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.     

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.     

Anesthesia for patients with carcinoid syndrome

Carcinoid syndrome, although rare, can create serious problems to the anesthetist, both by the nature and variability of clinical manifestations and by the complications that can occur peroperatively. Recent research has led to a better understanding of the pathophysiology of the disease process. However, modern medicine is far from unraveling the precise nature and physiological effects of all the peptide mediators produced by these tumors. The severity of symptoms does not predict the severity of perioperative complications, so that patients with minor preoperative symptoms may have significant intraoperative complications. While urinary 5-HIAA levels provide a good indicator of disease progression, they cannot predict the degree or type of physiological response to intraoperative tumor manipulation. Indeed, urinary 5-HIAA may be normal both in the presence of a clinical diagnosis of carcinoid syndrome and in the face of a peroperative carcinoid crisis. The keys to successful anesthetic management of patients with carcinoid syndrome are good communication between endocrinologist, anesthetist, and surgeon and preoperative optimization of the patient. This includes appropriate investigation and treatment of the effects of carcinoid peptides and the prevention of their release from tumors. If possible, advice should be sought from centers with experience at managing this group of patients. Octreotide has largely replaced the use of other drugs both for symptomatic control and acute treatment of the symptoms associated with carcinoid syndrome. However, other drugs, such as aprotinin, still have a significant place in the symptomatic control and treatment of peroperative complications, as serotonin is only one of a large variety of peptides responsible for the clinical effects of this disease. Anesthetic technique should be aimed at minimizing carcinoid mediator release, in response to stress it induction of anesthesia and tracheal intubation and during tumor manipulation. It is equally important to prepare for carcinoid crisis by, for example, ordering drugs, which are otherwise uncommonly used in the theater setting, ahead of time. Cardiovascular instability, particularly hypotension, is common, so that full monitoring and vigilance is vital to predict its onset. The current surgical view of management is that, while curative resection of carcinoid tumors less than 2 cm in diameter with no evidence of invasion or metastatic spread is appropriate, patients with disseminated disease should be medically managed unless symptom control is poor. The exceptions to this are those patients with early and correctable carcinoid cardiac disease and those who require palliative procedures such as defunctioning obstructed bowel. Survival rates in patients following excision of gastric and appendical carcinoid tumors approach those of the general population as a whole and the chance of metastasis is extremely low. Only two series have been published in the anesthetic literature on anesthesia for patients with carcinoid syndrome, although there are many single-case reports. Despite the rarity of this syndrome, further formal studies into the anesthetic management of this condition should be encouraged.     

Memory association and personality as predictors of alcohol use: mediation and moderator effects

We investigated the nature of the effects of memory associations on alcohol use and abuse. First, we determined if effects of memory associations on drinking problems are mediated entirely through the frequency of alcohol consumption or, alternatively, if such effects are more direct. Second, personality traits were assessed to evaluate whether they were confounded with memory association in their effects or whether they might moderate the effects of memory associations on alcohol use and abuse. The results showed that memory association measures directly and independently predicted alcohol consumption; these measures indirectly predicted problems from drinking, including drunk driving. None of the assessed personality variables moderated the predictive effects of memory association. The results are consistent with the view that memory associations influence behavior through cognitive processes that are not affected by personality traits or by cognitions emanating from such traits.     

''Candyflipping'': synergistic discriminative effect of LSD and MDMA

Tonsils have a privileged situation in the immune system in that they are in touch with the environment. Melatonin is a hormone that is influenced by the circadian environmental variations of dark-light and is a modulator of the immune system. We have studied a group of thirty five children with recurrent acute tonsillitis that were submitted for tonsillectomy. Tonsillar lymphocyte subsets were determined before and after culture through flow cytometry in a tonsillar mononuclear suspension. After the culture, the lymphocyte subsets of type B suffered a decrease that was restored in the presence of melatonin or phytohemaglutinin, and even increased above the values of the control when the culture was accomplished in the presence of both substances. This process was specific for B cells, no occurrence for T lymphocytes or natural killer cells. Melatonin is found in the crossroads of the interaction of the microorganisms, pollens or inert substances with the tonsillar lymphocytes in the production of the immune defences. Further study is required on tonsillar pathology to explain its physiopathology and its possible therapeutic role.     

A relatively small change in sodium chloride concentration has a strong effect on adhesion of ocular bacteria to contact lenses

We studied the effect of the nitric oxide synthase (NOS) inhibitor asymmetric dimethyl arginine (ADMA) and the inactive enantiomer N G-methyl-D-arginine (D-NMMA) on Pseudomonas aeruginosa infection of the respiratory mucosa in nasal turbinate organ cultures. We also investigated the effect of P. aeruginosa culture filtrate on the expression of inducible NOS (iNOS) messenger RNA (mRNA) by an epithelial cell line (A549). Organ cultures were preincubated with ADMA (0.1 to 4 x 10(-4) M) or D-NMMA (2 x 10(-4) M) for 30 min prior to bacterial infection. Infected organ cultures (8 h) had significantly (P <= 0.05) greater epithelial damage and fewer ciliated and unciliated cells than did control cultures. There was an increased level of nitrite in the medium feeding infected organ cultures as compared with control cultures. ADMA significantly (P <= 0.05) reduced both bacterially induced epithelial damage and loss of ciliated cells in a concentration-dependent manner. D-NMMA did not influence the effect of P. aeruginosa infection of the mucosa. ADMA, but not D-NMMA, significantly (P <= 0.04) reduced total bacterial numbers adherent to the respiratory mucosa. P. aeruginosa culture filtrates (24 h and 36 h) significantly (P = 0.02) increased iNOS with respect to glyceraldehyde-3-phosphate dehydrogenase mRNA expression. These results show that P. aeruginosa stimulates iNOS expression by a cell line and NO production by an organ culture. ADMA reduces mucosal damage and loss of ciliated cells, which suggests that NO may be a mediator of epithelial damage caused by P. aeruginosa.