Blockade of ionotropic receptor responses by progesterone in the ganglion cells of Aplysia

To compare nongenomic effects of progesterone on various receptor responses of neurons, Aplysia ganglion cells were pretreated with 30 microM progesterone for 5 min and various receptor responses were tested using a conventional voltage-clamp method. Progesterone reduced nicotinic receptor-activated Na(+)-currents, nicotinic receptor-activated Cl(-)-currents, gamma-aminobutyric acid receptor-activated Cl(-)-currents, and dopamine receptor-activated Na(+)-currents. These depressant effects are similar at two different agonist concentrations. On the other hand, progesterone affected neither muscarinic receptor-activated K(+)-currents nor dopamine receptor-activated K(+)-currents. The former four types of receptors are known to be ionotropic while the latter two types of receptors are known to be metabotropic. Therefore, progesterone selectively inhibited all the types of ionotropic receptor responses, presumably in a noncompetitive manner.     

Hypoplastic myelodysplastic syndromes can be distinguished from acquired aplastic anaemia by bone marrow stem cell expression of the tumour necrosis factor receptor

It is often difficult to distinguish hypoplastic myelodysplastic syndrome (h-MDS) from acquired aplastic anaemia (AA), because of the considerable clinical, cytological and histological similarities between these two disorders. The distinction between AA and h-MDS is important because there is a higher risk of progression to acute leukaemia in patients with h-MDS compared with AA. Recent studies suggest that tumour necrosis factor-alpha (TNF-alpha) plays an important role in the development of AA. In order to determine the potential importance of TNF-alpha in the differential diagnosis of hypoplastic bone marrow (BM) disorders, we examined whether analysis ofTNF-receptor expression could be used as a tool to differentiate AA from h-MDS. Flow cytometric analysis revealed that BM stem cells (CD34+) from AA patients have markedly greater TNF receptor (R) 1 and TNFR2 expression than those from patients with MDS and h-MDS. We suggest that the BM stem cells with a high expression of TNFR in patients with AA may be potently sensitive to TNF-alpha stimulation of differentiation. Thus, we propose that quantification of TNFR expression in BM stem cellsmay be a useful method to distinguish AA from h-MDS.     

Introduction of international normalized ratio of prothrombin time to evaluate multiple depletions of coagulation factors

Prothrombin time(PT) is utilized in worldwide as a global coagulation test reflected multiple depletions of coagulation factors in diseases such as severe liver dysfunction and Disseminated Intravascular Coagulation(DIC). However, standardization of regents and result reporting methods are not established yet except International Normalized Ration(INR) for control of oral anticoagulant therapy(OAT). We evaluated whether INR is capable for defect of multiple coagulation factors except OAT, using absorbed plasma and different origins of thromboplastin; human recombinant, human placenta, cultured human cell and rabbit brain. PTs of individual 90 samples(group MC) absorbed with BaSO4 and/or bentnite and 60 samples(group W) from patients with OAT were measured with 20 normal plasmas with respective reagents. Sensitivities of four reagents to plasma of group W and MC were determined respectively against human recombinant thromboplastin(ISI = 1.03). Both of human thromboplastin showed that sensitivity to absorbed plasma was very close to OAT plasma, whereas reductions of sensitivity to 84% and 66% for absorbed plasma were revealed in both of rabbit thromboplastins. Correlations of INRs calculated by two different sensitivities, one is to absorbed plasma and another to OAT plasma, indicated that discrepancy of sensitivities was emphasized as large slopes(1.50 and 2.76) of regression lines and large intercepts in rabbit thromboplastins, although slopes closed to 1.0 with small intercepts in both of human thromboplastins. We concluded that use of human thromboplastin was the first priority to introduce INR system for evaluation of multiple coagulation factors depletions.     

Glucocorticoids induced the production and gene expression of IL-1alpha through AP-1 and partially NF-kappaB activation in murine epidermal cells

To investigate the mechanism of the glucocorticoids-induced augmentation of skin response, we have recently reported the modulatory effect of glucocorticoids on the regulation of cytokines production in keratinocytes stimulated with various chemicals in vitro through both NF-kappaB and AP-1 activation. Further to clarify the mechanism in the glucocorticoids-induced augmentation of cytokines production from keratinocytes, we examined the effect of glucocorticoids to keratinocytes without chemical stimulation. Glucocorticoids 10(-4) M inhibited the production of IL-1alpha from Pam 212 cells. However, lower concentration (10(-8)-10(-10) M) of glucocorticoids significantly enhanced the production of IL-1alpha by Pam 212 cells at both the protein and mRNA levels. In contrast, glucocorticoids had no effect on the production of either TNF-alhpa, IL-6, nor GM-CSF by Pam 212 cells cultured for 6 h. Electrophoretic mobility shift assays (EMSA) revealed that 10(-10)-10(-12) M glucocorticoids induced the NF-kappaB activation in Pam 212 cells, however, augmented AP-1 activation by 10(-8)-10(-10) M of glucocorticoids was observed in Pam 212 cells. Furthermore, pyrrolidine dithiocarbamate (PDTC) partially inhibited the IL-1alpha production and completely inhibited NF-kappaB expression by Pam 212 cells. On the other hand, MAP-kinase inhibitors (PD98059, SB202190) completely abrogated not only AP-1 activation but the low concentration glucocorticoids-induced IL-1alpha production. These data indicated that lower concentration of glucocorticoids induced the augmentation of IL-1alpha production from keratinocytes mediated through the AP-1 pathway and partially through NF-kappaB pathway.     

Reverse pharmacological effect of loop diuretics and altered rBSC1 expression in rats with lithium nephropathy

BACKGROUND: Renal urinary concentration is associated with enhanced expression of rBSC1, a rat sodium cotransporter, in the thick ascending limb of Henle. Increased expression of rBSC1 was reported recently in nephrogenic diabetes insipidus induced by lithium chloride (Li nephropathy). However, the pathophysiological implication of altered rBSC1 expression has not yet been investigated. METHODS: Li nephropathy was induced in rats by an oral administration of 40 mmol lithium/kg dry food. In rats with reduced urinary osmolality to less than 300 mOsm/kg H2O, we examined the expression of rBSC1 mRNA and protein, plasma arginine vasopressin (AVP) and RNA expression of kidney-specific water channel, aquaporin-2 (AQP2), of collecting ducts. Rats with Li nephropathy were treated with furosemide (3 mg/kg body weight), which blocks the activity of rBSC1, and changes in urine concentration, plasma AVP, medullary accumulation of Li ions, and apical AQP2 expression were determined. RESULTS: Rats with Li nephropathy showed increased rBSC1 RNA and protein expression and reduced AQP2 RNA. In these rats, furosemide, which induces dilution of urine and polyuria in normal rats, resulted in a progressive and significant rise in urine osmolality from 167 +/- 11 (mean +/- SD) at baseline to 450 +/- 45 mOsm/kg H2O at three hours after administration, and significant oliguria. In the same rats, plasma AVP decreased significantly from 5.7 to 3.0 pg/mL. In addition, recovery of apical AQP2 expression was noted in a proportion of epithelial cells of the collecting ducts. Although Li+ in the renal medulla was slightly lower in rats with Li nephropathy treated with furosemide, statistical significance was not achieved. CONCLUSIONS: Our results suggest that dehydration or high plasma AVP results in an enhanced rBSC1 expression in Li nephropathy, and that rBSC1 expression is closely associated with the adverse effects of Li ions on collecting duct function.     

Image analysis of remesothelialization following chemical wounding of cultured human peritoneal mesothelial cells: the role of hyaluronan synthesis

BACKGROUND: To understand what happens during the wound healing process of the mesothelium, we have developed an in vitro wounding model of cultured human peritoneal mesothelial cells (HPMCs) utilizing an image acquisition and analysis system. Using this system, cell mobility and hyaluronan synthesis were quantified and their interrelationship discussed. METHODS: 1N NaOH was used to create circular wounds in cultured HPMC monolayers, which were then exposed for 30 minutes to the peritoneal dialysis solutions or fetal calf serum (FCS)-free M199 culture medium, followed by incubation with 0.3% FCS/M199 culture medium for up to 96 hours. Digitalized microscopic date was captured every 30 minutes to quantify the wound healing process. In separate experiments, the HPMC monolayers were stained with biotin-conjugated hyaluronan-binding protein (B-HABP) at a regular time interval. RESULTS: Centripetal migration of the HPMCs into the wound area was the predominant process involved in wound repair with proliferation playing a secondary role. Two noticeable observations were made from the digital video movies: (1) cell mobility varied and was dependent upon the morphology and location of the cell relative to the wound edge, and (2) cell migration continued even after wound closure. Staining for B-HABP was confined to the remesothelialized area when wound closure was complete at 24 hours. At 48 hours after wound closure, the stained area was even more visible, although somewhat diffuse; thereafter, staining was reduced to almost background levels. CONCLUSION: The cell culture model of wound healing used in our study has enabled us to demonstrate quantitative image data of the cellular processes that occur during wound healing. We have been able to continuously observe cell migration, proliferation, and transformation. Synthesis and subsequent decomposition of hyaluronan appears to be related to the mobility of the wounded and intact HPMCs in this model system.     

Increased urinary excretion of monocyte chemoattractant protein-1 in proteinuric renal diseases

Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that is produced mainly by tubular epithelial cells in kidney and contributes to renal interstitial inflammation and fibrosis. More recently, we have demonstrated that urinary MCP-1 excretion is increased in proportion to the degree of albuminuria (proteinuria) and positively correlated with urinary N-acetylglucosaminidase (NAG) levels in type 2 diabetic patients. Based on these findings, we have suggested that heavy proteinuria, itself, probably aggravates renal tubular damage and accelerates the disease progression in diabetic nephropathy by increasing the MCP-1 expression in renal tubuli. In the present study, to evaluate whether urinary MCP-1 excretion is increased in the proteinuric states not only in diabetic nephropathy but also in other renal diseases, we examined urinary MCP-1 levels in IgA nephropathy patients with macroalbuminuria (IgAN group; n = 6), and compared the results with the data obtained from type 2 diabetic patients with overt diabetic nephropathy (DN group; n = 23) and those without diabetic nephropathy (non-DN group; n = 27). Urinary MCP-1 excretion levels in non-DN, DN, IgAN groups were 157.2 (52.8-378.5), 346.1 (147.0-1276.7), and 274.4 (162.2-994.5) ng/g creatinine, median (range), respectively. Expectedly, urinary MCP-1 and NAG excretion levels in DN and IgAN groups were significantly elevated as compared with non-DN group. Therefore, we suggest that MCP-1 expression in renal tubuli is enhanced in proteinuric states,irrespective of the types of renal disease, and that increased MCP-1 expression probably contributes to renal tubular damage in proteinuric states.     

Contribution of scatter and attenuation compensation to SPECT images of nonuniformly distributed brain activities.

Correction of scatter and attenuation is essential for quantitative SPECT. In this work, we evaluated the accuracy gained from a method of transmission-dependent convolution subtraction (TDCS) in the quantitation of activity that is highly concentrated in the striatum (STR). METHODS: SPECT data were acquired from an (123)I-containing phantom with a constant activity in the STR but differing background (BKG) activities, so as to simulate various STR/BKG ratios (19.7:1, 9.7:1, 4.8:1, 1.9:1, and 1:1). In a study of healthy humans (n = 6), a transmission scan followed by an emission scan was performed 24 h after injection of (123)I-2beta-carbomethoxy-3beta-(4-iodophenyl)-tropane ((123)I-beta-CIT). All SPECT data was reconstructed with ordered-subset expectation maximization. TDCS was applied for scatter correction. Values of activity in the STR and occipital lobe (for BKG) were used to calculate binding potential V(3)" (= [STR - BKG]/BKG). The effect of SPECT collimator dependency on scatter correction was also evaluated for 6 collimators from 3 different SPECT cameras in the phantom experiment. RESULTS: Scatter correction in the phantom experiment increased the measured values of STR activity (36.2%), resulting in a substantial increase in V(3)" (66.1%). Scatter and attenuation corrections with recovery correction showed an overall bias of -7.3% for the STR, -4.0% for BKG activity, and -7.8% for V(3)". TDCS corrections of phantom activities were relatively uniform for the 6 different collimators, with variabilities of <5.5% for the STR and <3.0% for BKG activities. TDCS correction of human (123)I-beta-CIT images was of a similar, although slightly larger, magnitude than for the phantom data, with increased V(3)" values of 9.4 +/- 2.3 and 4.9 +/- 0.6, with and without scatter correction, respectively. CONCLUSION: The TDSC method significantly improved the accuracy of SPECT images with a nonuniform distribution of activity highly concentrated in central regions. The value of V(3)" was significantly increased in phantom and human data, with most of the improvement derived from an increase in STR activity. This scatter correction method was approximately equally useful with data from the 6 different collimators and is recommended for more accurate quantitation of nonuniformly distributed brain activities.     

Influence of modest endotoxemia on postoperative antithrombin deficiency and circulating secretory immunoglobulin a levels

OBJECTIVE: To evaluate the influence of modest endotoxemia on postoperative antithrombin deficiency and cholestasis. SUMMARY BACKGROUND DATA: It has not been determined whether endotoxin translocation in small amounts is a physiological phenomenon or whether it is a potential health hazard. METHODS: Blood endotoxin, antithrombin III (ATIII), secretory immunoglobulin A (sIgA), which was selected as a marker of cholestasis, C-reactive protein (CRP), and alpha-1-antitrypsin (AAT) concentrations were measured from the 20 patients undergoing curative gastrectomy for gastric cancer preoperatively and postoperatively. Portal and systemic blood samples were taken for the analysis of endotoxin and interleukin-6 (IL-6) concentrations during surgery in these patients. RESULTS: Although plasma endotoxin levels showed a significant increase during surgery, we did not find a correlation with ATIII, sIgA, CRP, and IL-6 levels. Systemic blood endotoxin levels during surgery correlated with a postoperative rise of serum AAT levels. Plasma ATIII levels transiently decreased on the first and third postoperative day, and sIgA levels were shown to increase on the seventh postoperative day. There was a weak relationship between the extent of postoperative endotoxemia and a reduction in ATIII concentrations. CONCLUSIONS: The influence of modest endotoxemia on postoperative antithrombin deficiency and cholestasis was limited, and increased translocational endotoxemia during abdominal surgery may be a physiological phenomenon to trigger off an acute-phase protein response.