Lymphotoxin alpha/beta and tumor necrosis factor are required for stromal cell expression of homing chemokines in B and T cell areas of the spleen

Mice deficient in the cytokines tumor necrosis factor (TNF) or lymphotoxin (LT) alpha/beta lack polarized B cell follicles in the spleen. Deficiency in CXC chemokine receptor 5 (CXCR5), a receptor for B lymphocyte chemoattractant (BLC), also causes loss of splenic follicles. Here we report that BLC expression by follicular stromal cells is defective in TNF-, TNF receptor 1 (TNFR1)-, LTalpha- and LTbeta-deficient mice. Treatment of adult mice with antagonists of LTalpha1beta2 also leads to decreased BLC expression. These findings indicate that LTalpha1beta2 and TNF have a role upstream of BLC/CXCR5 in the process of follicle formation. In addition to disrupted follicles, LT-deficient animals have disorganized T zones. Expression of the T cell attractant, secondary lymphoid tissue chemokine (SLC), by T zone stromal cells is found to be markedly depressed in LTalpha-, and LTbeta-deficient mice. Expression of the SLC-related chemokine, Epstein Barr virus-induced molecule 1 ligand chemokine (ELC), is also reduced. Exploring the basis for the reduced SLC expression led to identification of further disruptions in T zone stromal cells. Together these findings indicate that LTalpha1beta2 and TNF are required for the development and function of B and T zone stromal cells that make chemokines necessary for lymphocyte compartmentalization in the spleen.     

Riboflavin-mediated delivery of a macromolecule into cultured human cells

The effects of perivascular nerve stimulation and phenylephrine on osmolyte release were studied in the intact perfused rat liver and isolated liver parenchymal cells (PC) and nonparenchymal cells. In the perfused liver, electrical stimulation of perivascular nerves (20 Hz/2 ms/20 V) led to a phentolamine-sensitive increase of cell hydration by 6.5% +/- 1.2% (n = 3) and a transient phentolamine-sensitive stimulation of taurine and inositol, but not betaine, release. These nerve effects were mimicked by phenylephrine, but not prostaglandin F2alpha, and were not affected by sodium nitroprusside (SNP) or ibuprofen. Nerve stimulation-induced taurine, but not inositol, release was inhibited by 4, 4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS) (50 micromol/L). Single-cell fluorescence studies with isolated liver PC, Kupffer cells (KC), sinusoidal endothelial cells (SEC), and hepatic stellate cells (HSC) revealed that phenylephrine induced an increase in cytosolic free Ca2+ only in PC and HSC, but not in KC and SEC, whereas extracellular uridine triphosphate (UTP) produced Ca2+ transients/oscillations in all liver cell types studied. Phenylephrine had no effect on osmolyte release from isolated KC and SEC, but increased taurine (but not inositol) release from PC and inositol (but not taurine) efflux from HSC. The data suggest that: 1) liver cell hydration and-consecutively-osmolyte content are modulated by hepatic nerves via an alpha-adrenergic mechanism, which does not involve eicosanoids or hemodynamic changes; 2) that PC and HSC are the primary targets for nerve-dependent alpha-adrenergic activation, whereas 3) KC and SEC probably do not express alpha-adrenoceptors coupled to Ca2+ mobilization or osmolyte efflux.     

The effect of benzodiazepines and flumazenil on motor cortical excitability in the human brain

In the present study, the effects of benzodiazepines (diazepam) were evaluated in terms of cortical excitability changes, as tested with transcranial magnetic simulation (TMS). In particular, analyzed were drug-induced changes regarding two selected parameters of TMS: (1) the cortical excitability threshold and (2) the silent period duration (SP). For this purpose, we evaluated the effects of long-term therapy with diazepam in the patients affected by anxiety disorders and the changes induced by single oral doses of diazepam in both healthy controls and patients. In addition, we tested cortical excitability changes in two 'extreme conditions' where a considerable concentration of serum benzodiazepine-like activity was reached, as represented by diazepam overdose and idiopathic recurrent stupor (IRS). In both groups of patients, a significant increment of motor threshold was found, while in the overdose patients, the SP was also increased. The administration of flumazenil in these two conditions was followed by a prompt reversal effect, consisting of a return to normal cortical excitability parameters. The long-term usage of diazepam in patients with anxiety disorders is associated with significantly increased threshold; the increased value of these parameters was temporarily further enhanced by the administration of a single oral dose of diazepam, which, in normal control subjects, is not associated with changes of cortical excitability. The results of this study reveal that different physio-pathological conditions induced by the influence of benzodiazepine and its antagonist are reflected in excitability changes which attest to the involvement and modification of cortical GABAergic activity.     

Guanidinium-based potentiometric SO2 gas sensor

An SO2 gas sensor was developed by using a hydrogen sulfite-selective electrode positioned behind a gas-permeable membrane (GPM). The hydrogen sulfite-selective electrode was prepared by incorporating a multicyclic guanidinium ionophore in a plasticized poly(vinyl chloride) membrane. This gas sensor presents important advantages over the conventional Severinghaus-type SO2 gas sensor that contains a pH electrode immersed in an internal solution behind the GPM. The Severinghaus gas sensor suffers interferences from weak acids that can cross the GPM as gases and change the pH of the internal solution. In contrast, in the proposed sensor, the excellent selectivity of the HSO3- electrode and the ability of the GPM to discriminate gaseous from nongaseous species combine to generate the most selective potentiometric SO2 gas sensor reported to date.     

The impact of diaphragm management on prolonged ventilator support after thoracoabdominal aortic repair

PURPOSE: The relationship of the division of the diaphragm during thoracoabdominal aortic repair to prolonged ventilator support has not been studied. The purpose of this study was (1) to determine whether preservation of diaphragm integrity has a significant effect on postoperative ventilator duration and (2) to elucidate other pulmonary risk factors related to thoracoabdominal aortic surgery and to study the relationship of these factors to the intact diaphragm technique. METHODS:Between February 1991 and January 1997, we repaired 397 descending and thoracoabdominal aortic aneurysms. Descending thoracic aneurysms were not included in the study because their repair does not include the diaphragm. A total of 256 patients participated in this study. The diaphragm was divided in 150 patients and left intact in 106 patients. Examined as potential risk factors were patient demographics, history and physical findings, aneurysm extent, urgency of the procedure, acute dissection, cross-clamp time, homologous and autologous blood product consumption, and adjunctive operative techniques. FEV1 also was considered in the 197 patients for whom preoperative spirometry was available. Prolonged mechanical ventilation was defined as ventilator support for >72 hours. Data were analyzed by univariate contingency table and multiple logistic regression methods. RESULTS: Increasing age (odds ratio [OR], 1.02/y; P <.02), current smoking (OR, 2.6; P <.0008), total cross-clamp time (OR, 1.0/min; P <.008), units packed red blood cells transfused (OR, 1.06/unit; P <.008), and division of the diaphragm (OR, 2.03; P <.02) were significant, independent predictors of prolonged ventilation. Sixty-seven percent of patients (71 of 106) whose diaphragms were preserved were extubated in <72 hours compared with 52% of patients (78 of 150) who underwent diaphragm division (OR, 0.53; P <.02). CONCLUSION: Independently of well known pulmonary risk factors, an intact diaphragm during thoracoabdominal aortic repair results in a higher probability of early ventilator weaning.     

Evaluation of proteinase-activated receptor-1 (PAR1) agonists and antagonists using a cultured cell receptor desensitization assay: activation of PAR2 by PAR1-targeted ligands

We developed a calcium signaling-based assay, using cultured human embryonic kidney cells (HEK), that evaluates simultaneously, the activation/desensitization or blockade of the proteinase-activated receptors, PAR1 and PAR2. Using this assay, we analyzed the actions of a number of previously described putative PAR1-targeted peptide agonists and antagonists. We found that most of the previously described PAR1-targeted agents can also activate/desensitize PAR2, and most of these peptides can also activate a calcium signaling pathway in a target cell that possesses PAR2 along with PAR1. Furthermore, we used this assay to develop a PAR1 receptor-activating probe [Ala-parafluoroPhe-Arg-Cha-Cit-Tyr-NH2 (Cit-NH2)], which displays a high degree of specificity for PAR1 over PAR2, and we used the assay to quantitate the ability of trypsin to disarm the activation of PAR1 by thrombin. The abilities of the PAR1-targeted agents to desensitize or block PAR1 in the HEK cell assay were compared with their activities in a human platelet aggregation assay. Our data illustrate the usefulness of the HEK cell assay for evaluating the PAR1/PAR2 selectivity of PAR-activating agonists. The PAR1-selective agonist that we developed using the assay should prove useful for studying the effects of selectively activating PAR1 in vivo.     

Environmental upregulation of the atrial natriuretic peptide gene in the living fossil, Limulus polyphemus

Northern blot analysis revealed that atrial natriuretic peptide (ANP) gene expression occurs in heart, hematocytes and gills of the invertebrate Limulus polyphemus, the horseshoe crab. In low salinity and on land ANP prohormone messenger RNA in Limulus' heart was 32-fold less compared to that in a vertebrate heart (i.e., rat, Rattus norvegicus). ANP gene expression doubled (P < 0.05) in Limulus' heart and gills with change from land and low salinity to medium salinity and osmolality. ANP gene expression was 10-fold higher in Limulus' gills in seawater (i.e., high salinity). The products of this ANP gene expression (i.e., ANP, long acting natriuretic peptide, vessel dilator and kaliuretic peptide) were released and increased in the circulation, i.e., hemolymph, of Limulus proportional to the increase in salinity and osmolality (P = <0.01). These results suggest that modification of ANP gene expression enables animals to adapt to freshwater, seawater, and land.     

Elementary [Ca2+]i signals generated by electroporation functionally mimic those evoked by hormonal stimulation

Tryptophan residues in chitosanase from Streptomyces sp. N174 (Trp28, Trp101, and Trp227) were mutated to phenylalanine, and thermal unfolding experiments of the proteins were done in order to investigate the role of tryptophan residues in thermal stability. Four types of mutants (W28F, W101F, W227F and W28F/W101F) were produced in sufficient quantity in our expression system using Streptomyces lividans TK24. Each unfolding curve obtained by CD at 222 nm did not exhibit a two-state transition profile, but exhibited a biphasic profile: a first cooperative phase and a second phase that is less cooperative. The single tryptophan mutation decreased the midpoint temperature (Tm) of the first transition phase by about 7 degrees C, and the double mutation by about 11 degrees C. The second transition phase in each mutant chitosanase was more distinct and extended than that in the wild-type. On the other hand, each unfolding curve obtained by tryptophan fluorescence exhibited a typical two-state profile and agreed with the first phase of transition curves obtained by CD. Differential scanning calorimetry profiles of the proteins were consistent with the data obtained by CD. These data suggested that the mutation of individual tryptophan residues would partly collapse the side chain interactions, consequently decreasing Tm and enhancing the formation of a molten globule-like intermediate in the thermal unfolding process. The tryptophan side chains are most likely to play important roles in cooperative stabilization of the protein.     

Relaxin and decidualization in mice: a reappraisal

The nature of the physiological stimulus inducing decidualization in the endometrium is unknown. In this study we attempted to verify a recent report that relaxin can induce decidualization in intact mice primed with a high dose of estradiol valerate (5 micrograms) and a low dose (10 micrograms) of medroxyprogesterone acetate. In our study, neither s.c. nor intrauterine relaxin, nor intraluminal arachis oil, (an established deciduogenic stimulus) were able to induce decidualization. In addition, while oil was able to induce decidualization (increased uterine weight, and positive Pontamine Sky Blue and stromal alkaline phosphatase reactions) in ovariectomized mice treated with a regimen of estradiol and medroxyprogesterone acetate designed to produce optimum uterine sensitivity, no decidualization occurred in response to either s.c. or intraluminal relaxin. This study fails to provide any support for a role for relaxin as a deciduogenic stimulus.