COVID-19 lockdown, personal protective equipment, hyper-hygiene and allergy.

Summary: At the beginning of SARS-CoV 2 pandemic, in the absence of "targeted" therapies, the national health authorities have introduced some measures aimed at reducing the spread of infection in the community (lockdown, social distancing, personal protective equipment (PPE), personal hygiene and disinfection of living environments). All the containment measures have led to both positive and negative effects in patients with allergic diseases. We believe that further studies should be undertaken to investigate the possible correlations between SARS-CoV-2 infection and allergy, from a broader perspective. In particular, the risk factors for the development of undesirable effects should be investigated, especially in healthcare professionals forced to use PPE and sanitizing agents for a long time. However, since the COVID-19 pandemic probably will not be short-lived, the use of such protective aids will necessarily be widespread even in the general population. Therefore, further studies on the materials used for the production of PPE and sanitizing agents would be necessary to reduce their sensitizing and, in some cases, toxic potential.

New insights into vitamin D regulation: is there a role for alkaline phosphatase?

PURPOSE: The diagnosis of vitamin D deficiency is based on the determination of total plasma 25-hydroxyvitamin D (25-OHD) concentrations, but the regulation of vitamin D 25-hydroxylation is not a major consideration and very little information is available on this activity. To check what factors could interfere with the activity of vitamin D-25-hydroxylase and thus alter the 25-OHD concentrations, we looked for potential correlations between 25-OHD and results of liver function tests in healthy adults. METHODS: This single-centre study was retrospective and consisted of evaluating the correlations between 25-OHD and the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), and bone alkaline phosphatase (BALP) in 349 healthy subjects aged from 18 to 65 years. In particular, in Group 1 (n = 119), we looked for correlations between 25OHD and all liver function tests and in Group 2 (n = 230) the correlation between 25OHD and BALP. RESULTS: In Group 1, we found no correlation between 25OHD and AST (r = - 0.03; p = 0.8), ALT (r = - 0.02; p = 0.91), GGT (r = - 0.08; p = 0.68), direct bilirubin (r = - 0.02; p = 0.89), indirect bilirubin (r = - 0.24; p = 0.21), and total bilirubin (r = - 0.24; p = 0.21) but one between 25OHD and ALP (r = - 0.2; p = 0.007); in Group 2, we found a significant negative correlation between 25-OHD and BALP (r = - 0.2; p = 0.0008). CONCLUSIONS: The correlations that we found suggest that ALP and BALP might be involved in the regulation of vitamin D-25-hydroxylase activity, but further studies are mandatory to confirm our assumptions.

The presence of smear layer affects the antimicrobial action of root canal sealers.

AIM: To assess the chemical and microstructural characteristics of dentine after the use of two irrigation protocols and correlate this with the antimicrobial properties of hydraulic calcium silicate cement (HCSC) sealers and changes to the dentine structure/chemistry after sealer placement. METHODOLOGY: Two irrigation protocols - Protocol A using 2% NaOCl used 5 mL/5 min and Protocol B with 2% NaOCl (5 mL/5 min) followed by 17% EDTA (5 mL/3 min) - were used to prepare dentine. The chemical and microstructural changes following irrigation were assessed by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and Fourier transform infrared (FT-IR) spectroscopy (n = 5) on dentine obtained from the mid-root and coronal parts of extracted human teeth. Four sealers (AH Plus, BioRoot, MTA Fillapex, TotalFill) were characterized by SEM/EDS (n = 3). The ability of the sealers to eradicate intratubular Enterococcus faecalis biofilms was assessed by live/dead dye and confocal laser scanning microscopy to measure the percentage of living cells. The effect of combined irrigation and root filling on dentine was assessed by SEM and EDS analysis (n = 5). Statistical analysis was undertaken using one-way anova and a number of post hoc tests to detect intergroup differences. The F-test was used for comparison of variances in the microbiology testing. RESULTS: The use of NaOCl alone left the smear layer intact, with traces of chlorine remaining on dentine. The use of BioRoot sealer restored the calcium levels of dentine which had been depleted by the irrigation with EDTA. BioRoot exhibited antimicrobial properties against intratubular bacteria even in the presence of smear layer (Protocol A). Smear layer removal improved the bactericidal effect of all sealers and Ca2+ leaching. The use of a chelating agent was important for the intratubular sealer penetration for AH Plus but not the other sealers. CONCLUSION: The removal of smear layer was necessary for penetration of AH Plus into the dentinal tubules. BioRoot was a more effective sealer in reducing the bacterial load in the dentinal tubules than the other materials tested and the presence of smear layer did not affect its activity.

Evaluation of endocrine therapy and patients preferences in early breast cancer: results of Elena study.

PURPOSE: The development of the adjuvant therapy requires that clinicians and patients should discuss the magnitude of benefit of treatment for individual patient, estimating the pros and cons and the personal preferences. The aim of the present study was to determine the preferences of women treated with adjuvant hormonal therapy (HT) for breast cancer. METHODS: The analyses were conducted into three different groups of early breast cancer patients to evaluate the survival benefit needed to make treatment worthwhile before starting HT (A), after a few months from the beginning (B) and after several years of HT (C). The questionnaires, showing hypothetical scenarios based on potential survival times and rates without HT, were used to determine the lowest gains women judged necessary to make the treatment worthwhile. RESULTS: A total of 452 patients were included in the study: 149 in group A, 150 in group B and 153 in group C. In group C, 65% of patients were receiving HT with aromatase inhibitors (with or without a LHRH analogue). In the groups A, B, C 8%, 20% and 26%, respectively, received adjuvant chemotherapy. Overall, 355 women (79%) had children. The responses were quite similar between the three groups. A median gain of 10 years was judged necessary to make adjuvant HT worthwhile based on the hypothetical scenario of untreated mean survival time of 5 and 15 years. Median gain of 20% more women surviving was judged necessary to make adjuvant HT worthwhile based on an untreated 5-year survival rate expectation of 60%. Cognitive dysfunction was considered the side effect least compatible with the continuation of treatment in all three groups. CONCLUSIONS: This is a large study of patient preferences on HT. Compared with other studies with similar design, the patients included in the present study required larger benefits to make adjuvant therapy worthwhile.

Bimodal effect of D-aspartate on brain aging processes: insights from animal models.

Nowadays it is widely recognized that D-amino acids are present in bacteria as well as in eukaryotes, including mammals. In particular, free D-serine and D-aspartate are found in the brain of mammals. Notably, D-aspartate occurs at substantial levels in the embryo brain to then consistently decrease at post-natal phases. Temporal regulation of D-aspartate content depends on the post-natal onset of D-aspartate oxidase expression, the only known enzyme able to catabolize this D-amino acid. Pharmacological evidence indicates that D-aspartate binds and activates NMDA receptors (NMDARs). To decipher the physiological function of D-aspartate in mammals, in the last years, genetic and pharmacological mouse models with abnormally higher levels of this D-amino acid have been generated. Overall, these animal models have pointed out a significant neuromodulatory role for D-aspartate in the regulation of NMDAR-dependent functions. Indeed, increased content of D-aspartate are able to increase hippocampal NMDAR-dependent long-term potentiation (LTP) and spatial memory of adult mice. However, if exposure to elevated levels of D-Asp lasts for the entire lifetime of mice, enhancement of synaptic plasticity turns into a dramatic worsening, thus triggering an acceleration of the NMDAR-dependent aging processes in the hippocampus. Nonetheless, administration of D-Asp to old mice can restore the physiological age-related decay of hippocampal NMDA-related LTP. Besides its effect on hippocampus-dependent processes in mouse models, different points of evidence are indicating, today, a potential role for D-Asp in neurologic and psychiatric disorders associated with aberrant signalling of NMDARs.

Warm fish with cold hearts: thermal plasticity of excitation-contraction coupling in bluefin tuna.

Bluefin tuna have a unique physiology. Elevated metabolic rates coupled with heat exchangers enable bluefin tunas to conserve heat in their locomotory muscle, viscera, eyes and brain, yet their hearts operate at ambient water temperature. This arrangement of a warm fish with a cold heart is unique among vertebrates and can result in a reduction in cardiac function in the cold despite the elevated metabolic demands of endothermic tissues. In this study, we used laser scanning confocal microscopy and electron microscopy to investigate how acute and chronic temperature change affects tuna cardiac function. We examined the temporal and spatial properties of the intracellular Ca2+ transient (Δ[Ca2+]i) in Pacific bluefin tuna (Thunnus orientalis) ventricular myocytes at the acclimation temperatures of 14°C and 24°C and at a common test temperature of 19°C. Acute (less than 5 min) warming and cooling accelerated and slowed the kinetics of Δ[Ca2+]i, indicating that temperature change limits cardiac myocyte performance. Importantly, we show that thermal acclimation offered partial compensation for these direct effects of temperature. Prolonged cold exposure (more than four weeks) increased the amplitude and kinetics of Δ[Ca2+]i by increasing intracellular Ca2+ cycling through the sarcoplasmic reticulum (SR). These functional findings are supported by electron microscopy, which revealed a greater volume fraction of ventricular SR in cold-acclimated tuna myocytes. The results indicate that SR function is crucial to the performance of the bluefin tuna heart in the cold. We suggest that SR Ca2+ cycling is the malleable unit of cellular Ca2+ flux, offering a mechanism for thermal plasticity in fish hearts. These findings have implications beyond endothermic fish and may help to delineate the key steps required to protect vertebrate cardiac function in the cold.

Physiological, biochemical and molecular responses to water stress and rehydration in Mediterranean adapted tomato landraces.

Mediterranean tomato landraces adapted to arid environments represent an option to counteract drought, and to address the complexity of responses to water deficit and recovery, which is a crucial component of plant adaptation mechanisms. We investigated physiological, biochemical and molecular responses of two Mediterranean tomato landraces, 'Locale di Salina' (Lc) and 'Pizzutello di Sciacca' (Pz) under two dehydration periods and intermediate rehydration in greenhouse pot experiments. Relationship between CO2 assimilation (A) and stomatal conductance under severe water stress (gs  < 0.05 mol·m-2 ·s-1 ) indicated the occurrence of stomatal and non-stomatal limitations of photosynthesis. Gas exchange promptly recovered within 2-3 days of rehydration. ABA and gs showed a strict exponential relationship. Both leaf ABA and proline peaked under severe water stress. Lc showed higher accumulation of ABA and higher induction of the expression of both NCED and P5CS genes than Pz. Poly(ADP-ribose) polymerase increased during imposition of stress, mainly in Lc, and decreased under severe water stress. The two landraces hardly differed in their physiological performance. Under severe water stress, gs showed low sensitivity to ABA, which instead controlled stomatal closure under moderate water stress (gs  > 0.15 mol·m-2 ·s-1 ). The prompt recovery after rehydration of both landraces confirmed their drought-tolerant behaviour. Differences between the two landraces were instead observed at biochemical and molecular levels.

Carbon dioxide pneumoperitoneum prevents mortality from sepsis.

BACKGROUND: Carbon dioxide (CO2) pneumoperitoneum has been shown to attenuate the inflammatory response after laparoscopy. This study tested the hypothesis that abdominal insufflation with CO2 improves survival in an animal model of sepsis and investigated the associated mechanism. METHODS: The effect of CO2, helium, and air pneumoperitoneum on mortality was studied by inducing sepsis in 143 rats via intravenous injection of lipopolysaccharide (LPS). To test the protective effect of CO2 in the setting of a laparotomy, an additional 65 animals were subjected to CO2 pneumoperitoneum, helium pneumoperitoneum, or the control condition after laparotomy and intraperitoneal LPS injection. The mechanism of CO2 protection was investigated in another 84 animals. Statistical significance was determined via Kaplan-Meier analysis for survival and analysis of variance (ANOVA) for serum cytokines. RESULTS: Among rats with LPS-induced sepsis, CO2 pneumoperitoneum increased survival to 78%, as compared with using helium pneumoperitoneum (52%; p < 0.05), air pneumoperitoneum (55%; p = 0.09), anesthesia control (50%; p < 0.05), and LPS-only control (42%; p < 0.01). Carbon dioxide insufflation also significantly increased survival over the control condition (85% vs 25%; p < 0.05) among laparotomized septic animals, whereas helium insufflation did not (65% survival). Carbon dioxide insufflation increased plasma interleukin-10 (IL-10) levels by 35% compared with helium pneumoperitoneum (p < 0.05), and by 34% compared with anesthesia control (p < 0.05) 90 min after LPS stimulation. Carbon dioxide pneumoperitoneum resulted in a threefold reduction in tumor necrosis factor-alpha (TNF-alpha) compared with helium pneumoperitoneum (p < 0.05), and a sixfold reduction with anesthesia control (p < 0.001). CONCLUSION: Abdominal insufflation with CO2, but not helium or air, significantly reduces mortality among animals with LPS-induced sepsis. Furthermore, CO2 pneumoperitoneum rescues animals from abdominal sepsis after a laparotomy. Because IL-10 is known to downregulate TNF-alpha, the increase in IL-10 and the decrease in TNF-alpha found among the CO2-insufflated animals in our study provide evidence for a mechanism whereby CO2 pneumoperitoneum reduces mortality via IL-10-mediated downregulation of TNF-alpha.

Laparoscopic surgery and the parasympathetic nervous system.

BACKGROUND: Laparoscopic surgery preserves the immune system and has anti-inflammatory properties. CO2 pneumoperitoneum attenuates lipopolysaccharide (LPS)-induced cytokine production and increases survival. We tested the hypothesis that CO2 pneumoperitoneum mediates its immunomodulatory properties via stimulation of the cholinergic pathway. METHODS: In the first experiment, rats (n = 68) received atropine 1 mg/kg or saline injection 10 min prior to LPS injection and were randomization into four 30-min treatment subgroups: LPS only control, anesthesia control, CO2 pneumoperitoneum, and helium pneumoperitoneum. In a second experiment, rats (n = 40) received atropine 2 mg/kg or saline 10 min prior to randomization into the same four subgroups described previously. In a third experiment, rats (n = 96) received atropine 2 mg/kg or saline 10 min prior to randomization into eight 30-min treatment subgroups followed by LPS injection: LPS only control; anesthesia control; and CO2 or helium pneumoperitoneum at 4, 8, and 12 mmHg. In a fourth experiment, rats (n = 58) were subjected to bilateral subdiaphragmatic truncal vagotomy or sham operation. Two weeks postoperatively, animals were randomized into four 30-min treatment subgroups followed by LPS injection: LPS only control, anesthesia control, CO2 pneumoperitoneum, and helium pneumoperitoneum. Blood samples were collected from all animals 1.5 h after LPS injection, and cytokine levels were determined by enzyme-linked immunosorbent assay. RESULTS: Serum tumor necrosis factor-alpha (TNF-alpha) levels were consistently suppressed among the saline-CO2 pneumoperitoneum groups compared to saline-LPS only control groups (p < 0.05 for all four experiments). All chemically vagotomized animals had significantly reduced TNF-alpha levels compared to their saline-treated counterparts (p < 0.05 for all), except among the CO2 pneumoperitoneum-treated animals. Increasing insufflation pressure with helium eliminated differences (p < 0.05) in TNF-alpha production between saline- and atropine-treated groups but had no effect among CO2 pneumoperitoneum-treated animals. Finally, vagotomy (whether chemical or surgical) independently decreased LPS-stimulated TNF-alpha production in all four experiments. CONCLUSION: CO2 pneumoperitoneum modulates the immune system independent of the vagus nerve and the cholinergic pathway.

Mammalian liver contains an activity which mimics bacterial chloramphenicol acetyltransferase.

Rat liver extracts contain an activity which mimics Escherichia coli chloramphenicol acetyltransferase (CAT); the latter is commonly used to report transcriptional activation of chimeric genes transfected into cultured cells. Although the activities are indistinguishable by the standard thin-layer chromatography assay, alternate methods can discriminate between them. The rat CAT-like activity appears to be an integral membrane protein. It was observed in the microsomal fraction of both liver and kidney. Similarly CAT-like activities were detected in mouse, rabbit and pig liver. In addition, liver homogenates which contain the CAT-like activity also contain a heat-labile inhibitor of (authentic) bacterial CAT.

ADP-ribosylation reactions in Sulfolobus solfataricus, a thermoacidophilic archaeon.

An ADP-ribosylating system was detected in a crude homogenate from Sulfolobus solfataricus, a thermophilic archaeon, optimally growing at 87 degrees C. The archaeal ADP-ribosylation reaction was time-, temperature- and NAD-dependent. It proved to be highly thermostable, with about 30% decrease of 14C incorporation from [14C]NAD on incubation at 80 degrees C for up to 24 h. The main reaction product was found to be mono-ADP-ribose. Testing both [adenine-14C(U)]NAD and [adenine-14C(U)]ADPR as substrates, it was found that acceptor proteins were modified by ADP-ribose both enzymatically, via ADP-ribosylating enzymes, and via chemical attachment of free ADP-ribose, likely produced by NAD glycohydrolase activity. The synthesis of ADP-ribose-protein complexes was shown to involve mainly acceptors with molecular masses in the 40-100 kDa range, as determined by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulphate.

The role of the spleen in laparoscopy-associated inflammatory response.

BACKGROUND: Carbon dioxide (CO(2)) pneumoperitoneum alters the inflammatory response in animal models of sepsis. The spleen is a key organ in inflammation and its removal was predicted to modify this effect. METHODS: The acute phase inflammatory response to lipopolysaccharide (LPS) challenge in male rats was examined for the effects of splenectomy (spx) and the technique of removal (open or laparpscopic). A series of experiments compared LPS-only controls with LPS injection 2 or 9 days following open spx, lap CO2 spx, open sham, or lap CO2 sham. The method of splenectomy was studied by randomization to control, open spx, lap CO2 spx, lap helium (He) spx, or lap air spx with LPS challenge on postoperative day 2. Serum levels of tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (INF-gamma) and, interleutin (IL) 10 were collected at multiple time points, assayed by commercial enzyme-linked immunosorbent assay, analyzed by analysis of variance. RESULTS: Levels of TNF-alpha at 1.5 were significantly lower following open sham than following lap sham (p < 0.05). Splenectomy drastically reduced INF-gamma and TNF-alpha levels compared to controls (p < 0.05) on postoperative day 2. No method of spx preserved TNF-alpha or INF-gamma responses. Recovery of TNF-alpha response on day 9 was delayed in the spx groups. CONCLUSIONS: Splenectomy dramatically reduces TNF-alpha and INF-gamma responses to LPS challenge, although by different mechanisms. Pneumoperitoneum-mediated modulation of the septic inflammatory response is partially dependent on the spleen.

A role for D-aspartate oxidase in schizophrenia and in schizophrenia-related symptoms induced by phencyclidine in mice.

Increasing evidence points to a role for dysfunctional glutamate N-methyl-D-aspartate receptor (NMDAR) neurotransmission in schizophrenia. D-aspartate is an atypical amino acid that activates NMDARs through binding to the glutamate site on GluN2 subunits. D-aspartate is present in high amounts in the embryonic brain of mammals and rapidly decreases after birth, due to the activity of the enzyme D-aspartate oxidase (DDO). The agonistic activity exerted by D-aspartate on NMDARs and its neurodevelopmental occurrence make this D-amino acid a potential mediator for some of the NMDAR-related alterations observed in schizophrenia. Consistently, substantial reductions of D-aspartate and NMDA were recently observed in the postmortem prefrontal cortex of schizophrenic patients. Here we show that DDO mRNA expression is increased in prefrontal samples of schizophrenic patients, thus suggesting a plausible molecular event responsible for the D-aspartate imbalance previously described. To investigate whether altered D-aspartate levels can modulate schizophrenia-relevant circuits and behaviors, we also measured the psychotomimetic effects produced by the NMDAR antagonist, phencyclidine, in Ddo knockout mice (Ddo(-)(/-)), an animal model characterized by tonically increased D-aspartate levels since perinatal life. We show that Ddo(-/-) mice display a significant reduction in motor hyperactivity and prepulse inhibition deficit induced by phencyclidine, compared with controls. Furthermore, we reveal that increased levels of D-aspartate in Ddo(-/-) animals can significantly inhibit functional circuits activated by phencyclidine, and affect the development of cortico-hippocampal connectivity networks potentially involved in schizophrenia. Collectively, the present results suggest that altered D-aspartate levels can influence neurodevelopmental brain processes relevant to schizophrenia.

Identification of glycoproteins that are receptors for peanut agglutinin on immature (cortical) mouse thymocytes.

Binding of peanut agglutinin is being widely used as a marker for immature mouse thymocytes and for the separation of these cells from the mature thymocytes. Two cell surface glycoproteins that bind peanut agglutinin were detected on unfractionated as well as immature thymocytes by lectin overlay and affinity chromatography: one of Mr between 170 000 and 180 000, and the other, a minor component, of Mr 110000, both of which are partially sialylated. No receptors for peanut agglutinin were detected on the mature cells, whereas desialylation experiments revealed the presence of a glycoprotein of Mr 110000. These findings were corroborated by electrophoretic analysis of cell surface glycoproteins of the isolated thymocyte subpopulations labeled in their carbohydrate moieties.

A murine model for evaluating metastatic potential: characterization of a 90-110-kDa metastasis-binding protein.

Reliable discriminatory tests to predict metastatic disease would clearly facilitate the management of cancer in the elderly. We have recently identified a 90-110-kilodalton (kDa) cell surface glycoprotein that is differentially expressed in benign and malignant murine adrenal carcinoma cells. In view of the proteins highly glycosylated nature, we have tested its ability to bind to a panel of agarose-bound lectins. Wheat germ agglutinin (WGA), a lectin specific for terminal sialic acid and N-acetylglucosamine (G1cNAc), had a strong affinity for the metastasis-related protein but failed to detect such a glycoprotein in nonmetastatic cells. Treatment of cells with sialidase to remove terminal sialic acids did not affect the affinity of the protein for the lectin, indicating the presence of terminal G1cNAc. We show by in situ that this metastatic binding protein (MBP) is regionally concentrated on the surface of invasive cells but absent in cells unable to invade. We postulate that MBP plays an active role in cell migration through interactions with beta-1,4 galactosytransferase and basement membrane glycoproteines.

Increase in beta-galactosidase activity in a non-isothermal bioreactor utilizing immobilized cells of Kluyveromyces fragilis: fundamentals and applications.

The beta-galactosidase activity of Kluyveromyces fragilis cells immobilized in a kappa carrageenan gel was studied in a bioreactor functioning under isothermal and non-isothermal conditions. We observed an increase in enzyme activity which was found to be proportional to the intensity of the temperature gradient applied across the biocatalytic membrane, as well as to the average temperature of the bioreactor. The efficiency of such a non-isothermal bioreactor was calculated with respect to the yield of a bioreactor working under comparable isothermal conditions and was evaluated in terms of reduction of processing times in industrial applications. The possibility that enzyme activity in living cells is affected by non-isothermal conditions naturally existing owing to metabolic heat production is also discussed.

Heat shock proteins: facts, thoughts, and dreams.

The most primitive mechanism of cellular protection involves the expression of a polypeptide family named heat shock or stress proteins (hsps). Some of these hsps are present in unstressed cells and play an important role in the folding and translocation of polypeptides across membranes. Thus, they have been termed molecular chaperones. Hsps are expressed in response to an array of stresses, including hyperthermia, oxygen radicals, heavy metals, ethanol, and amino acid analogues. In addition, the heat shock response is induced during clinically relevant situations such as ischemia/reperfusion and circulatory and hemorrhagic shock. All of the above stresses have in common that they disturb the tertiary structure of proteins and have adverse effects on cellular metabolism. Pretreatment of cells with a mild stress, sufficient to induce the expression of hsps, results in protection to subsequent insults. This phenomenon has been coined "stress tolerance" and is apparently caused by the resolubilization of proteins that were denatured during the stress. In addition, cellular structures (microfilaments and centrosomes) and processes (transcription, splicing, and translation) are stabilized or repaired during a second stress in stress tolerant cells and organisms. There is a great body of evidence indicating a direct role of hsps in the stabilization of these events. The intrinsic capacity of hsps to protect cells has potential relevance as a natural mechanism of organ protection during harmful environmental conditions and operative procedures, and in the combat against pathogens.

Expression of HSP70 is impaired at the transcriptional level in stressed murine neuroblastoma cells.

Although the expression of heat shock or stress proteins (hsps) is a well conserved response to stress, the accumulation of these proteins is different between various cell-types. Particularly, cells of neuronal origin show a reduced expression of Hsp70 after stress. The possible mechanism of this reduced Hsp70 expression was studied in thermally stressed murine neuroblastoma cells (N18). These cells showed no detectable levels of Hsp70 or Hsp70 mRNA after heat shock. Hsp70 transcription was not detectable after stress. However, heat shock transcription factor 1 (HSF1) is active in these cells under stress conditions. Cells transiently transfected with the chloramphenicol acetyltransferase (CAT) gene under control of the human heat shock promoter showed a stress-dependent expression of CAT, suggesting that the cells contain the factors necessary for the expression of Hsp70. Integration of the foreign human heat shock promoter into genomic DNA did not affect its transcriptional inducibility. These results suggest that the impairment of Hsp70 expression in N18 cells is due to the environment (chromatin structure, methylation pattern) of the Hsp70 locus.

Expression of the connexin 43 gene is increased in the kidneys and the lungs of rats injected with bacterial lipopolysaccharide.

At the molecular level, the inflammatory response is characterized by changes in gene expression of various organ systems. One gene by which expression has been observed to be altered in the liver during inflammation is connexin (Cx) 32. Cx genes encode the polypeptide subunits of the hemichannels that comprise gap junctions. In the present study, an increase in the expression of a different Cx gene, Cx43, was observed in the kidney and lung of rats injected with a sublethal dose (1 mg/kg) of bacterial lipolysaccharide (LPS). To elucidate the possible mechanism by which the Cx43 expression is increased during inflammation, the 5' flanking region of the gene was cloned and coupled to a reporter gene (human growth hormone). This construct was transfected into cells of renal origin (NRK), which express Cx43 constitutively. The Cx43 promoter activity was indeed found in the cloned region, which contained 725 base pairs upstream of the transcriptional initiation site of the Cx43 gene. The Cx43 promoter activity was found to be increased by incubation of the transfected cells with serum obtained from LPS-treated rats. Moreover, direct incubation of the transfected cells with LPS or interleukin 1beta, but not with other cytokines, was observed to increase the Cx43 promoter activity. These results suggest the expression of Cx43 after administration of LPS is part of the inflammatory response. Moreover, the expression of this gene seems to be mediated by proinflammatory mediators.