Amino Acid Transport into Cultured Tobacco Cells: I. LYSINE TRANSPORT

Lysine transport into suspension-cultured Wisconsin-38 tobacco cells was observed. Uptake was linear (up to 90 minutes) with respect to time and amount of tissue only after 4 to 6 hours preincubation in calcium-containing medium. The observed cellular accumulation of lysine was against a concentration gradient and not due to exchange diffusion. Transport was stimulated by low pH and characterized by a biphasic uptake isotherm with two K(m) values for lysine. System I (K(m) approximately 5 x 10(-6) molar; V(max) approximately 180 nanomoles per gram fresh weight per hour) and system II (K(m) approximately 10(-4) molar; V(max) approximately 1900 nanomoles per gram fresh weight per hour) were inhibited by N-ethylmaleimide and a variety of respiratory inhibitors. This inhibition was not due to increased efflux. In antagonism experiments, system I was inhibited most effectively by basic amino acids, followed by the sulfur amino acids. System I was only slightly inhibited by the neutral and aromatic amino acids and was not inhibited by the acidic amino acids aspartic and glutamic acids. Transport by system II was inhibited by all of the tested amino acids (including aspartic and glutamic acids) and analogs; however, this system was not inhibited by d-arginine. Neither system was strongly inhibited by d-lysine or the lysine analog S-2-aminoethyl-l-cysteine. Arginine was shown to be a competitive inhibitor of both systems with values for K(i) similar to the respective K(m) values.These studies suggest the presence of at least two amino acid permeases in W-38 tobacco cells.     

Visual detection of granulocyte surface antigens using the avidin-biotin complex

A visual test for detection of granulocyte surface markers using the avidin-biotin complex (ABC) has been developed. That this assay is highly specific, reproducible, and sensitive was determined by studying the expression of HLA antigens on granulocytes with monoclonal antibodies. Further, using granulocyte specific alloantisera, the results of the ABC test compared well to data from leukoagglutination assays and indirect immunofluorescence tests. The assay is particularly advantageous in that granulocytes can be stored, only small amounts of cells and sera are needed, and heterogeneous cell populations can easily be studied.     

Serologic survey of selected zoonotic disease agents in black-tailed jack rabbits from western Texas

A serologic survey for the agents of Rocky Mountain spotted fever (RMSF) (Rickettsia rickettsii), Borrelia spp. including the causative agent for Lyme disease (Borrelia burgdorferi), and plague (Yersinia pestis) was conducted on blood samples collected from 30 and 46 black-tailed jack rabbits (Lepus californicus) from an urban environment in Lubbock, Texas (USA) during winter 1987 and the following spring 1988, respectively. Antibody titers to the agents of RMSF and borreliosis were detected in sera of 28 and 1% of the jack rabbits, respectively. Neither organisms (rickettsiae and/or spirochetes) nor their associated antigens were detected in any of the tissue or whole blood samples; plague antibodies were not detected in the 76 jack rabbits sampled. Four of 18 ticks (Dermacentor parumapertus) removed from 12 jack rabbits were positive for RMSF using the fluorescent antibody test. The black-tailed jack rabbit is a common wildlife species living in close proximity to higher density human populations in many areas of the southwestern United States. Our results indicate the potential importance of urban populations of this mammal as reservoirs for at least one important zoonotic disease, RMSF, in western Texas.     

TRH-enkephalin interactions in the amygdaloid complex during gastric stress ulcer formation in rats

The formation of gastric stress ulcers was studied as a function of interactions between thyrotropin releasing hormone (TRH) and endogenous opioids in the central amygdalar nucleus (CEA) in rats. Bilateral microinjections of TRH (1 or 10 micrograms) into the CEA produced dose-related aggravations in cold restraint stress (CRS, 3 h at 4 degrees C)-induced gastric ulcer formation. Similar stress ulcer facilitating effects were also seen with intra-CEA injections of the opioid antagonists, naloxone (1 or 10 micrograms). On the other hand, the enkephalin analog, D-Ala2-metenkephalinamide (DAMEA, 1, 10 or 20 micrograms) produced dose-dependent attenuations in gastric stress pathology, the effects being most marked with the latter two doses. Pretreatment of rats with intra-CEA naloxone (1 microgram) (a) antagonized the gastric cytoprotective effects of DAMEA (20 micrograms) and (b) further aggravated the ulcerogenic response of TRH (1 microgram), without influencing significantly the TRH (10 micrograms) effect. Further, when DAMEA (20 micrograms) was administered intra-CEA just after TRH (10 micrograms), the stress ulcer facilitating effects of the latter was neutralized. The results indicate that TRH-enkephalin interactions are possible at the level of the CEA during CRS-induced gastric ulcer formation.     

Analysis of leukotrienes, prostaglandins, and other oxygenated metabolites of arachidonic acid by high-performance liquid chromatography

High-performance liquid chromatography procedures were developed which separate leukotrienes (LTs), hydroxy-fatty acids (HETEs), prostaglandins (PGs), the stable metabolite of prostacyclin (6-keto-PGF1 alpha), the stable metabolite of thromboxane A2 (TXB2), 12-hydroxyheptadecatrienoic acid (HHT), and arachidonic acid (AA). Two methods employing reverse-phase columns are described. One method uses a radial compression system, the other a conventional steel column. Both systems employ methanol and buffered water as solvents. The radial compression system requires 60 min for separation of the AA metabolites, while the conventional system requires 100 min. Both methods provide good separation and recovery of 6-keto-PGF1 alpha, TXB2, PGE2, PGF2 alpha, PGD2, LTC4, LTB4, LTD4, LTE4, HHT, 15-, 12-, and 5-HETE; and AA. The 5S,12S-dihydroxy-6-trans, 8-cis, 10-trans, 14-cis-eicosatetraenoic acid (5S,12S-diHETE), a stereoisomer of LTB4, coelutes with LTB4. To determine the applicability of the methods to biologic systems, AA metabolism was studied in two models, guinea pig lung microsomes and rat alveolar macrophages. Both HPLC systems demonstrated good recovery and resolution of eicosanoids from the two biological systems. A simple evaporation technique for HPLC sample preparation, which avoids the use of chromatographic and other time-consuming methodology, is also described.     

Influence of pan-caspase inhibitors on coxsackievirus B3-infected CD19<Superscript>+</Superscript> B lymphocytes

Coxsackievirus B3 (CVB3), together with other enteroviruses of the picornavirus family, is associated with a wide variety of acute and chronic forms of human diseases. Using the murine model of CVB3-caused myocarditis, this pathogen can be detected not only in solid organs but also in different types of immune cells, preferentially in B lymphocytes. Therefore, these cells could represent a non-cardiac virus reservoir and may play an important role with regard to viral dissemination in the infected host. In addition, the infection of specific immune cells might modulate the severity of tissue injury and the pattern of virus-caused pathology in susceptible or resistant individuals. In the present study it could be demonstrated that CVB3 was capable to infect productively a certain percentage of murine CD19⁺ B cells. In vivo studies revealed that CVB3 invaded murine CD19⁺ B cells during an acute infection. Three days p. i. approximately 0.5–1.0% of these cells were productively infected. This proportion could be decreased up to 45%, if 3 days p. i. mice were intravenously treated with the pan-caspase inhibitors Z-VAD-FMK or Q-VD-OPH. These data were compared with results obtained from CVB3-infected human Raji cells.     

An 18 kDa Scaffold Protein Is Critical for Staphylococcus epidermidis Biofilm Formation

Virulence of the nosocomial pathogen Staphylococcus epidermidis is crucially linked to formation of adherent biofilms on artificial surfaces. Biofilm assembly is significantly fostered by production of a bacteria derived extracellular matrix. However, the matrix composition, spatial organization, and relevance of specific molecular interactions for integration of bacterial cells into the multilayered biofilm community are not fully understood. Here we report on the function of novel 18 kDa Small basic protein (Sbp) that was isolated from S. epidermidis biofilm matrix preparations by an affinity chromatographic approach. Sbp accumulates within the biofilm matrix, being preferentially deposited at the biofilm–substratum interface. Analysis of Sbp-negative S. epidermidis mutants demonstrated the importance of Sbp for sustained colonization of abiotic surfaces, but also epithelial cells. In addition, Sbp promotes assembly of S. epidermidis cell aggregates and establishment of multilayered biofilms by influencing polysaccharide intercellular-adhesin (PIA) and accumulation associated protein (Aap) mediated intercellular aggregation. While inactivation of Sbp indirectly resulted in reduced PIA-synthesis and biofilm formation, Sbp serves as an essential ligand during Aap domain-B mediated biofilm accumulation. Our data support the conclusion that Sbp serves as an S. epidermidis biofilm scaffold protein that significantly contributes to key steps of surface colonization. Sbp-negative S. epidermidis mutants showed no attenuated virulence in a mouse catheter infection model. Nevertheless, the high prevalence of sbp in commensal and invasive S. epidermidis populations suggests that Sbp plays a significant role as a co-factor during both multi-factorial commensal colonization and infection of artificial surfaces.     

Altered Pattern of Immunoglobulin Hypermutation in Mice Deficient in Slip-GC Protein

We recently identified a novel germinal center GTPase, SLIP-GC, that localizes to replication factories in B cells and that, when reduced, induces DNA breaks in lymphoma B cell lines in an activation-induced deaminase (AID)-dependent manner. Herein, we generated mice deficient in SLIP-GC and examined the impact of SLIP-GC deficiency in immunoglobulin hypermutation and class switch recombination, both AID-dependent mechanisms. SLIP-GC-deficient mice experienced a substantial increase in mutations at G:C base pairs at the region downstream of JH4 in the immunoglobulin heavy chain locus. This change was reflected in the overall mutation frequency, and it was associated with an increase in transitions from G:C base pairs, a hallmark of AID-mediated deamination during replication. In addition, G:C transitions at non-immunoglobulin loci also increased in these mice. Given the intracellular localization of SLIP-GC to sites of replicating DNA, these results suggest that SLIP-GC protects replicating DNA from AID-mediated deamination of cytosines in both strands.