Oral immunization with ATP-dependent protease-deficient mutants protects mice against subsequent oral challenge with virulent Salmonella enterica serovar typhimurium

We evaluated the efficacy of mutants with a deletion of the stress response protease gene as candidates for live oral vaccine strains against Salmonella infection through infection studies with mice by using a Salmonella enterica serovar Typhimurium mutant with a disruption of the ClpXP or Lon protease. In vitro, the ClpXP protease regulates flagellum synthesis and the ClpXP-deficient mutant strain exhibits hyperflagellated bacterial cells (T. Tomoyasu et al., J. Bacteriol. 184:645-653, 2002). On the other hand, the Lon protease negatively regulates the efficacy of invading epithelial cells and the expression of invasion genes (A. Takaya et al., J. Bacteriol. 184:224-232, 2002). When 5-week-old BALB/c mice were orally administered 5 x 10(8) CFU of the ClpXP- or Lon-deficient strain, bacteria were detected with 10(3) to 10(4) CFU in the spleen, mesenteric lymph nodes, Peyer's patches, and cecum 1 week after inoculation and the bacteria then decreased gradually in each tissue. Significant increases of lipopolysaccharide-specific immunoglobulin G (IgG) and secretory IgA were detected at week 4 and maintained until at least week 12 after inoculation in serum and bile, respectively. Immunization with the ClpXP- or Lon-deficient strain protected mice against oral challenge with the serovar Typhimurium virulent strain. Both the challenged virulent and immunized avirulent salmonellae were completely cleared from the spleen, mesenteric lymph nodes, Peyer's patches, and even cecum 5 days after the challenge. These data indicate that Salmonella with a disruption of the ATP-dependent protease ClpXP or Lon can be useful in developing a live vaccine strain.     

Therapy with nebulized beta2 agonists (procaterol) in asthmatic children: pulmonary function and plasma levels after inhalation]

BACKGROUND: Relationship between post administrative changes in plasma drug levels and bronchodilation remains unknown. In this study, we measured plasma levels of procaterol, a beta2-agonist, when being inhaled through nebulizers in children with bronchial asthma to examine relationship between improvement of pulmonary function and the plasma levels. METHOD: Six asthmatic children with the mean age of 9.8 years, inhaled 0.3 ml of 0.01% procaterol solution through a nebulizer. We examined changes in pulmonary function and plasma procaterol levels before and after inhalation. RESULTS: Procaterol was detected in the plasma 2 minutes after inhalation when it already rose to the maximum level, and kept the steady until showing a decline in 30 minutes. The measured highest value was 87.8+/-45.1 pg/ml. FEV 1.0 remarkably increased 2 minutes after inhalation and was maintained until 60 minutes after inhalation. Other lung function parameters also improved. There was no significant change in the heart rate, but serum potassium concentrations significantly dropped in all patients 60 minutes after inhalation. CONCLUSION: Plasma procaterol levels promptly rose to the peak at 2 minutes after inhalation and decreased 30 minutes later. Improvement of pulmonary function started promptly at minutes after inhalation and it became a peak 60 minutes later.     

Thin-filament-binding domains of cardiac and fast skeletal muscle troponin I isoforms as studied by epitope tagging

We examined the binding domains of cardiac and fast skeletal muscle troponin I (CTnI and FTnI, respectively) to myofibrils (MFs). Deletion mutants containing CTnI amino acid residues 1–79, 43–207 and 80–207 (CTnI-head, CTnI-tail-1 and CTnI-tail-2, respectively) and FTnI amino acid residues 1–54 and 55–182 (FTnI-head and FTnI-tail, respectively) were transiently expressed in cardiac and fast skeletal muscle cells. To monitor the intracellular localization of these exogenously introduced truncated TnIs, epitope tagging was used. CTnI-tail-1 was incorporated into cardiac MFs specifically, but CTnI-tail-2 was not assembled onto any MFs examined. This suggests that there is no potent actin filament-binding site in CTnI-tail-2. Since CTnI-tail-1 has an amino acid extension (CTnI residues 43–79) whose sequence is longer than that of CTnI-head-2; it appears that this sequence extension is important in binding to cardiac MFs. FTnI-tail, containing the inhibitory domain of actomyosin ATPase, showed intensive and specific incorporation into fast MFs. FTnI-tail was a homologous fragment of CTnI-tail-2, but the binding patterns of these two domains differed greatly from each other. It is possible that the absence of potent binding affinity of CTnI-tail-2 corresponding to the inhibitory domain of actomyosin ATPase is advantageous for continuous cardiac muscle contraction, since a potent inhibitory activity is a serious obstacle to cardiac muscle contraction. It can be assumed that distinctive binding ability of functional domains of TnI-tails reflect unique adaptations to muscles with different physiological properties.     

Expression and localization of chromogranin A gene and protein in human submandibular gland

Human saliva chromogranin A (CgA) is clinically promising as a psychological stress marker. However, expression of CgA is poorly understood in humans, although salivary gland localization of CgA in other mammals, such as rodents and horses, has been demonstrated. In the present study, we investigated the expression and localization of CgA in the human submandibular gland (HSG) using various methods. CgA was consistently localized in serous and ductal cells in HSG, as detected by immunohistochemistry and in situhybridization. Reactivity was stronger in serous cells than in ductal cells. In addition, strong immunoreactivity for CgA was observed in the saliva matrix of ductal cavities. Western blotting gave one significant immunoreactive band of 68 kDa in the adrenal gland, HSG and saliva. Finally, CgA was detected in secretory granules of serous and ductal cells by immunoelectron microscopy. In conclusion, CgA in humans is produced by HSG and secreted into saliva.     

Inclusion body hepatitis due to adenovirus in pigeons

Inclusion body hepatitis (IBH) occurred on a pigeon farm of Japan. Basophilic and/or eosinophilic inclusion bodies were found in nuclei of many hepatocytes of all the four pigeons examined pathologically. The same inclusions were seen in epithelial cells of the uriniferous tubules and enterocytes of the small intestine. Ultrastructurally, the hepatic inclusions had viral particles morphologically identical to adenoviruses. The inoculum prepared from the affected liver produced neither cytopathic effects in chick embryo fibroblasts nor pock lesions on chorioallantoic membranes of developing chick embryos.     

Stress-responsive protein kinases in redox-regulated apoptosis signaling

Both extra- and intracellular stimuli elicit a wide variety of responses, such as cell survival, proliferation, differentiation, and apoptosis, through regulation of cell signaling. Recent studies have revealed that stress-responsive signal transduction pathways are strictly regulated by the intracellular redox state. The redox state of the cell is a consequence of the precise balance between the levels of oxidizing and reducing equivalents, such as reactive oxygen species (ROS) and endogenous antioxidants. The generation of ROS fluctuates in response to alterations of both external and internal environment and, in turn, triggers specific signaling cascades, including mitogen-activated protein kinases, which determine cell survival or cell death. This review focuses on the regulatory mechanisms of stress-responsive protein kinases and their involvement in oxidative stress-induced apoptosis. It also provides recent findings on the molecular mechanisms by which redox signaling cross-talks with stress-responsive protein kinase cascades.     

Differences in regional wash-in and wash-out time constants for xenon-CT ventilation studies

Xenon-enhanced computed tomography (Xe-CT) has been used to measure regional ventilation by determining the wash-in (WI) and wash-out (WO) rates of stable Xe. We tested the common assumption that WI and WO rates are equal by measuring WO-WI in different anatomic lung regions of six anesthetized, supine sheep scanned using multi-detector-row computed tomography (MDCT). We further investigated the effect of tidal volume, image gating (end-expiratory EE versus end-inspiratory EI), local perfusion, and inspired Xe concentration on this phenomenon. RESULTS: WO time constant was greater than WI in all lung regions, with the greatest differences observed in dependent base regions. WO-WI time constant difference was greater during EE imaging, smaller tidal volumes, and with higher Xe concentrations. Regional perfusion did not correlate with WI-WO. We conclude that Xe-WI rate can be significantly different from the WO rate, and the data suggest that this effect may be due to a combination of anatomic and fluid mechanical factors such as Rayleigh-Taylor instabilities set up at interfaces between two gases of different densities.