 isoforms is determined by controversial interpretations of their function in general and with respect to single isoforms. Continuing a series of publications about brain P450 isoforms, we now present evidence for the expression of cytochrome P450 3A11 and 3A13 in mouse brain. Immunocytochemical and non-radioactive in situ hybridization studies revealed identical distribution of their proteins and mRNAs throughout the brain especially in neuronal populations, and to some extent in astrocytes. The cerebral expression of these P450 isoforms was confirmed by Western blot and RNAse protection assay analysis. The well-known testosterone-metabolizing capacity and the inducibility of cytochrome P450 3a isoforms by xenobiotics as well as their presence in steroid hormone-sensitive areas and neurons (e.g. hippocampus) clarify the significance of these isoforms for impairment of steroid hormone actions by P450-inducing environmental substances. Therefore, investigation of inducible cerebral P450 isoforms which are able to metabolize xenobiotics as well as steroid hormones might help us to understand neuroendocrine regulation of brain’s plasticity.

Modulation Of Sulfur Mustard-induced Inflammation And Gene Expression By Olvanil In The Hairless Mouse Vesicant Model

Cutaneous exposure to sulfur mustard [bis(2-chloroethyl) sulfide (SM)] produces a delayed inflammatory skin response that is followed by severe dermal injury. Assessment of anti-inflammatory therapies against SM-induced skin injury has mainly relied on qualitative histopathological evaluation. The goal of this study was to identify proinflammatory biomarkers in the hairless mouse vesicant model that could be used as additional indicators of SM-induced skin injury for evaluating anti-inflammatory treatment. SM-induced inflammation was determined at 2, 6, and 24 hr postexposure by changes in edema. Ribonuclease protection assay (RPA) was used to determine changes in gene expression of inflammatory mediators. At 2, 6, and 24 hr postexposure, a time-dependent increase in edema was observed in SM-exposed skin, which was significant at 6 and 24 hr when compared to unexposed controls. Ribonuclease protection assay analysis revealed a two-fold or greater increase in monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2), MIP-1α, tumor necrosis factor-α, and interleukin (IL)-1β following exposure to SM when compared to unexposed controls. A significant time-dependent increase was observed in MCP-1, MIP-1α, and IL-1β over the 24 hr time period. At 24 hr postexposure, skin treated with the anti-inflammatory drug olvanil showed a significant decrease in SM-induced edema. Additionally, mRNA levels of MCP-1, MIP-2, and IL-1β were decreased when compared to skin exposed to SM alone. In this study, we identified molecular biomarkers at the mRNA level, up-regulated in skin exposed to SM, which can be partially suppressed by olvanil. Further characterization of the mRNA and protein expression patterns of proinflammatory biomarkers may enable the use of other classes of anti-inflammatory drugs or therapeutic treatments against SM dermal injury.

Hypercytokinemia is Not Originated from Peripheral Mononuclear Cells at Acute Phase of Kawasaki Disease

Immune activation and generalized vasculitis are two central features of Kawasaki disease (KD). Recent observations indicated that serum levels of cytokines including interferon(INF)- γ, tumor necrosis factor (TNF)- α and interleukin (IL)-1 are remarkably increased, and that the administration of intravenous γ-globulin (IVGG) reduces the levels of elevated cytokines. Objective: We examined whether the plasma exchange (PE) for intractable KD cases reduces the elevated serum cytokines, and whether hypercytokinemia is originated from peripheral mononuclear cells at acute phase. Materials and Methods: We first compared the serum levels of cytokines between pre- and post-treatment of IVGG (8 cases) and PE (8 cases), using ELISA and cytometric bead array (CBA) system (Becton & Dickinson). For 4 cases among them, we investigated the messenger RNA levels of several cytokines in peripheral mononuclear cells at acute phase, using ribonuclease protection assay (RPA) system (Becton & Dickinson). Results: In sera of the children intractable to IVGG, increased levels of cytokines were detected such as INF-γ, IL-6, IL-10 and soluble TNF receptor (sTNFR). After PE therapy, serum levels of them markedly decreased to the normal ranges (INF-γ: 9.1 ± 13.4 →4.0 ± 6.4 pg/ml, IL-6: 67.5 ± 74.3→6.6 ± 8.4 pg/ml, IL-10: 14.1 ± 5.1→7.4 ± 2.1 pg/ml, sTNFR: 2521.9 ± 728.2→1338.2 ± 575.9 pg/ml). However, RPA analysis demonstrated that messenger RNA levels of several cytokines at acute phase were undetectable in both pre- and post-treatment of PE in peripheral mononuclear cells. Discussion: These findings suggested that the effectiveness of IVGG and PE treatment for KD will be attributed to the reduction of proinflammatory cytokine levels in serum, and that the increased levels of proinflammatory cytokines were originated not from the circulating mononuclear cells, but presumably from in situ lymphocytes and macrophages located at inflammatory lesion such as vasculitis lesion.

Neonatal exposure to genistein induces estrogen receptor (ER)alpha expression and multioocyte follicles in the maturing mouse ovary: evidence for ERbeta-mediated and nonestrogenic actions.

Outbred CD-1 mice were treated neonatally on Days 1-5 with the phytoestrogen, genistein (1, 10, or 100 micro g per pup per day), and ovaries were collected on Days 5, 12, and 19. Ribonuclease protection assay analysis of ovarian mRNA showed that estrogen receptor beta (ERbeta) predominated over ERalpha in controls and increased with age. Genistein treatment did not alter ERbeta expression, however, ERalpha expression was higher on Days 5 and 12. ERbeta was immunolocalized in granulosa cells, whereas ERalpha was immunolocalized in interstitial and thecal cells. Genistein treatment caused a dramatic increase in ERalpha in granulosa cells. Genistein-treated ERbeta knockout mice showed a similar induction of ERalpha, which is seen in CD-1 mice, suggesting that ERbeta does not mediate this effect. Similar ERalpha induction in granulosa cells was seen in CD-1 mice treated with lavendustin A, a tyrosine kinase inhibitor that has no known estrogenic actions, which suggests that this property of genistein may be responsible. As a functional analysis, genistein-treated mice were superovulated and the number of oocytes was counted. A statistically significant increase in the number of ovulated oocytes was observed with the lowest dose, whereas a decrease was observed with the two higher doses. This increase in ovulatory capacity with the low dose coincided with higher ERalpha expression. Histological evaluations on Day 19 revealed a dose-related increase in multioocyte follicles (MOFs) in genistein-treated mice. Tyrosine kinase inhibition was apparently not responsible for MOFs because they were not present in mice that had been treated with lavendustin; however, ERbeta must play a role, because mice lacking ERbeta showed no MOFs. These data taken together demonstrate alterations in the ovary following neonatal exposure to genistein. Given that human infants are exposed to high levels of genistein in soy-based foods, this study indicates that the effects of such exposure on the developing reproductive tract warrant further investigation.

Eighteen-Month-Old Fischer 344 Rats Fed a Spinach-Enriched Diet Show Improved Delay Classical Eyeblink Conditioning and Reduced Expression of Tumor Necrosis Factor α (TNFα) and TNFβ in the Cerebellum

Diets high in antioxidant properties are known to reverse some deficits in neuronal and cognitive function that occur in aging animals. Antioxidants are also known to reduce levels of proinflammatory factors in the CNS. We report here that 6 weeks of a spinach-enriched diet ameliorates deficits in cerebellar-dependent delay classical eyeblink learning and reduces the proinflammatory cytokines tumor necrosis factor α (TNFα) and TNFβ in the cerebelli of eyeblink-trained animals. Eighteen-month-old Fischer 344 rats were given spinach-enriched lab chow or regular lab chow for 6 weeks. The rats were then given 6 d of 30 trials per day training using a 3 kHz tone conditioned stimulus and airpuff unconditioned stimulus. Rats were killed 3 weeks after eyeblink training. Cytokine expression was measured using RNase protection assay analysis in the eyeblink-trained animals and in a group of young control animals given regular lab chow diet. Old animals on the spinach-enriched lab chow diet learned delay eyeblink conditioning significantly faster than old animals on the regular diet. Cerebelli from older animals on the spinach-enriched diet had significantly less TNFα and TNFβ than cerebelli from older animals on the control diet.

Eighteen-month-old Fischer 344 rats fed a spinach-enriched diet show improved delay classical eyeblink conditioning and reduced expression of tumor necrosis factor alpha (TNFalpha ) and TNFbeta in the cerebellum.

Diets high in antioxidant properties are known to reverse some deficits in neuronal and cognitive function that occur in aging animals. Antioxidants are also known to reduce levels of proinflammatory factors in the CNS. We report here that 6 weeks of a spinach-enriched diet ameliorates deficits in cerebellar-dependent delay classical eyeblink learning and reduces the proinflammatory cytokines tumor necrosis factor alpha (TNFalpha) and TNFbeta in the cerebelli of eyeblink-trained animals. Eighteen-month-old Fischer 344 rats were given spinach-enriched lab chow or regular lab chow for 6 weeks. The rats were then given 6 d of 30 trials per day training using a 3 kHz tone conditioned stimulus and airpuff unconditioned stimulus. Rats were killed 3 weeks after eyeblink training. Cytokine expression was measured using RNase protection assay analysis in the eyeblink-trained animals and in a group of young control animals given regular lab chow diet. Old animals on the spinach-enriched lab chow diet learned delay eyeblink conditioning significantly faster than old animals on the regular diet. Cerebelli from older animals on the spinach-enriched diet had significantly less TNFalpha and TNFbeta than cerebelli from older animals on the control diet.

The human integrin β4B and β4C variants are not expressed in a tissue-specific manner

The human integrin β4B and β4C variants are not expressed in a tissue-specific manner

The placental expression of the porcine pregnancy-associated glycoprotein (pPAG) gene family examined in situ and in vitro

The objective of the study was to define the expression of the porcine pregnancy-associated glycoprotein (pPAG) gene family, including pPAG1, pPAG2, pPAG3, pPAG4 and pPAG6 that belong to the aspartic proteinase family. Porcine pPAG2, PAG4 and pPAG6 are members of a subfamily (pPAG2-like), which all have highly conserved sequences to pepsins, within two catalytic domains, suggesting enzymatic activity of these molecules. In contrast, pPAG1 and pPAG3 have catalytic sites with critical amino acid substitutions that likely render these molecules enzymatically inactive. The expression of pPAG mRNA was examined by using in situ hybridisation (ISH) in placental tissues or cultured cells and by ribonuclease protection assay (RPA). The pPAG protein family, secreted in vitro during long-term cultures, was examined using Western blotting. The trophoblastic pPAG mRNA expression starts around implantation and is continued in chorionic epithelium (trophectoderm) throughout pregnancy. ISH performed on porcine placental sections with pPAG antisense cDNA probes revealed an expression of pPAG transcripts, locally restricted only to trophectoderm. The pPAG2-like mRNA expression occurred in different trophectoderm cells. Some trophoblast cells were bigger than others and were involved in local rearrangements of maternal epithelium layer, especially in developing placental folds. A high similarity of dominating pPAG2-like transcript expression was confirmed by RPA analysis. Cultures of trophoblast cells revealed their differentiation to multinucleated forms that were not observed in situ. This confirms a strong inhibitory effect of the maternal microenvironment of uterine lumen on mononuclear trophoblast within porcine placental units that was not present during the development of multinucleated trophoblast cells in vitro. Long-term cultures of chorionic explants revealed a very efficient system of pPAG protein production in vitro. Western blotting of secretory pPAG proteins indicated similar immunologic epitope(s) of these molecules and pregnancy-stage dependent profile of chorionic secretion. Thus, some of the subpopulation(s) of porcine trophoblast cells expressing pPAG2-like transcripts and their secretory products can play an important role(s) in the mechanism(s) of the confrontation between trophoblast/trophectoderm cells and maternal endometrial epithelium during implantation, placenta formation and successful pregnancy maintenance in the pig.

Genetic Characterization of Infectious Hematopoietic Necrosis Virus of Coastal Salmonid Stocks in Washington State

Infectious hematopoietic necrosis virus (IHNV) is a pathogen that infects many Pacific salmonid stocks from the watersheds of North America. Previous studies have thoroughly characterized the genetic diversity of IHNV isolates from Alaska and the Hagerman Valley in Idaho. To enhance understanding of the evolution and viral transmission patterns of IHNV within the Pacific Northwest geographic range, we analyzed the G gene of IHNV isolates from the coastal watersheds of Washington State by ribonuclease protection assay (RPA) and nucleotide sequencing. The RPA analysis of 23 isolates indicated that the Skagit basin IHNV isolates were relatively homogeneous as a result of the dominance of one G gene haplotype (S). Sequence analysis of 303 bases in the middle of the G gene (midG region) of 61 isolates confirmed the high frequency of a Skagit River basin sequence and identified another sequence commonly found in isolates from the Lake Washington basin. Overall, both the RPA and sequence analysis showed that the Washington coastal IHNV isolates are genetically homogeneous and have little genetic diversity. This is similar to the genetic diversity pattern of IHNV from Alaska and contrasts sharply with the high genetic diversity demonstrated for IHNV isolates from fish farms along the Snake River in Idaho. The high degree of sequence and haplotype similarity between the Washington coastal IHNV isolates and those from Alaska and British Columbia suggests that they have a common viral ancestor. Phylogenetic analyses of the isolates we studied and those from different regions throughout the virus's geographic range confirms a conserved pattern of evolution of the virus in salmonid stocks north of the Columbia River, which forms Washington's southern border.

The cardiac sodium channel mRNA is expressed in the developing and adult rat and human brain

Expression of the rat (RH-I/SkM2) and human (hH1/SCN5A) tetrodotoxin-resistant (TTX-R), voltage-sensitive sodium channels is thought to be specific to cardiac tissue. We detected RH-I/SkM2 mRNA in newborn rat brain using both RNase protection assay analysis and in situ hybridization and in adult rat brain using RNase protection assay analysis. This expression was observed primarily in developing limbic structures of the cerebrum and diencephalon, and in the medulla of the brain stem. Using RT-PCR analysis, we detected hH1/SCN5A mRNA in both fetal and adult human brain. Interestingly, mutations in the human cardiac sodium channel are known to lead to cardiac abnormalities, which result in arrhythmias and frequently in sudden cardiac death. If these mutant channels were also expressed in limbic regions of the brain, alterations in channel function could have drastic effects on the brain’s signaling ability, possibly promoting seizure activity.

Latency-Associated Sense Transcripts Are Expressed during in Vitro Human Cytomegalovirus Productive Infection

Published studies have identified novel sense transcripts in latent human cytomegalovirus (HCMV) infection. These cytomegalovirus latency-associated transcripts (CLTs) have start sites upstream from the MIE gene productive start site (PSS). We evaluated the expression of the sense CLTs in an in vitro HCMV productive infection. Transcripts with initiation sites upstream from the PSS were detected in infected human fibroblasts using reverse transcription–polymerase chain reaction and 5′ rapid amplification of cDNA ends (RACE) analysis. DNA sequencing of 5′ RACE PCR products confirmed that the start sites were consistent with sense CLT expression. Furthermore, ribonuclease protection assay analysis showed that transcripts initiating at the latent start site-1 were most abundant at late times postinfection after transcription from the PSS had decreased. In addition, transcription consistent with sense CLT expression was identified in HCMV-infected dTHP-1 cells, monocyte-derived macrophages, and endothelial cells, as well as in clinical isolate-infected human fibroblasts. These findings clearly demonstrate the expression of sense CLTs during in vitro HCMV infection.

Developmental and regional expression patterns of Type I Nitric Oxide Synthase mRNA and protein in fetal sheep brain during the last third of gestation

Type I NOS (nNOS) catalytic activity represents the activity of full-size protein and truncated protein variants originated from many different spliced mRNA variants. Splice mRNA variants are thought to be important in determining the differential organ and subcellular expression of Type I NOS. The present study was directed to increase our understanding of the developmental regulation of Type I NOS in fetal brain. In four discrete areas of the fetal brain, we measured steady-state mRNA levels and catalytic activity and protein mass in the soluble and particulate fractions. Under general anesthesia, we collected sensory-motor cortex, striatum, hippocampus and cerebellum from sheep fetuses at 105, 115, 125 and 135 days gestation (32 fetuses). NOS protein in the soluble and particulate fractions was characterized using Western blot (molecular weight) and arginine to citrulline conversion (enzymatic activity). At the mRNA level, steady state levels were determined using probes directed against exon 2 and exon 21/22 by ribonuclease protection assay (RPA). Our data show that NOS catalytic activity is regulated in a region, subcellular and temporal manner. NOS activity was higher in the soluble fraction in all brain regions and significantly higher levels were found in the soluble fraction of striatum and particulate fraction of hippocampus ( P<0.05 by ANOVA). Western blot analysis revealed three distinct molecular weight bands for Type I NOS (155, 144 and 136 kDa). The bands were present in all brain regions and in both cellular compartments with the 155 kDa band being the most abundant molecular form. Truncated protein variants accounted for 25% and 15% of total Type I NOS protein in the soluble fraction and particulate fraction respectively. RPA analysis showed a differential regulation of mRNA variants with exon 2 frame deletions in striatum and hippocampus. A coordinated increase with advancing gestational age of catalytic activity, the full-length protein, the protein variants and steady state mRNA levels was observed in cortex and striatum as demonstrated by higher levels at 125 and 135 days gestation ( P<0.05 by ANOVA). NOS enzymatic activity was Ca 2+ and calmodulin dependent. However, in the particulate fraction 20% of the NOS activity was resistant to calmodulin inhibition. In summary, fetal brain Type I NOS mRNA variants are differentially regulated according to brain regions. Our data suggests that exon 2 deleted mRNA variants have low translation efficiency as indicated by the lack of parallel expression of truncated Type I NOS protein variants.

Multiprobe RNase protection assay analysis of mRNA levels for the Escherichia coli oxidative DNA glycosylase genes under conditions of oxidative stress.

Escherichia coli formamidopyrimidine DNA glycosylase (Fpg), MutY DNA glycosylase, endonuclease VIII, and endonuclease III are oxidative base excision repair DNA glycosylases that remove oxidized bases from DNA, or an incorrect base paired with an oxidized base in the case of MutY. Since genes encoding other base excision repair proteins have been shown to be part of adaptive responses in E. coli, we wanted to determine whether the oxidative DNA glycosylase genes are induced in response to conditions that cause the type of damage their encoded proteins remove. The genes fpg, mutY, nei, and nth encode Fpg, MutY, endonuclease VIII, and endonuclease III, respectively. Multiprobe RNase protection assays were used to examine the transcript levels of these genes under conditions that induce the SoxRS, OxyR, and SOS regulons after a shift from anaerobic to aerobic growth and at different stages along the growth curve. Transcript levels for all four genes decreased as cells progressed from log-phase growth to stationary phase and increased after cells were shifted from anaerobic to aerobic growth. None of the genes were induced by hydrogen peroxide, paraquat, X rays, or conditions that induce the SOS response.

A noninvasive method for quantifying and distinguishing inflammatory skin reactions

Background: The ribonuclease protection assay (RPA) represents a technology that allows detection of small amounts of intact RNA. Recent progress in understanding cytokine networks in the skin suggests that measurements of cytokine mRNA levels could provide a method to distinguish various reactions such as irritant contact dermatitis and allergic contact dermatitis that can occur in the skin. Objective: We attempted to differentiate and quantitate irritant and immunologic skin reactions by measuring mRNA levels. Methods: We have used the technique of tape stripping human skin to remove superficial cell layers and have extracted RNA from these skin samples. This RNA was used for RPA analysis. Results: By means of RPA analysis, we have demonstrated distinct cytokine profiles that appear to discriminate, for example, irritant from immunologic skin reactions. Conclusion: We have shown that multiple cytokine mRNA levels can be defined in these RNA samples obtained from the skin. This approach assesses not only the cytokine gene profiles, but at the same time may quantify the severity of common irritant versus allergic skin reactions. (J Am Acad Dermatol 1999;41:687-92.)

Alternative splicing of synaptotagmins involving transmembrane exon skipping

The synaptotagmin gene family currently includes 12 members. Analysis of the three known genomic synaptotagmin sequences reveals conserved exon-intron patterns which delineate the synaptotagmin structural domains. We used expressed sequence tag, reverse transcription PCR and RNAse protection assay analysis of synaptotagmin messenger RNAs to demonstrate the occurrence of alternative splicing events involving a number of exons. Exon-skipped messages where transmembrane sequences have been removed or altered were found to be abundantly expressed by synaptotagmins 1, 4, 6 and 7. Although the expression of most synaptotagmins predominates in neural tissue, we find that by contrast, synaptotagmin 6 is more abundant in thymus.

Loss of expression of the candidate tumor suppressor gene ZAC in breast cancer cell lines and primary tumors.

Loss of chromosome 6q21-qter is the second most frequent loss of chromosomal material in sporadic breast neoplasms suggesting the presence of at least one tumor suppressor gene on 6q. We recently isolated a cDNA encoding a new zinc finger protein which we named ZAC according to its functional properties, namely induction of apoptosis and control of cell cycle progression. ZAC is expressed in normal mammary gland and maps to 6q24-q25, a recognized breast cancer hot spot on 6q. In the present report, we investigated the possible inactivation of ZAC in breast cancer cell lines and primary tumors. We detected no mutation in ZAC coding region in a panel of 45 breast tumors with allelic imbalance of 6q24-q25. However, a survey of eight breast cancer cell lines showed a deeply reduced (three cell lines) or complete loss of (five cell lines) ZAC expression. Treatment of three of these cell lines with the methylation-interfering agent 5-azacytidine induced ZAC re-expression. In addition, Northern blot and RNase protection assay analysis of ZAC expression in 23 unselected primary breast tumors showed a reduced expression in several samples. Together with its functional properties and chromosomal localization, these findings substantiate ZAC as a good candidate for the tumor suppressor gene on 6q24-q25.