Cysteine proteases of parasites: A remarkable diversity of function

Summary Papain family cysteine proteases function primarily intracellularly in higher eukaryotes, but are often extracellular proteases in protozoan helminths. The utility of this class of enzymes is reflected in the diversity of functions they perform in both parasite life cycles and the pathogenesis of parasitic diseases. Examples include secretion of proteases into the gut of parasitic worms for hemoglobin degradation, release from the surface of nematodes to degrade cuticular proteins during molting, and facilitating excystment of protozoa.     

Gallstones Containing Bacteria are Biofilms: Bacterial Slime Production and Ability to Form Pigment Solids Determines Infection Severity and Bacteremia

Objective Gallstone bacteria provide a reservoir for biliary infections. Slime production facilitates adherence, whereas β-glucuronidase and phospholipase generate colonization surface. These factors facilitate gallstone formation, but their influence on infection severity is unknown. Methods Two hundred ninety-two patients were studied. Gallstones, bile, and blood (as applicable) were cultured. Bacteria were tested for β-glucuronidase/phospholipase production and quantitative slime production. Infection severity was correlated with bacterial factors. Results Bacteria were present in 43% of cases, 13% with bacteremia. Severe infections correlated directly with β-glucuronidase/phospholipase (55% with vs 13% without, P < 0.0001), but inversely with slime production (55 vs 8%, slime <75 or >75, P = 0.008). Low slime production and β-glucuronidase/phospholipase production were additive: Severe infections were present in 76% with both, but 10% with either or none (P < 0.0001). β-Glucuronidase/phospholipase production facilitated bactibilia (86% with vs 62% without, P = 0.03). Slime production was 19 (±8) vs 50 (±10) for bacteria that did or did not cause bacteremia (P = 0.004). No bacteria with slime >75 demonstrated bacteremia. Conclusions Bacteria-laden gallstones are biofilms whose characteristics influence illness severity. Factors creating colonization surface (β-glucuronidase/phospholipase) facilitated bacteremia and severe infections; but abundant slime production, while facilitating colonization, inhibited detachment and cholangiovenous reflux. This shows how properties of the gallstone biofilm determine the severity of the associated illness. Presented at the annual meeting of the Society for Surgery of the Alimentary Tract, held May 20–24, 2006 in Los Angeles, California.     

The immunopathology of M cells

Conclusions Mucosal epithelia have two functions, which at first glance seem at variance with each other — maintenance of a barrier and uptake of antigens. The M cell is a unique solution to this problem. However, it also provides many mucosal and systemic pathogens with a convenient portal of invasion, and may also be the point of uptake of detrimental antigens, such as carcinogens and those that induce autoimmune disease. Much remains unknown about the functions of M cells and the precise nature of their interactions with pathogens. Determinants of pathogen tropism for M cells, in particular anatomic sites, must be elucidated before we can proceed to the task of modulating M cell antigenic uptake to enhance absorption and presentation of vaccines and to reduce systemic penetration of allergens and pathogenic microorganisms. Research in this area would be considerably advanced by the development of techniques to induce and maintain differentiated M cells in vitro, and the production of monoclonal antibodies specific for cells of this lineage. Irrespective of these gaps in our current knowledge, it has become increasingly apparent that the M cell may be of paramount importance in the initiation of colonization by many, if not all, important human and animal mucosal pathogens.     

Epidermal injury stimulates prenylation in the epidermis of hairless mice

Isoprenylation is the covalent attachment of isoprenyl groups, intermediates of the cholesterol biosynthesis pathway, to carboxy terminal cysteine residues of proteins. Numerous proteins are isoprenylated including small GTP binding proteins, trimeric G proteins, and nuclear lamins, and these prenylated proteins regulate a variety of cell functions, including cell growth, cytokinesis, and differentiation. Here, we quantitated protein prenylation and determined which proteins are prenylated in the epidermis of hairless mice by radiolabeling with ³ H-mevalonolactone following acute or chronic epidermal injury. In normal epidermis, four major radiolabeled bands, with molecular weights of 17–26, 48, 54, and 68 kDa, were observed. The levels of each of these bands increased by 24–63% 16 h following acute epidermal injury induced by topical acetone treatment or tape stripping, returning to normal by 24 h. On 2D gel electrophoresis, there were no major differences between the patterns of labeling following barrier disruption. Subacute epidermal injury induced by either acetone or tape stripping twice a day for 7 days and chronic injury induced by feeding an essential fatty acid-deficient (EFAD) diet, also resulted in a significant increase in protein prenylation. As with acute injury, SDS-PAGE and 2D gel electrophoresis did not reveal marked differences in the pattern of protein prenylation. These results demonstrate that the prenylation of proteins in the epidermis is stimulated by injury, suggesting that one or more of these prenylated species may be important in epidermal proliferation or differentiation. Received: 29 May 1996     

The effects of clonidine and yohimbine on human information processing

The effects of clonidine and yohimbine on human information processing were tested in six normal volunteers ages 18–30 years. Subjects were tested in a pre-post design with sessions conducted at weekly intervals. Three drug conditions were: Placebo (lactose), 0.2 mg clonidine, and 30 mg yohimbine. Two choice reaction time (RT) tasks were used. One was a stimulus evaluation-response selection task (SERS) that has been shown to be sensitive tod-amphetamine, methylphenidate and scopolamine. The other task was to assess stimulus pre-processing and used spatial frequency as a discriminative stimulus. The principle finding was that clonidine slowed RT; this effect was significant for both tasks. In contrast, yohimbine tended to speed RT, but the effects were significant only for the spatial frequency task on some analyses while not for others. RTs to high spatial frequency stimuli were speeded more than for low spatial frequency. The effects of these two NE drugs were compared with findings withd-amphetamine and scopolamine and interpreted within the framework of a serial information processing model proposed by Callaway (1983). Specifically, it is suggested that yohimbine and clonidine affect an early pre-processing stage.     

Aberrations of growth factor control in metastatic follicular thyroid cancerin vitro

The aggressiveness of follicular thyroid cancer (FTC) varies widely, and metastasis is the primary cause of death. Uncontrolled proliferation of cancer cells may be associated with loss of growth factor control. We investigated the effects of stimulating (epidermal growth factor [EGF]; thyreotropin [TSH] in low concentrations) and inhibiting growth factors (transforming growth factor beta 1 [TGF beta 1]; TSH in high concentrations) on invasion and growth of FTC cell lines from the thyroid tumor (FTC133) and from the lymph node (FTC236) and lung (FTC238) metastases of the same patient. Invasion—penetration through an 8m pore membrane, covered by Matrigel (basement membrane)—and growth were measured using the MTT-method. EGF (10 ng/ml) and TSH in low concentrations (1 mU/ml) stimulated invasion and growth of all FTC cell lines, but the amplitude of stimulation differed significantly. The parental cell line FTC133 was considerably more responsive to growth factor stimulation than the metastatic clones. Invasion of FTC133 was enhanced by 42% (EGF;p<0.02) and 21% (TSH;p<0.01), invasion of FTC236 by 8% (EGF;p<0.02) and 8% (TSH;p<0.01), and invasion of FTC238 by 9% (EGF;p<0.02) and 8% (TSH;p<0.01). Conversely, invasion and growth of FTC133 were significantly more inhibited by TGF beta 1 (10 ng/ml) and supraphysiologic concentrations of TSH (100 mU/ml) than the cell lines from the lymph node and lung metastases. At day 7, invasion of FTC133 was inhibited by 32% (TGF beta 1;p<0.02) and 21% (TSH;p<0.01), invasion of FTC236 by 18% (TGF beta 1;p<0.02) and 11% (TSH;p<0.01), and invasion of FTC238 by 16% (TGF beta 1;p<0.02) and 12% (TSH;p<0.01). Moreover, we analyzed growth factor independence in minimally supplemented or unsupplemented medium. Growth, but no invasion was evident when cells were cultured completely unsupplemented over 7 days. These results suggest that metastatic FTCs may have developed by escaping from the normal control of TSH and other growth factors.     

Alpha1-adrenoceptor-mediated inhibition of cellular cAMP accumulation in neonatal rat ventricular myocytes

Summary We studied adrenergic regulation of cellular cAMP in neonatal rat ventricular myocytes. Since CAMP content depends on synthesis, breakdown and egress, the contribution of each of these mechanisms was assessed. In the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, cAMP accumulation stimulated by the -adrenoceptor agonist (–)-isoprenaline was diminished when the mixed + adrenoceptor agonist (–)-noradrenaline was coincubated with (–)-isoprenaline. Moreover, adenylyl cyclase activation stimulated by (–)-isoprenaline was decreased by (–)-noradrenaline and by the selective a₁-adrenoceptor agonists (–)-phenylephrine and methoxamine, suggesting that -adrenoceptor agonism regulates CAMP metabolism through its effect on the synthetic pathway. Evidence for ₁-adrenoceptor mediation of this response was enhancement of (–)-noradrenaline-induced cAMP generation by the selective ₁-adrenoceptor antagonist terazosin (10 nmol/l). The selective ₂-adrenoceptor antagonist yohimbine (10 nmol/l) had no effect. The ₁-adrenoceptor mediated depression of (–)-isoprenaline-stimulated CAMP generation and adenylyl cyclase activation was prevented by terazosin and in separate experiments markedly enhanced by pertussis toxin pretreatment, suggesting involvement of a guanine-nucleotide regulatory protein in this process.Occupation of the ₁-adrenoceptor by (–)-noradrenaline did not accelerate the rate of CAMP breakdown in the absence of phosphodiesterase inhibition. Furthermore, there was no enhancement of total phosphodiesterase activity by (–)-noradrenaline in the presence of (–)-propranolol. By contrast, pertussis toxin pretreatment augmented phosphodiesterase activity. Neither pertussis toxin nor (–)-noradrenaline increased CAMP egress.We conclude that in rat neonatal cardiac myocytes agonist occupation of the ₁-adrenoceptor inhibits -adrenoceptor stimulated CAMP accumulation most likely by coupling to a guanine nucleotide inhibitory protein.Supported by a grant from the Department of the Veterans Affairs Research Service and Program Project Grant HL 25847 from the National Heart, Lung and Blood Institute     

What might be the impact on neurology of the analysis of brain metabolism by in vivo magnetic resonance spectroscopy?

In vivo nuclear magnetic resonance spectroscopy (MRS) of the human brain is a recently developed technique which allows to assay noninvasively in vivo key molecules of brain metabolism. After a review of the origin of the signals detected by phosphorus and proton MRS of human brain, the impact of MRS on clinical neurology is examined. MRS of the brain does not purport to be a metabolic biopsy, but unique applications for brain MRS are (1) quantitating the oxidative state of the brain and defining neuronal death, (2) assessing and mapping neuron damage, (3) evaluating membrane alterations, and (4) characterizing encephalopathies. In the near future brain MRS will be performed routinely after conventional MRI, as a valuable metabolic (and functional) complement to the anatomical evaluation of cerebral pathologies, particularly the toxic, metabolic and infectious encephalopathies.     

Novel pharmacologic therapy of heart failure

Opinion statement The prevalence of congestive heart failure is progressively increasing and despite recent advances in therapeutics, there is a continuing need for novel effective therapies. New, investigational treatment strategies include inotropic drugs, neurohormonal antagonists, anticytokine and anti-inflammatory strategies, hormonal therapies, and nutritional supplements. Current positive inotropes (eg, dobutamine and phosphodiesterase inhibitors) provide symptomatic relief, but newer agents may have a better adverse effect profile. Angiotensin-converting enzyme (ACE) inhibitors should remain first-line treatment with angiotensin receptor blockers used in ACE inhibitor-intolerant patients. Many new neurohormonal antagonists have recently been investigated and eplerenone has demonstrated clinical benefit. New hormonal, anticytokine, and anti-inflammatory therapies have shown benefit in small trials, but results in larger trials have been disappointing. Other approaches are currently being tested in large trials that will clarify their role. Nutritional supplements need to be tested in a large prospective trial before they can be recommended.