Devising negative pressure within intercuff space reduces microaspiration.

BACKGROUND: Microaspiration past the tracheal tube cuffs causes ventilator-associated pneumonia. The objective of the current study was to evaluate whether creating negative pressure between the tracheal double cuffs could block the fluid passage past the tracheal tube cuffs. METHODS: A new negative pressure system was devised between the double cuffs through a suction hole in the intercuff space. Blue-dyed water was instilled above the cuff at negative suction pressures of - 54, - 68, - 82, - 95, - 109, - 122, and - 136 cmH2O, and the volume leaked was measured in an underlying water trap after 10 min. Leakage tests were also performed during positive pressure ventilation, and using higher-viscosity materials. The actual negative pressures delivered at the hole of double cuffs were obtained by placing microcatheter tip between the intercuff space and the artificial trachea. RESULTS: No leakage occurred past the double cuff at - 136 cmH2O suction pressure at all tracheal tube cuff pressures. The volume leaked decreased significantly as suction pressure increased. When connected to a mechanical ventilator, no leakage was found at - 54 cmH2suction pressure. Volume of the higher-viscosity materials (dynamic viscosity of 63-108 cP and 370-430 cP) leaked was small compared to that of normal saline (0.9-1.1 cP). The pressures measured in the intercuff space corresponded to 3.8-5.9% of those applied. CONCLUSIONS: A new prototype double cuff with negative pressure in the intercuff space completely prevented water leakage. The negative pressure transmitted to the tracheal inner wall was a small percentage of that applied.

Improved Blood Suppression of Motion-Sensitized Driven Equilibrium in High-Resolution Whole-Brain Vessel Wall Imaging: Comparison of Contrast-Enhanced 3D T1-Weighted FSE with Motion-Sensitized Driven Equilibrium and Delay Alternating with Nutation for Tailored Excitation.

BACKGROUND AND PURPOSE: High-resolution vessel wall MR imaging is prone to slow-flow artifacts, particularly when gadolinium shortens the T1 relaxation time of blood. This study aimed to determine the optimal preparation pulses for contrast-enhanced high-resolution vessel wall MR imaging. MATERIALS AND METHODS: Fifty patients who underwent both motion-sensitized driven equilibrium and delay alternating with nutation for tailored excitation (DANTE) preparation pulses with contrast-enhanced 3D-T1-FSE were retrospectively included. Qualitative analysis was performed using a 4-grade visual scoring system for black-blood performance in the small-sized intracranial vessels, overall image quality, severity of artifacts, and the degree of blood suppression in all cortical veins as well as transverse sinuses. Quantitative analysis of the M1 segment of the MCA was also performed. RESULTS: The qualitative analysis revealed that motion-sensitized driven equilibrium demonstrated a significantly higher black-blood score than DANTE in contrast-enhanced 3D-T1-FSE of the A3 segment (3.90 versus 3.58, P < .001); M3 (3.72 versus 3.26, P = .004); P2 to P3 (3.86 versus 3.64, P = .017); the internal cerebral vein (3.72 versus 2.32, P < .001); and overall cortical veins (3.30 versus 2.74, P < .001); and transverse sinuses (2.82 versus 2.38, P < .001). SNRlumen, contrast-to noise ratiowall-lumen, and SNRwall in the M1 vessel were not significantly different between the 2 preparation pulses (all, P > .05). CONCLUSIONS: Motion-sensitized driven equilibrium demonstrated improved blood suppression on contrast-enhanced 3D-T1-FSE in the small intracranial arteries and veins compared with DANTE. Motion-sensitized driven equilibrium is a useful preparation pulse for high-resolution vessel wall MR imaging to decrease venous contamination and suppress slow-flow artifacts when using contrast enhancement.

In-situ synchrotron radiation photoemission spectroscopy study of the initial atomic layer deposition of Al2O3 film on Si(001) substrate.

In-situ synchrotron radiation photoemission spectroscopy and X-ray photoemission spectroscopy have been used to investigate the initial stages of Al2O3 growth on a Si(001) substrate by atomic layer deposition (ALD). The core level spectra of Si 2p, O 1s, and Al 2p as well as the valence band spectra were measured at every half reaction in the trimethylaluminum (TMA)-H2O ALD process. The line shape changes and binding energy shifts of the core level spectra reveal that Al2O3 is predominantly formed with a small amount of Si oxide in the initial stages without the formation of Al silicate. All core level spectra were alternately shifted toward higher and lower binding energies sides at every half ALD reaction. This can be explained by the band bending effect induced by different chemical species on the surface during the TMA-H2O ALD reaction. The valence band spectra showed that four cycles of ALD reactions were necessary to complete the electronic structure of the Al2O3 film with a valence band offset of 3.73 eV.

Antiproliferative role of dopamine: loss of D2 receptors causes hormonal dysfunction and pituitary hyperplasia.

The function of dopamine (DA) in the nervous system is paralleled by its neuroendocrine control of pituitary gland functions. Here, we document the neuroendocrine function of dopamine by studying the pituitary gland of mice lacking DA D2 receptors (D2R). These mice present a striking, progressive increase in lactotroph number, which ultimately leads to tumors in aged animals. Females develop tumors much earlier than males. An estrogen-mediated lactotroph proliferation cannot account for this sexual dimorphism, since D2R-null females are hypoestrogenic and, thus, have estrogen levels similar to males. In contrast, prolactin levels are six times higher in females than in males. We show that active prolactin receptors are present in the pituitary and their expression increases in concomitance with tumor expansion. These results point to prolactin as an autocrine proliferative factor in the pituitary gland. Additionally, they demonstrate an antiproliferative function for DA regulated through D2 receptor activation.

Effect of Hydroxyethyl Starch on Acute Kidney Injury After Living Donor Hepatectomy.

BACKGROUND: Concerns about the adverse effects of hydroxyethyl starch (HES) on renal function have been raised in recent studies involving critically ill patients. We aimed to evaluate the effect of HES on acute kidney injury (AKI) after living donor right hepatectomy. METHODS: We performed a 1:3 propensity score matching analysis of the medical records of 1641 living donors who underwent a donor right hepatectomy. They were divided into the control group (n = 60), who received only crystalloids, and the colloid group (n = 1,581), who received HES 130/0.4 and crystalloids. Postoperative AKI was determined by AKI Network (AKIN) and Risk, Injury, Failure, Loss, End-stage (RIFLE) criteria. RESULTS: A 1:3 propensity score matching was performed in 206 donors, 54 donors in the control group and 152 donors in the colloid group. For the matched colloid group, the median amount of 7.65 mL/kg (interquartile range, 6.64-9.20) of colloid and 58.19 mL/kg (interquartile range, 45.63-71.51) of crystalloid were given. The median amount of administered crystalloid in the control group was 56.48 mL/kg (interquartile range, 47.94-76.12) after propensity score matching. The incidences of AKI were not different between the control and colloid groups (P = .460 by AKIN criteria; P = .999 by RIFLE criteria). CONCLUSION: Intraoperative administration of HES may not be associated with AKI after living donor hepatectomy. This result can provide useful information on perioperative fluid management in living liver donors.

Minimization of MC1R selectivity by modification of the core structure of alpha-MSH-ND.

BACKGROUND: Melanocortin, through its distinct receptor subtypes, has many different effects. Receptor-selective ligands are required to reduce the undesirable effects of melanocortin. To investigate which conformation is preferable to a given melanocortin receptor subtype, a structural and functional analysis of the ligand-receptor interactions was made by studying the biological activity, the nuclear magnetic resonance structures, and the patterns of the ligand-receptor interaction for each receptor subtype by homology modeling analysis. RESULTS: Among the several analogues examined, [Gln(6)]alpha-melanocyte-stimulating hormone (MSH)-ND was found to have 10000 times less biological activity than alpha-MSH-ND for the MC1R, whereas, the potencies of both oligopeptides were comparable in both the melanocortin-3 receptor (MC3R) and MC4R. [Gln(6)]alpha-MSH-ND exhibited a type I' beta-turn that was similar to the type I beta-turn structure of alpha-MSH-ND. However, a remarkable structural difference was observed with respect to the side chain orientations of the sixth and seventh residues of [Gln(6)]alpha-MSH-ND, which were found to be mirror images of alpha-MSH-ND. By homology modeling analysis, the His(6) of alpha-MSH-ND was found to interact with the TM2 regions of all three receptors (Glu(94) of MC1R, Glu(94) of MC3R, and Glu(100) of MC4R), but [Gln(6)]alpha-MSH-ND did not. The phenyl ring of the D-Phe(7) residue of [Gln(6)]alpha-MSH-ND revealed an interaction with the TM3 regions of both the MC3R and MC4R (Ser(122) of MC3R or Ser(127) of MC4R). However, in the MC1R, these serine residues corresponded to Val(122), which contains two methyl groups that induce steric hindrance with D-Phe(7) of [Gln(6)]alpha-MSH-ND. This is a possible explanation for the biological activity of [Gln(6)]alpha-MSH-ND for the MC1R being significantly lower than that for either the MC3R or MC4R. CONCLUSIONS: Minimization of the MC1R selectivity whilst preserving its comparable potency for both the MC3R and MC4R could be achieved by modifying the D-Phe(7) orientation of alpha-MSH-ND, while maintaining the 'type I beta-turn'-like structure.

Molecular cloning and characterization of a protein tyrosine phosphatase enriched in testis, a putative murine homologue of human PTPMEG.

Protein tyrosine phosphorylation is regulated by protein tyrosine kinase and protein tyrosine phosphatase activities. These two counteracting proteins are implicated in cell growth and transformation. Using polymerase chain reaction with degenerate primers, we have identified a novel mouse protein tyrosine phosphatase (PTP). This cDNA contains a single open reading frame of the predicted 926 amino acids. Those predicted amino acids showed significant identity with human megakaryocyte protein-tyrosine phosphatase by 91% in nucleotide sequences and 94% in amino acid sequences. We have identified that expression of this PTP is highly enriched in the testis in mouse and human and has been termed here as a 'testis-enriched phosphatase' (TEP). Northern analysis detected two mRNA species of 3.7 and 3.2kb for this PTP in mouse testis and the expression of TEP is regulated during development. The recombinant phosphatase domain possesses protein tyrosine phosphatase activity when expressed in Escherichia coli. Immunohistochemical analysis of the cellular localization of TEP on mouse testis sections showed that this PTP is specifically expressed in spermatocytes and spermatids within seminiferous tubules, suggesting an important role in spermatogenesis.

Dopamine D2 receptors in signal transduction and behavior.

The dopamine D2 receptor belongs to the family of seven transmembrane domain G-protein-coupled receptors and is highly expressed in the central nervous system and the pituitary gland. The binding of dopamine to the D2 receptor is crucial for the regulation of diverse physiological functions, such as the control of locomotor activity and the synthesis of peptide hormones. Two alternatively spliced transcripts are generated from the D2 receptor gene and code for the D2L and D2S isoforms, which are 444 and 415 amino acids in length, respectively. These isoforms exhibit similar pharmacological characteristics and are expressed in the same cell types, with a ratio that normally favors expression of the longer isoform. The D2L isoform differs from D2S by the insertion of 29 amino acids in the putative third intracellular loop of the receptor. This loop is involved in the coupling of the receptor to different G proteins. Experiments have shown that the D2 isoforms have different G-protein-coupling affinities, suggesting that these receptors might serve different functions in vivo. Additionally, this difference in coupling affinity could be a mechanism to amplify the signal transduced by the binding of dopamine to D2 receptors. Important insights into D2 receptor function in vivo have been obtained by knocking out the D2 gene in mice. The Parkinsonian-like phenotype of D2-null mice demonstrates the importance of the D2 receptor for locomotor function.

Differential profiles of salivary proteins with affinity to Streptococcus mutans lipoteichoic acid in caries-free and caries-positive human subjects.

Streptococcus mutans is a representative oral pathogen that causes dental caries and pulpal inflammation. Its lipoteichoic acid (Sm.LTA) is known to be an important cell-wall virulence factor involved in bacterial adhesion and induction of inflammation. Since Sm.LTA-binding proteins (Sm.LTA-BPs) might play an important role in pathogenesis and host immunity, we identified the Sm.LTA-BPs in the saliva of caries-free and caries-positive human subjects using Sm.LTA-conjugated beads and LTQ-Orbitrap hybrid Fourier transform mass spectrometry. Sm.LTA was conjugated to N-hydroxysuccinimidyl-Sepharose(®) 4 Fast Flow beads (Sm.LTA-beads). Sm.LTA retained its biological properties during conjugation, as determined by the expression of nitric oxide and interferon-γ-inducible protein 10 in a murine macrophage cell line and activation of Toll-like receptor 2 (TLR2) in CHO/CD14/TLR2 cells. Sm.LTA-BPs were isolated from pooled saliva prepared from 10 caries-free or caries-positive human subjects each, electrophoresed to see their differential expression in each group, and further identified by high-resolution mass spectrometry. A total of 8 and 12 Sm.LTA-BPs were identified with statistical significance in the pooled saliva from the caries-free and caries-positive human subjects, respectively. Unique Sm.LTA-BPs found in caries-free saliva included histone H4, profilin-1 and neutrophil defensin-1, and those in caries-positive saliva included cystatin-C, cystatin-SN, cystatin-S, cystatin-D, lysozyme C, calmodulin-like protein 3 and ß-actin. The Sm.LTA-BPs found in both groups were hemoglobin subunits α and ß, prolactin-inducible protein, protein S100-A9, and SPLUNC2. Collectively, we identified Sm.LTA-BPs in the saliva of caries-free and caries-positive subjects, which exhibit differential protein profiles.

Ultrasound-guided oblique subcostal transversus abdominis plane block for analgesia after laparoscopic cholecystectomy: a randomized, controlled, observer-blinded study.

BACKGROUND: The oblique subcostal transversus abdominis plane (OSTAP) block has been described as an effective analgesic method for upper abdominal surgery. We evaluated the postoperative analgesia of the OSTAP block and compared it with that of the transversus abdominis plane (TAP) block in patients undergoing laparoscopic cholecystectomy (LC). METHODS: Patients scheduled for elective LC were randomized to receive either standard care or to undergo an OSTAP or TAP block. All blocks were performed with ultrasound guidance, and 20 mL of 0.375% ropivacaine was injected bilaterally. The postoperative pain score and consumption of rescue analgesics were evaluated. RESULTS: The OSTAP block reduced postoperative verbal numerical rating scale pain scores (median [Interquartile range, IQR]) compared to standard care at 10 min (2 [1-4] vs. 7 [5-8]), 30 min (2 [1-5] vs. 6 [5-8]), 1 h (2 [1-3] vs. 5 [4-6]), and 3 h (2 [2-3] vs. 4 [3-5]). Pain scores were also lower in the OSTAP group than in the TAP group at 10 min (2 [1-4] vs. 4 [2-6]), 1 h (2 [1-3] vs. 3 [3-4]), 3 h (2 [2-3] vs. 3 [3-4]), 6 h (2 [2-3] vs. 3 [3-5]), and 24 h (1 [1-2] vs. 2 [2-3]) postoperatively. The total fentanyl requirement was reduced in the OSTAP group (p = 0.005). CONCLUSION: The OSTAP block can provide better analgesia than the TAP block or standard care during the postoperative 24 h period in patients undergoing LC.

Alpha-amylase is a human salivary protein with affinity to lipopolysaccharide of Aggregatibacter actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans lipopolysaccharide (Aa.LPS) is a major virulence factor associated with aggressive periodontitis. Although the recognition of Aa.LPS is potentially initiated by salivary proteins in the oral cavity, Aa.LPS-binding proteins (Aa.LPS-BPs) in saliva are poorly characterized. The purpose of this study was to capture and identify Aa.LPS-BPs in human saliva using a LTQ-Orbitrap hybrid Fourier transform mass spectrometry. Aa.LPS conjugated onto N-hydroxysuccinimidyl-Sepharose(®) 4 Fast Flow beads (Aa.LPS-beads) activated Toll-like receptor 4 and produced nitric oxide and Interferon gamma-inducible protein-10, implying that the conjugation process did not alter the biological properties of Aa.LPS. Aa.LPS-BPs were subsequently isolated from the nine human saliva samples from healthy individuals with the Aa.LPS-beads followed by identification with the mass spectrometry. Aa.LPS-BPs include α-amylase, serum albumin, cystatin, lysozyme C, submaxillary gland androgen-regulated protein 3B, immunoglobulin subunits, polymeric immunoglobulin receptor, deleted in malignant brain tumors 1, prolactin-inducible protein, lipocalin-1, and basic salivary proline-rich protein 2. Specific binding was validated using a pull-down assay with α-amylase which was captured at the highest frequency. Alpha-amylase demonstrated to interfere with the adherence and biofilm formation of A. actinomycetemcomitans. Even heat-inactivated α-amylase showed the interference to the same extent. Conclusively, we identified unique Aa.LPS-BPs that provide useful information to understand bacterial pathogenesis and host innate immunity in the oral cavity.

ZNF313 is a novel cell cycle activator with an E3 ligase activity inhibiting cellular senescence by destabilizing p21(WAF1.).

ZNF313 encoding a zinc-binding protein is located at chromosome 20q13.13, which exhibits a frequent genomic amplification in multiple human cancers. However, the biological function of ZNF313 remains largely undefined. Here we report that ZNF313 is an ubiquitin E3 ligase that has a critical role in the regulation of cell cycle progression, differentiation and senescence. In this study, ZNF313 is initially identified as a XIAP-associated factor 1 (XAF1)-interacting protein, which upregulates the stability and proapoptotic effect of XAF1. Intriguingly, we found that ZNF313 activates cell cycle progression and suppresses cellular senescence through the RING domain-mediated degradation of p21(WAF1). ZNF313 ubiquitinates p21(WAF1) and also destabilizes p27(KIP1) and p57(KIP2), three members of the CDK-interacting protein (CIP)/kinase inhibitor protein (KIP) family of cyclin-dependent kinase inhibitors, whereas it does not affect the stability of the inhibitor of CDK (INK4) family members, such as p16(INK4A) and p15(INK4B). ZNF313 expression is tightly controlled during the cell cycle and its elevation at the late G1 phase is crucial for the G1-to-S phase transition. ZNF313 is induced by mitogenic growth factors and its blockade profoundly delays cell cycle progression and accelerates p21(WAF1)-mediated senescence. Both replicative and stress-induced senescence are accompanied with ZNF313 reduction. ZNF313 is downregulated during cellular differentiation process in vitro and in vivo, while it is commonly upregulated in many types of cancer cells. ZNF313 shows both the nuclear and cytoplasmic localization in epithelial cells of normal tissues, but exhibits an intense cytoplasmic distribution in carcinoma cells of tumor tissues. Collectively, ZNF313 is a novel E3 ligase for p21(WAF1), whose alteration might be implicated in the pathogenesis of several human diseases, including cancers.

Emergency rescue primary stenting for atherosclerotic basilar artery occlusion with acute thrombosis. A case report.

SUMMARY: We demonstrate endovascular stent deployment for the treatment of atherosclerotic basilar artery occlusion with acute thrombosis. Application of a microstent without previous balloon dilatation resulted in vessel reopening and good clinical improvement. Emergency primary stent application can be technically feasible and improve the outcome in acute basilar artery occlusion and clinical status.

Effect of light-enhanced bleaching on in vitro surface and intrapulpal temperature rise.

PURPOSE: This study investigated the effect of the presence, absence, and aging of a heat-enhancing compound (colorant) added to bleaching gel on the temperature rise of the gel itself, as well as the temperature rise within the pulp chamber, when a tooth was exposed to a variety of light-curing units in vitro. MATERIALS AND METHODS: An extracted human upper central incisor was fitted with thermocouples placed in the pulp chamber as well as on the facial tooth surface. A temperature-controlled simulated intrapulpal fluid flow was provided to the tooth, and bleaching agent (Opalesence XTRA, Ultradent) containing heat-enhancing colorant, aged colorant, or no colorant was applied to the facial surface. The tooth and light-curing unit were placed in a thermostatically controlled oven at 37 degrees C, and real-time gel and intrapulpal temperature values were recorded digitally. Light-curing units used were a plasma arc light (PAC) (PowerPac, ADT), a conventional quartz tungsten halogen source (QTH) (Optilux 501, Demetron/Kerr), the QTH light used in high-power (bleaching) mode, and an argon ion laser (AccuCure 3000, LaserMed). An exposure scenario simulating light-enhanced bleaching of 10 upper teeth was developed. Temperature rise over the pre-exposure, baseline value associated with the last light exposure in the bleaching sequence was calculated for each curing and bleaching combination. Five replications for each test condition were made. Temperature rise values were compared using analysis of variance (ANOVA) at a preset alpha of 0.05. RESULTS: When fresh colorant-containing bleach was used, the PAC light increased bleach temperature 39.3 degrees C above baseline. With no added colorant, temperature rise was 37.1 degrees C. The QTH light in bleach mode resulted in gel temperature 24.8 degrees C above baseline, whereas the temperature increase was only 11.5 degrees C when no colorant was used. Conventional QTH light use increased fresh bleach temperature by 17.7 degrees C, whereas an increase of only 11.1 degrees C was measured without colorant. The argon ion laser produced equivalent temperature rise regardless of the presence or freshness of the colorant, approximately 9.4 degrees C. Intrapulpal temperatures were all significantly lower than those recorded in the bleaching gel and ranged from 5 degrees to 8 degrees C. As a rule, the presence of fresh heat-enhancing colorant in the bleaching gel resulted in a significant intrapulpal temperature increase (approximately 1 degrees C) over that reached using other lights. The PAC and the QTH light used in bleach mode induced greater intrapulpal temperature rise than the laser. CLINICAL SIGNIFICANCE: Freshness of bleaching agent incorporating light-activated, heat-enhancing colorant influences temperature rise of bleaching gel and also may increase intrapulpal temperature values. Use of intense lights does elevate bleach temperature and also results in increased intrapulpal temperature that may further impact on patient sensitivity and pulpal health resulting from this treatment.

Cobalt-induced occupational asthma associated with systemic illness.

We report a case of occupational asthma caused by cobalt associated with systemic symptoms. He was a non-atopic, ex-smoker and had worked in a glassware factory for 14 months. A skin prick test with CoSO4 up to 100 mg/ml showed a negative result. A bronchoprovocation test with CoSO4 demonstrated an isolated asthmatic response with systemic symptoms such as fever, arthralgia and myalgia. Although an initial methacholine bronchial challenge test showed a negative result, the following methacholine bronchial challenge test which was done 24 hours after the challenge testing demonstrated an increased airway hyperresponsiveness at 2.5 mg/ml which recovered 7 days later. An intradermal skin test with 10 mg/ml and 100 mg/ml CoSO4 solution demonstrated positive responses respectively(13 x 12/40 x 32, 20 x 15/40 x 37 , histamine 16 x 14/64 x 50). A patch test including cobalt showed a negative result. Bronchoalveolar lavage fluid after the cobalt inhalation testing and other laboratory findings showed no evidence of hypersensitivity pneumonitis. These results suggested that cobalt could induce occupational asthma with systemic illness in an exposed worker.

DNA methylation patterns of the rat gamma-glutamyl transpeptidase gene in embryonic, adult and neoplastic liver.

The methylation status of the rat gamma-glutamyl transpeptidase (GGT) gene was investigated during liver development and hepatocarcinogenesis. The analysis with the restriction enzymes MspI/HpaII revealed that, during ontogeny, there is a progressive methylation of the GGT gene that coincides with a progressive decrease in GGT activity. Thus, there is an inverse correlation between methylation and expression of the GGT gene, suggesting a role for DNA methylation in the regulation of the gene during normal differentiation. The methylation patterns of the GGT gene in liver tumours induced by aflatoxin B1 exhibit heterogeneity. Nevertheless, a band of 5.7 kb was observed in all the DNA samples from aflatoxin B1-induced tumours which was not present in control liver DNA. The specificity of the DNA methylation changes was assessed using nafenopin, which induces hepatic tumours without elevation of GGT activity. We conclude that, during hepatocarcinogenesis, there is a modification of the DNA methylation pattern of the GGT gene, but there is no simple correlation with GGT activity. In no case was the GGT gene methylation in hepatocarcinogenesis found to be equivalent to the pattern observed in fetal liver. Thus if methylation is involved in the regulation of GGT gene transcription, the mechanisms must be different in fetal liver and hepatocarcinoma.

Repetitive 5-azacytidine treatments of Fao cells induce a stable and strong expression of gamma-glutamyl transpeptidase.

The role of DNA methylation in the expression of the rat gamma-glutamyl transpeptidase (GGT) gene was assessed in the Fao cell line using a hypomethylating agent, 5-azacytidine. Ten repetitive treatments of the cells, with 8 microM 5-azacytidine for 24 h, led to 13- and 80-fold increases, respectively, in GGT activity and in GGT mRNA level. The DNA methylation patterns generated by the isoschizomeric restriction enzymes Hpa II and Msp I indicated that the GGT gene, highly methylated in Fao cells, became strongly demethylated after 5-azacytidine treatments. Thus, DNA demethylation increases the expression of the GGT gene. 5-Azacytidine treatments also increased, but to a lesser extent, mRNAs level for actin, albumin, mitochondrial aspartate aminotransferase, aldolase B mRNAs (12- to 16-fold) as well as for tubulin, gluthathione transferase, and tyrosine aminotransferase mRNAs (2- to 5-fold). The GGT gene expression was further studied in B4 cells, cloned from the demethylated Fao cell population. This clone B4 exhibited a stable and strong GGT activity and a highly demethylated GGT gene. Among the three GGT mRNA I, II, or III, transcribed from three different promoters of the single rat GGT gene, only mRNA III was detected in Fao cells and was increased in clone B4, indicating that the demethylation acts on the promoter for mRNA III. The analysis of the differentiation state of B4 cells, as compared to Fao cells, showed a loss of the regulation of GGT and aspartate aminotransferase genes by dexamethasone, as well as a loss of the gluconeogenic pathway. Interestingly, B4 cells have retained many other specific functions of hepatic differentiation and have acquired alpha-fetoprotein expression; thus this clone exhibits the characteristics of a hepatic fetal phenotype.

Recurrent herpes zoster myelitis.

Recurrent zoster myelitis is quite rare. We present a previously healthy 27-year-old woman who developed recurrent attacks of myelopathy shortly after the characteristic skin rashes of herpes zoster. Magnetic resonance imaging studies demonstrated each lesion in the spinal cord at the same segments as the skin lesions. She had two attacks at opposite sites at the same spinal cord level and complete recovery after being treated with intravenous acyclovir. We suspect that direct invasion of varicella zoster virus was the cause of recurrent myelopathy in our patient.

G protein-mediated mitogen-activated protein kinase activation by two dopamine D2 receptors.

Two isoforms of dopamine D2 receptor, D2L (long) and D2S (short), differ by the insertion of 29 amino acids specific to D2L within the putative third intracellular loop of the receptor, which appears to be important in selectivity for G-protein coupling. We have generated D2L- and D2S-expressing Chinese hamster ovary (CHO) cells, and regulation of the mitogen-activated protein kinase (MAPK) pathway was examined in these cells. Both D2L and D2S mediated a rapid and transient activation of MAPK with dominant activation of p42-kDa MAPK. Pertussis toxin treatment completely abrogated stimulation of MAPK mediated by D2L and D2S, demonstrating that both receptors couple to pertussis toxin-sensitive G proteins in this signaling. Stimulation of MAPK mediated by both D2L and D2S receptor was markedly attenuated by coexpression of the C-terminus of beta-adrenergic receptor kinase (betaARKct), which selectively inhibits Gbetagamma-mediated signal transduction. Further analysis of D2L- and D2S-mediated MAPK activation demonstrated that D2L-mediated MAPK activation was not significantly affected by PKC depletion or partially affected by genistein. In contrast, D2S-mediated MAPK activation was potentially inhibited by PKC depletion and genistein was capable of completely inhibiting D2S-mediated MAPK activation. Together, these results suggest that D2L- and D2S-mediated MAPK activation is predominantly Gbetagamma subunit-mediated signaling and that protein kinase C and tyrosine phosphorylations are involved in these signaling pathways.