Selective association and transport of Campylobacter jejuni through M cells of rabbit Peyer's patches

M cells in the Peyer's patches may facilitate transport of pathogens such as Campylobacter jejuni from the intestine. We evaluated this hypothesis by using electron microscopy to examine Peyer's patches in ligated adult rabbit ileal loops inoculated with 5-mL suspensions of 10(9) cfu/mL of Campylobacter jejuni. Peyer's patches taken at intervals from 15 min to 2 h after inoculation of loops in anaesthetized rabbits provided evidence that Campylobacter jejuni selectively adhered to M cells as opposed to absorptive epithelial cells and was transported, apparently intact, into the M cell follicle. Although intercellular organisms were seen within the follicle, many others were phagocytosed by lymphoid cells. The proximity of the lymphatic and blood circulatory systems to the M cell follicle makes this a probable route for systemic spread of Campylobacter jejuni.     

Single-channel studies on linear gramicidins with altered amino acid side chains. Effects of altering the polarity of the side chain at position 1 in gramicidin A.

The modulation of gramicidin A single-channel characteristics by the amino acid side chains was investigated using gramicidin A analogues in which the NH2 terminal valine was chemically replaced by other amino acids. The replacements were chosen such that pairs of analogues would have essentially isosteric side chains of different polarities at position 1 (valine vs. trifluorovaline or hexafluorovaline; norvaline vs. S-methyl-cysteine; and norleucine vs. methionine). Even though the side chains are not in direct contact with the permeating ions, the single-channel conductances for Na+ and Cs+ are markedly affected by the changes in the physico-chemical characteristics of the side chains. The maximum single-channel conductance for Na+ is decreased by as much as 10-fold in channels formed by analogues with polar side chains at position 1 compared with their counterparts with nonpolar side chains, while the Na+ affinity is fairly insensitive to these changes. The relative conductance changes seen with Cs+ were less than those seen with Na+; the ion selectivity of the channels with polar side chains at position 1 was increased. Hybrid channels could form between compounds with a polar side chain at position 1 and either valine gramicidin A or their counterparts with a nonpolar side chain at position 1. The structure of channels formed by the modified gramicidins is thus essentially identical to the structure of channels formed by valine gramicidin A. The polarity of the side chain at position 1 is an important determinant of the permeability characteristics of the gramicidin A channel. We discuss the importance of having structural information when interpreting the functional consequences of site-directed amino acid modifications.     

Characterization of calcium-activated, phospholipid-dependent protein kinase from rat serosal mast cells and RBL-1 cells

Evidence is presented that rat serosal mast cells and cells of the rat basophilic leukemia line, RBL-1, each contain a calcium-activated, phospholipid-dependent protein kinase. The enzymes are very similar in their activation requirements to the calcium-dependent enzymes termed protein kinase Cs in brain. The enzyme is selectively stimulated by diolein and phosphatidylserine and is inhibited by several local anesthetics. The Ka for Ca2+ is 1.0 X 10(-3) M and 1.5 X 10(-4) M in mast cells and RBL-1 cells, respectively. The enzyme in mast cells is rapidly activated and apparently changed in its intracellular distribution when intact mast cells are stimulated with 48/80, A-23187, and anti-IgE and 12-O-tetradecanoyl-13-acetate in combination.     

Factors Affecting High-Oxygen Survival of Heterotrophic Microorganisms from an Antarctic Lake

We sought to determine factors relating to the survival of heterotrophic microorganisms from the high-dissolved-oxygen (HDO) waters of Lake Hoare, Antarctica. This lake contains perpetual HDO about three times that of normal saturation (40 to 50 mg liter⁻¹). Five isolates, one yeast and four bacteria, were selected from Lake Hoare waters by growth with the membrane filter technique with oxygen added to yield dissolved concentrations 14 times that in situ, 175 mg liter⁻¹. One bacterial isolate was obtained from the microbial mat beneath the HDO waters. This organism was isolated at normal atmospheric oxygen saturation. The bacteria were gram-negative rods, motile, oxidase positive, catalase positive, and superoxide dismutase positive; they contained carotenoids. The planktonic isolates grew in media containing 10 mg of Trypticase soy (BBL Microbiology Systems)-peptone (2:1) liter⁻¹ but not at 10 g liter⁻¹. Under low-nutrient levels simulating Lake Hoare waters (10 mg liter⁻¹), two of the planktonic isolates tested were not inhibited by HDO. Growth inhibition by HDO increased as nutrient concentration was increased. A carotenoid-negative mutant of one isolate demonstrated a decreased growth rate, maximal cell density, and increased cell lysis in the death phase under HDO compared with the parent strain. The specific activity of superoxide dismutase was increased by HDO in four of the five bacterial isolates. The superoxide dismutase was of the manganese type on the basis of inhibition and electrophoretic studies. The bacterial isolates from Lake Hoare possess several adaptations which may aid their survival in the HDO waters, as well as protection due to the oligotrophic nature of the lake.     

Expression and purification of glutamine synthetase cloned from Bacteroides fragilis

A glutamine synthetase (GS) gene, glnA, from Bacteroides fragilis was cloned on a recombinant plasmid pJS139 which enabled Escherichia coli glnA deletion mutants to utilize (NH4)2SO4 as a sole source of nitrogen. DNA homology was not detected between the B. fragilis glnA gene and the E. coli glnA gene. The cloned B fragilis glnA gene was expressed from its own promoter and was subject to nitrogen repression in E. coli, but it was not able to activate histidase activity in an E. coli glnA ntrB ntrC deletion mutant containing the Klebsiella aerogenes hut operon. The GS produced by pJS139 in E. coli was purified; it had an apparent subunit Mr of approximately 75,000, which is larger than that of any other known bacterial GS. There was very slight antigenic cross-reactivity between antibodies to the purified cloned B. fragilis GS and the GS subunit of wild-type E. coli.     

Effect of serotonin on murine macrophages: suppression of Ia expression by serotonin and its reversal by 5-HT2 serotonergic receptor antagonists

Serotonin (5-HT), a mediator released from platelets at sites of inflammation, suppressed IFN-gamma-induced Ia expression in mouse bone marrow macrophages maintained in vitro. (Mean percent suppression = 63.9% +/- 9.2, n = 40.) This suppression was not toxic or endotoxin-related, was concentration-dependent, and occurred at the physiologic concentrations of 5-HT present at inflammatory sites. The concentration of 5-HT producing the half-maximal effect was 2.5 to 5.5 X 10(-8) M. Related compounds, dopamine, histamine, and tryptamine, were much less potent in suppressing IFN-gamma-induced Ia, with maximally suppressing concentrations more than 100-fold higher than the maximally suppressing 5-HT concentration. L-5-hydroxytryptophan (5-HTP), the most potent analog tested, was 10-fold less potent than 5-HT in suppressing Ia expression. The concentration of 5-HTP producing the half-maximal effect = 4 X 10(-7) M. 5-HT suppression of IFN-gamma-induced Ia expression was antagonized by the 5-HT2 type receptor antagonists spiperone, ketanserin, and LY53857. Concentrations of these agents resulting in 50% inhibition of the serotonin effect were 1.5 X 10(-8) M, 7.5 X 10(-8) M, and 4.5 X 10(-12) M, respectively. 5-HT was most effective in suppressing IFN-gamma-induced Ia when added early in culture simultaneously with IFN-gamma. These data provide functional evidence that 5-HT suppression of IFN-gamma-induced Ia expression is mediated through a 5-HT receptor with some characteristics of the 5-HT2 type. 5-HT may play a physiologic role at sites of inflammation as a modulator of the effects of IFN-gamma on macrophage function.     

Effects of inspiratory resistance loading on lung fluid balance in awake sheep

Because pulmonary edema has been associated clinically with airway obstruction, we sought to determine whether decreased intrathoracic pressure, created by selective inspiratory obstruction, would affect lung fluid balance. We reasoned that if decreased intrathoracic pressure caused an increase in the transvascular hydrostatic pressure gradient, then lung lymph flow would increase and the lymph-to-plasma protein concentration ratio (L/P) would decrease. We performed experiments in six awake sheep with chronic lung lymph cannulas. After a base-line period, we added an inspiratory load (20 cmH2O) and allowed normal expiration at atmospheric pressure. Inspiratory loading was associated with a 12-cmH2O decrease in mean central airway pressure. Mean left atrial pressure fell 11 cmH2O, and mean pulmonary arterial pressure was unchanged; calculated microvascular pressure decreased 8 cmH2O. The changes that occurred in lung lymph were characteristic of those seen after other causes of increased transvascular hydrostatic gradient, such as increased intravascular pressure. Lung lymph flow increased twice base line, and L/P decreased. We conclude that inspiratory loading is associated with an increase in the pulmonary transvascular hydrostatic gradient, possibly by causing a greater fall in interstitial perimicrovascular pressure than in microvascular pressure.     

Subunit interactions of class I histocompatibility antigens

The kinetics of dissociation of iodinated beta 2-microglobulin (beta 2m) from the papain-solubilized class I histocompatibility antigen HLA-B7 have been investigated. In the presence of unlabeled beta 2m, most of the HLA dissociates according to a single rate constant, whereas in the absence of unlabeled beta 2m, the system approaches an equilibrium dependent upon the initial HLA concentration. When iodinated beta 2m is incubated with unlabeled HLA-B7, the rate of incorporation of beta 2m into the complex is much less dependent on the concentration than is expected for a simple association/dissociation system; instead, the system behaves as if the "activity" (in a thermodynamic sense) of the HLA heavy-chain intermediate cannot surpass a critical concentration. The dissociation rate for each class I specificity is a function of temperature, ionic strength, pH, and the status of the heavy chain (papain solubilized vs. detergent solubilized). High temperature, high ionic strength, and extremes of pH promote dissociation. The intact molecule dissociates about 10 times more slowly than the papain-solubilized molecule. In contrast, the rate of dissociation of all papain-solubilized class I antigens tested falls within the range of about a factor of 2. The presence of the carbohydrate has no effect on the rate of dissociation. The possibility that HLA class I antigen dissociation may occur in vivo within acidic internal vesicles is discussed.