Virucidal activity of glutaric acid and evidence for dual mechanism of action.

Rhinoviruses as a group are notably sensitive to inactivation in solutions with a pH of less than 5.3. Glutaric acid appears to possess virucidal activity in addition to the aciduant effect against rhinoviruses. A model system in which rhinovirus type 14 was incubated in the presence of glutaric acid (GA) (pH 4.0) at 0 degrees C was devised to separate intrinsic virucidal activity from the aciduant effect. Under these conditions, virucidal activity against rhinovirus type 14 was directly related to the concentration of GA present and the proportion of the acid in the diprotonated form. The virucidal activities of GA and several other compounds, including GA analogs and other mono- and dicarboxylic acids, were tested under the conditions described. In general, as the alkane bridge separating two carboxylic acid functions was lengthened, virucidal activity decreased. When 26 additional strains of rhinoviruses were tested in the model system, 19 were inactivated slowly enough to be compared. Of these, 63% were more susceptible to GA than to sodium acetate buffer and 26% were more susceptible to sodium acetate buffer. Eleven percent were resistant to both GA and sodium acetate buffer. The virucidal activity of GA for a majority of strains tested appeared to be due to combination of low pH and another mechanism of action presumably unrelated to pH.     

Spinal cord stimulation: Indications, mechanism of action, and efficacy

Unrelieved pain is costly to the economic fabric of our society; its direct costs to patients and their families is staggering. Spinal cord stimulation for the treatment of chronic pain is cost-effective when used in the context of a pain treatment continuum. Many theories on the mechanism of action of spinal cord stimulation have been suggested, including activation of gate control mechanisms, conductance blockade of the spinothalamic tracts, activation of supraspinal mechanisms, blockade of supraspinal sympathetic mechanisms, and activation or release of putative neuromodulators. Whatever theory or theories of mechanism are correct, spinal cord stimulation has efficacy in the treatment of failed back surgery syndrome, complex regional pain syndromes, intractable pain due to peripheral vascular disease, and intractable pain due to angina.     

Fenfluramine selectively and differentially decreases the density of serotonergic nerve terminals in rat brain: evidence from immunocytochemical studies

Fenfluramine is an amphetamine derivative which is used primarily as an anorectic agent in the treatment of obesity. High doses of fenfluramine have been reported to cause long-term decreases in brain serotonin (5-HT) levels and density of high-affinity 5-HT uptake sites, actions characteristic of a "neurotoxic" effect of the drug. In view of these neurochemical changes, we used immunocytochemistry to assess, in detail, the effects of fenfluramine treatment on the morphology and density of 5-HT-like immunoreactive neurons in rat brain. Twelve to 18 hr after high dose dl-fenfluramine HCl treatment (24 mg/kg s.c., twice daily for 4 days), there was a profound regional decrease in density of fine-caliber 5-HT-like immunoreactive fibers and terminals in brain. This effect was especially apparent in cerebral cortex, hippocampus, cerebellum and striatum and less striking decreases were noted in septum, locus ceruleus and hypothalamus. On the other hand, 5-HT-like immunoreactive somata in midbrain nuclei and fibers and terminals in spinal cord appeared unaffected after fenfluramine treatment. Remaining 5-HT-like immunoreactive fibers and terminals displayed morphology characteristic of degenerating axons (thickening, swollen varicosities and fragmentation). Two weeks after the 4-day treatment regimen, patterns of 5-HT-like immunostaining appeared similar to those noted immediately (i.e., 18 hr) after drug treatment; however, the presence of grossly deformed fibers and terminals seen shortly after drug treatment was lacking. Tyrosine hydroxylase-like immunoreactivity, used to assess changes in catecholamine-containing neurons, appeared unaffected by drug treatment. These data suggest that, in rats, high s.c. doses of fenfluramine may be neurotoxic to some 5-HT-like immunoreactive axons and terminals. The relevance of these observations to the continued therapeutic use in humans of smaller p.o. doses of fenfluramine remains to be determined.     

New chalcone derivatives: synthesis,antiviral activity and mechanism of action

In this work, twenty-eight chalcone derivatives containing a purine (sulfur) ether moiety were synthesized and their antiviral activities were evaluated. Biological results showed that compound 5d exhibited outstanding inactive activity against tobacco mosaic virus (TMV) in vivo (EC₅₀ = 65.8 μg mL⁻¹), which is significantly superior to that of ribavirin (EC₅₀ = 154.3 μg mL⁻¹). Transmission electron microscopy indicated that compound 5d can break the integrity of TMV particles. The results of microscale thermophoresis, fluorescence titration and molecular docking showed that compound 5d had stronger combining affinity (K_a = 1.02 ×10⁵ L mol⁻¹, K_d = 13.4 μmol L⁻¹) with TMV coat protein (TMV-CP), which is due to the formation of five hydrogen bonds between compound 5d and the amino-acid residues of TMV-CP. These findings revealed that compound 5d can effectively inhibit the infective ability of TMV. This work provides inspiration and reference for the discovery of new antiviral agents.

The chalcone derivatives containing a purine (sulfur) ether moiety were synthesized. The antiviral mechanism suggested that the antiviral activity of compound 5d may depend on its stronger binding affinity with TMV-CP.     

Angiotensin II stimulation of hydrogen ion secretion in the rat early proximal tubule. Modes of action, mechanism, and kinetics.

Physiologic concentrations of angiotensin II stimulate sodium transport by intestinal and renal early (S1) and late (S2) proximal tubule epithelial cells. We recently found that hydrogen ion secretion, which effects sodium bicarbonate absorption, was a transport function preferentially and potently increased by angiotensin II in S1 cells. S1 cells are normally responsible for half of the total renal hydrogen ion secretion. The mechanism by which angiotensin II regulates intestinal sodium transport is by potentiating sympathetic nerve activity and norepinephrine release. Direct control of hydrogen ion secretion by angiotensin II via receptors on epithelial cells has not been previously demonstrated. We now report that stimulation of in vivo hydrogen ion secretion in the rat early proximal tubule by angiotensin II was not mediated via change in nerve activity. Rather, enhanced hydrogen ion secretion by angiotensin II correlated with increased angiotensin II receptor density on epithelial cells in the early compared to late microdissected proximal tubule. Basolateral as well as luminal angiotensin II stimulated bicarbonate absorption. Angiotensin II reduced bicarbonate permeability and caused alteration in the apparent substrate affinity, but not maximal capacity, of the proximal hydrogen ion secretory system involving the Na+/H+ antiporter.     

Reduction of tryptophan hydroxylase activity and 5-hydroxytryptamine concentration in certain rat brain nuclei after p-chloroamphetamine.

High-pressure liquid chromatography and mass fragmentography have been used to determine tryptophan hydroxylase activity and 5-hydroxytryptamine (5-HT) concentration, respectively, in certain rat brain nuclei. (+/-)-p-Chloroamphetamine (100 mumol/kg i.p.) decreased enzyme activity in 5-HT terminals of the caudate, hippocampus and septum to a minimum level within 3 days. In hippocampus, but not in septum, the enzyme activity and 5-HT content remained maximally depleted (about 80%) for as long as 2 months after the p-chloroamphetamine injection. Of the 5-HT cell body areas investigated, only in the B9 nucleus was the tryptophan hydroxylase activity and 5-HT content still significantly reduced (about 25-50%) at 21 and 60 days after the drug injection. The enzyme activity and 5-HT content of the B7 and B8 nuclei were not significantly different from control values at these times. These results suggest that the primary site of action of p-chloroamphetamine is on nerve terminals followed by secondary effects on serotonergic cell bodies.     

Spinal cord stimulation of dorsal columns in a rat model of neuropathic pain: evidence for a segmental spinal mechanism of pain relief

Although spinal cord stimulation (SCS) of the dorsal columns is an established method for treating chronic neuropathic pain, patients still suffer from a substantial level of pain. From a clinical perspective it is known that the location of the SCS is of pivotal importance, thereby suggesting a segmental spinal mode of action. However, experimental studies suggest that SCS acts also through the modulation of supraspinal mechanisms, which might suggest that the location is unimportant. Here we investigated the effect of the rostrocaudal location of SCS stimulation and the effectiveness of pain relief in a rat model of chronic neuropathic pain. Adult male rats (n = 45) were submitted to a partial ligation of the sciatic nerve. The majority of animals developed tactile hypersensitivity in the nerve lesioned paw. All allodynic rats were submitted to SCS (n = 33) for 30 minutes (f = 50 Hz; pulse width 0.2 ms). In one group (n = 16) the electrodes were located at the level where the injured sciatic nerve afferents enter the spinal cord (T13), and in a second group (n = 17) the electrodes were positioned at more rostral levels (T11) as verified by X-ray. A repositioning experiment of electrodes from T12 to T13 was performed in 2 animals. Our data demonstrate that SCS of the dorsal columns at the level where the injured fibers enter the spinal cord dorsal horn result in a much better pain-relieving effect than SCS at more rostral levels. From this we conclude that SCS in treatment of neuropathic pain acts through a segmental spinal site of action.     

Ceftibuten--in-vitro activity against respiratory pathogens, beta-lactamase stability and mechanism of action

The in-vitro activity of ceftibuten against respiratory pathogenic bacteria was studied and compared with that of other oral beta-lactam agents. Ceftibuten displayed high activity against Haemophilus influenzae and Branhamella catarrhalis. There was reduced activity against Streptococcus pneumoniae (MIC90 16 mg/l). The protein binding of ceftibuten was 77% and the primary target site PBP 3. A high degree of stability to beta-lactamase hydrolysis was observed.     

Nadolol, propranolol, and thyroid hormones: evidence for a membrane-stabilizing action of propranolol

Ten normal subjects participated in a placebo-controlled, randomized, parallel study to determine the effects on thyroid hormones of chronic (4 wk) propranolol or nadolol, including observation for 2 wk after their discontinuation. Subjects took placebo for 1 wk, then propranolol or nadolol doses increased weekly to 240 mg/day by 3 wk. After 1 wk of placebo, after 2 wk of the highest dose of propranolol or nadolol, and 2, 4, 6, 9, and 13 days after their discontinuation, thyroid hormone levels were measured by radioimmunoassay and heart rate responses to exercise were assessed. Both drugs induced equal and high degrees of exercise tachycardia inhibition. Propranolol decreased 3,3'5-triiodothyronine (T3) levels, increased 3-3'-5'-triiodothyronine (rT3) levels, tended to increase thyroxine levels, but did not increase thyroid-stimulating hormone levels. After discontinuation of propranolol, rT3 levels slowly (day 6) returned to values after placebo, suggesting delayed recovery of 5'-deiodination. There was no evidence of any rebound in T3 levels after withdrawal of propranolol. Nadolol induced no significant changes in the thyroid hormones measured. The data agree with the known effects of propranolol on thyroid hormones in normal man and show that nadolol does not have these effects when given chronically at an equivalent beta-blocking dose. The likely explanation is that the membrane-stabilizing activity of propranolol alters thyroid physiology by interfering with 5'-deiodinase.     

Deposition in and release of vitamin D3 from body fat: evidence for a storage site in the rat

Vitamin D in all body tissues was radio-labeled by supplementing completely vitamin D-deficient weanling rats with oral vitamin D(3)-4-(14)C for 2 wk. All vitamin D was then withheld, and radioactivity and vitamin D content in a variety of organs and tissues were measured. Adipose tissue was found to contain by far the greatest quantity of radioactivity throughout the 3 month experimental period. Immediately after supplementation, half of the total radioactivity in adipose tissue corresponded to unaltered vitamin D(3), and the other half to polar metabolites and esters of vitamin D(3) and unidentified peak II. 1 month later there was approximately the same proportion but a decrease in the total quantity of each form. We conclude that adipose tissue is the major storage site for vitamin D(3) in its several forms. Unaltered vitamin D(3) was the principal storage form observed and presumably a source available for conversion to other metabolites during deprivation.     

Steroid modulation of the chloride ionophore in rat brain: structure-activity requirements, regional dependence and mechanism of action

Further in vitro studies of steroids active at the gamma-aminobutyric acidA (GABAA) receptor regulated Cl- channel labeled by [35S]-t-butylbicyclophosphorothionate ([35S]TBPS) reveal additional structural requirements necessary for activity. Evaluation of selected steroids for activity against TBPS-induced convulsions show similar requirements for activity. Interestingly, steroids (e.g., 5 alpha-pregnan-3 alpha, 20 alpha-diol) were identified that have high potency but limited efficacy as modulators of [35S]TBPS binding. These characteristics are reminiscent of the clinically useful benzodiazepines (BZs) such as clonazepam. However, interactions between the prototypical anesthetic-barbiturate, sodium pentobarbital, and steroids active at the Cl- channel suggest that they do not share a common site of action as allosteric modulators of [35S]TBPS and BZ receptor binding. The most potent steroid evaluated, 5 alpha-pregnan-3 alpha-ol-20-one, modulates [35S]TBPS binding at low concentrations (IC50 approximately 17 nM) in a regionally dependent manner. All [35S]TBPS binding sites appear to be functionally coupled to a steroid "modulatory site." Because several of the active steroids are metabolites of progesterone, their ability to inhibit the binding of [3H]promegestrone to the cytosolic progestin receptor in rat uterus was evaluated. Those steroids showing potent activity at the GABAA receptor-Cl- ionophore were inactive at the intracellular progestin receptor. Such specificity coupled with their high potency provide additional support for the hypothesis that some of these steroids may be involved in the homeostatic regulation of brain excitability via the GABAA-BZ receptor complex.     

MUC7 20-Mer: investigation of antimicrobial activity,secondary structure,and possible mechanism of antifungal action

This study was aimed at examining the spectrum of antimicrobial activity of MUC7 20-mer (N-LAHQKPFIRKSYKCLHKRCR-C; residues 32 to 51 of MUC7, the low-molecular-weight human salivary mucin, comprised of 357 residues) and comparing its antifungal properties to those of salivary histatin 5 (Hsn-5). We also examined the secondary structure of the 20-mer and the possible mechanism of its antifungal action. Our results showed that MUC7 20-mer displays potent killing activity against a variety of fungi and both gram-positive and gram-negative bacteria at micromolar concentrations. Time-dependent killing of Candida albicans and Cryptococcus neoformans by MUC7 20-mer and Hsn-5 indicated differences in killing rates between MUC7 20-mer and Hsn-5. The secondary structure prediction showed that MUC7 20-mer adopts an amphiphilic helix with distinguishable hydrophilic and hydrophobic faces (a characteristic that is associated with antimicrobial activity). In comparison to that of Hsn-5, the fungicidal activity of MUC7 20-mer against C. albicans seems to be independent of fungal cellular metabolic activity, as evidenced by its killing potency at a low temperature (4 degrees C) and in the presence of inhibitors of oxidative phosphorylation in the mitochondrial system. Fluorescence microscopy showed the ability of MUC7 20-mer to cross the fungal cell membrane and to accumulate inside the cells. The internalization of MUC7 20-mer was inhibited by divalent cations. Confocal microscopy of cells doubly labeled with MUC7 20-mer and a mitochondrion-specific dye indicated that mitochondria are not the target of MUC7 20-mer for either C. albicans or C. neoformans.     

Lipoprotein lipase: structure,function and mechanism of action

Summary Lipoprotein lipase (LPL) plays a central role in the hydrolysis of circulating triglycerides present in chylomicrons, and very low density lipoproteins. The active form of the enzyme is a non-covalent homodimer which contains multiple functional domains required for normal hydrolytic activity including a catalytic domain, as well as sites involved in co-factor, heparin and lipid binding. Recent studies involving site-directed mutagenesis, the elucidation of the three dimensional crystallographic structure of different lipases, as well as analysis of the molecular defects that result in the expression of the familial chylomicronemia syndrome have provided new insights into the structure-function relationship of LPL. As a result, our understanding of structural domains involved in catalysis, heparin, lipid binding, and enzyme-cofactor interaction as well as the mechanism of action of LPL as an acylglycerol hydrolase has been greatly enhanced.