Adaptive acid tolerance response in Listeria monocytogenes: isolation of an acid-tolerant mutant which demonstrates increased virulence.

The ability of Listeria monocytogenes to tolerate low-pH environments is of particular importance because the pathogen encounters such environments in vivo, both during passage through the stomach and within the macrophage phagosome. In our study, L. monocytogenes was shown to exhibit a significant adaptive acid tolerance response following a 1-h exposure to mild acid (pH 5.5), which is capable of protecting cells from severe acid stress (pH 3.5). Susceptibility to pH 3.5 acid is growth phase dependent. Stationary-phase Listeria cultures are naturally resistant to the challenge pH (pH 3.5), while exponential-phase cultures require adaptation at pH 5.5 to induce acid tolerance. Adaptation requires protein synthesis, since treatment with chloramphenicol prevents the development of acid tolerance. Induction of the acid tolerance response also protects L. monocytogenes against the effect of other environmental stresses. Acid-adapted cells demonstrate increased tolerance toward thermal stress, osmotic stress, crystal violet, and ethanol. Following prolonged exposure of L. monocytogenes to pH 3.5, we isolated mutants which constitutively demonstrate increased acid tolerance at all stages of the growth cycle. These mutants do not display full acid tolerance, but their resistance to low pH can be further increased following adaptation to mild-acid conditions. The mutants demonstrated increased lethality for mice relative to that of the wild type when inoculated by the intraperitoneal route. When administered as lower inocula, the mutants reached higher levels in the spleens of infected mice than did the wild type. The data suggest that low-pH conditions may have the potential to select for L. monocytogenes mutants with increased natural acid tolerance and increased virulence.     

Selective translocation of protein kinase C-delta in PC12 cells during nerve growth factor-induced neuritogenesis.

The specific intracellular signals initiated by nerve growth factor (NGF) that lead to neurite formation in PC12 rat pheochromocytoma cells are as of yet unclear. Protein kinase C-delta (PKC delta) is translocated from the soluble to the particulate subcellular fraction during NGF-induced-neuritogenesis; however, this does not occur after treatment with the epidermal growth factor, which is mitogenic but does not induce neurite formation. PC12 cells also contain both Ca(2+)-sensitive and Ca(2+)-independent PKC enzymatic activities, and express mRNA and immunoreactive proteins corresponding to the PKC isoforms alpha, beta, delta, epsilon, and zeta. There are transient decreases in the levels of immunoreactive PKCs alpha, beta, and epsilon after 1-3 days of NGF treatment, and after 7 days there is a 2.5-fold increase in the level of PKC alpha, and a 1.8-fold increase in total cellular PKC activity. NGF-induced PC12 cell neuritogenesis is enhanced by 12-O-tetradecanoyl phorbol-13-acetate (TPA) in a TPA dose- and time-dependent manner, and this differentiation coincides with abrogation of the down-regulation of PKC delta and other PKC isoforms, when the cells are treated with TPA. Thus a selective activation of PKC delta may play a role in neuritogenic signals in PC12 cells.     

Endocytotic uptake of fluorescent dextrans by pollen tubes grown in vitro

Summary Pollen tubes grow by tip growth, with high levels of exocytosis at the apex. The commercial availability of FITC labelled -linked dextrans provides a source of biologically inert tracers for endocytotic activity in pollen tubes. Growing tubes ofNicotiana andTradescantia were transferred to media containing 1% FD-4 for varying period of time before washing in control media and observation in a fluorescence microscope. Fluorescent material appeared to enter the pollen tubes only at the tip region, and to accumulate in vacuoles, starting with smaller vacuoles near the tip and spreading to the main vacuolated part of the tube. Mature tubes, with callose plugs, were only labelled up to the first complete plug from the tip, younger tubes without plugs were labelled into the pollen grain vacuole. The fluorescent material within the pollen tubes was shown to represent uptake of intact high molecular weight dextran by the following criteria: (i) free FITC and low molecular weight dextrans could not be detected in any of the media or pollen tubes using thin layer chromatography and (ii) pollen tube growth rates were unaffected by the fluorescent dextran, but were severely inhibited by low levels of free FITC. It was concluded that the dextrans entered the tubes by endocytosis, possibly in the tip region, and were then transferred to the vacuole system of the pollen tube.Abbreviations FITC fluorescein isothiocyanate - FD fluorescent dextran     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Kinetic modeling of a multiple immobilized enzyme system. I. Development and testing of the model

A kinetic model for the reaction sequence catalyzed by coimmobilized invertase and glucose oxidase with a sucrose substrate in a tubular reactor has been developed. The computerized mathematical model employs and orthogonal collection technique for solving oxidase were coimmobilized in poly(2-hydroxyethlmethacrylate) gels and used in a continuous flow packed-bed tubular reactor system. In addition to describing the development of the kinetic model, this article compares experimentally determined reactor effluent concentrations for various sucrose feed solutions to those predicted by the model. Variations between experimental and predicted reactor effluent concentrations were found to be on the micromolar level for sucrose feed concentrations as low as 1.38mM.     

Defective Glucose Transport Across Brain Tissue Barriers: A Newly Recognized Neurological Syndrome

Impaired glucose transport across brain tissue barriers causes infantile seizures, developmental delay and acquired microcephaly. Since the first report in 1991 (De Vivo et al, NEJM, 1991) 17 patients have been identified with the glucose transporter protein syndrome (GTPS). The diagnostic feature of the syndrome is an unexplained hypoglycorrhachia in the clinical setting of an infantile epileptic encephalopathy. We review our clinical experience by highlighting one illustrative case: a 6-year old girl who presented at age 2 months with infantile seizures and hypoglycorrhachia. The CSF/blood glucose ratio was 0.33. DNA sequencing identified a missense mutation in exon 7 (C1108T). Erythrocyte GLUT1 immunoreactivity was normal. The time course of 3-0-methyl-glucose (3OMG) uptake by erythrocytes of the patient was 46% that of mother and father. The apparent K_m was similar in all cases (2–4 mmol/L), but the apparent V_max in the patient was only 28% that of the parents (500 versus 1,766 fmol/s/10⁶RBC; p < 0.004). In addition, a 3-month trial of oral thioctic acid also benefited the patient and increased the V_max to 935 fmol/s/10⁶ RBC (p < 3 × 10^–7). Uptake of dehydroascorbic acid by erythrocytes of the patient was impaired to the same degree as that of 3OMG (V_max was 38% of that of the mother's), which supports previous observations of GLUT1 being multifunctional. These studies confirm the molecular basis of the GTPS and the multifunctional role of GLUT1. The need for more effective treatment is compelling.     

Carbonylation of glycolytic proteins is a key response to drug-induced oxidative stress and apoptosis

Recent work has highlighted the importance of protein post-translational modifications such as phosphorylation (enzymatic) and nitrosylation (nonenzymatic) in the early stages of apoptosis. In this study, we have investigated the levels of protein carbonylation, a nonenzymatic protein modification that occurs in conditions of cellular oxidative stress, during etopside-induced apoptosis of HL60 cells. Within 1 h of VP16 treatment, a number of proteins underwent carbonylation due to oxidative stress. This was inhibited by the antioxidant N-acetyl-L-cysteine. Among the proteins found to be carbonylated were glycolytic enzymes. Subsequently, we found that the rate of glycolysis was significantly reduced, probably due to a carbonylation mediated reduction in enzymatic activity of glycolytic enzymes. Our work demonstrates that protein carbonylation can be rapidly induced through cytotoxic drug treatment and may specifically inhibit the glycolytic pathway. Given the importance of glycolysis as a source of cellular ATP, this has severe implications for cell function.     

Is nebulized saline a placebo in COPD?

Background

Many trials of nebulized therapy have used nebulized saline as a "placebo". However, nebulized isotonic saline is sometimes used to assist sputum expectoration and relieve breathlessness in COPD patients. We designed this study to establish if nebulized saline had a placebo effect or a clinical effect.

Methods

40 patients were studied following hospital admission for exacerbated COPD (mean FEV1 30% predicted). Patients were randomised to single-blind administration of either 4 mls of nebulized isotonic saline using an efficient nebulizer (active group n = 20) or an inefficient nebulizer (placebo group n = 20). Spirometry and subjective breathlessness scores (Modified Likert Scale) were measured before nebulized treatment and 10 minutes after treatment.

Results

There was no significant change in FEV1 after active or placebo nebulized saline treatment. Patients reported a 4% improvement in mean breathlessness score following placebo (Wilcoxon test; p = 0.37) compared with 23% improvement following active nebulized saline (p = 0.0001). 65% of patients given active nebulized saline but only 5% of the placebo group reported that mucus expectoration was easier after the treatment.

Conclusions

This study lends support to the current use of nebulized saline to relieve breathlessness (possibly by facilitating sputum clearance) in COPD patients. Lung function was not affected. Nebulized saline can therefore be used as a placebo in bronchodilator studies involving COPD patients but it cannot be used as a placebo in trials assessing symptom relief.