Expression of membrane activation antigens on murine B lymphocytes stimulated with lipopolysaccharide

The expression of two membrane glycoproteins, RL388 antigen and transferrin receptor (TfR), was examined on murine B cells stimulated with lipopolysaccharide (LPS) in vitro. Immunofluorescent staining with monoclonal antibodies and flow cytofluorometric analysis were used to monitor the expression of these markers as a function of the time in culture, the state of membrane Ia antigen expression, the position in cell cycle, and the degree of B-cell differentiation. Freshly explanted splenic B cells expressed low levels of RL388 antigen and TfR. Following LPS stimulation, increased expression of RL388 antigen was detectable by 8 to 12 hr of culture, a time span characterized by increased Ia antigen expression, blast transformation, and G0 to G1 phase transition. The increased expression of TfR was apparent later and correlated with entry into late G1 phase and the onset of S phase. LPS-stimulated cell cultures treated with actinomycin D (G0/G1 block) exhibited increased expression of Ia antigen, but neither RL388 antigen nor TfR, whereas hydroxyurea treatment (G1/S block) allowed expression of all three markers. These results indicate that hyperexpression of RL388 antigen and TfR occurs during G1 phase and that these events are subsequent to Ia antigen hyperexpression. Finally, B cells in late G1 through M phase of the cell cycle simultaneously express high levels of RL388 antigen and TfR. These findings suggest that the expression patterns of RL388 antigen and TfR might be useful parameters for defining compartments of the murine B-cell cycle.     

Functional testing of hepatocytes following their recovery from cryopreservation

Various tests of function have been suggested for assessing hepatocytes recovered from cryopreservation. In this study we have investigated hepatocyte attachment during tissue culture and cellular density in order to assess function and compared them with two classical dye exposure tests. The ability of hepatocytes to exclude trypan blue dye (TB) and metabolize fluorescein diacetate (FDA) was demonstrated. In populations of freshly prepared hepatocytes 88.07% were able to exclude TB and 87.31% were able to metabolize FDA. However in populations of hepatocytes recovered after cryopreservation using 1.5 M dimethyl sulfoxide as cryoprotectant only 33.44% were able to exclude TB and 31.59% able to metabolize FDA. Both of these tests gave the same estimate of functional ability. Density gradient centrifugation of hepatocytes on Percoll 400 (Pharmacia, Uppsala, Sweden) separated two populations of hepatocytes; one (density ca.1.07 g/ml Percoll) in which most of the cells were able to exclude TB and the second (density ca. 1.02 g/ml Percoll) in which they were stained blue. The dense population was highly enriched in dye-excluding hepatocytes: freshly prepared hepatocytes, 92.4%, and cryopreserved hepatocytes, 88.66%. When samples of these cells (2 x 10(6) dye-excluding cells per dish) were tested for their ability to attach to tissue culture dishes only 17.28% of the cryopreserved hepatocytes were able to attach compared to 55.28% of the freshly prepared cells. We conclude that cryopreservation of hepatocytes produces a population of cells which are not metabolically identical to a population of freshly prepared hepatocytes even though they appear to have the same buoyant density and dye-excluding capabilities.(ABSTRACT TRUNCATED AT 250 WORDS)     

IgM-mediated enhancement of in vivo anti-sheep erythrocyte antibody responses: isotype analysis of the enhanced responses

The direct splenic anti-sheep erythrocyte (anti-SRBC) responses as well as the serum IgG1, IgG2a, IgG2b, and IgG3 anti-SRBC responses of CBA/CaJ mice were monitored 4-35 days after immunization with: (1) a suboptimal dose of SRBC, (2) a suboptimal dose of SRBC plus monoclonal IgM anti-SRBC, or (3) a high dose of SRBC. The direct plaque-forming cell (PFC) responses of mice in treatment group 2 were significantly higher than those in group 1 but similar to the responses in group 3. The serum anti-SRBC antibody responses of all IgG subclasses were significantly enhanced by IgM anti-SRBC and were generally even higher than the responses obtained with high doses of SRBC. The relative proportions of each serum IgG subclass were similar in all three groups. These data suggest that the enhancement of suboptimal anti-SRBC antibody responses by IgM anti-SRBC extends through IgM and all of the IgG subclasses and, further, that the isotype profile in antibody-enhanced responses is similar to that obtained with high doses of SRBC.     

Ecology of food microorganisms

The behavior of microorganisms in foods is governed by the constraints applied to the microflora by a variety of environmental and ecological factors. These include water activity, pH, E_h, chemical composition, the presence of natural or added antimicrobial compounds, and storage temperature, as well as processing factors such as the application of heat and physical manipulation. Control of these factors will govern whether the food spoils or not, whether any microbial health hazard arises, and whether desired microbial processes are successful or not. While much is known about the effects of individual environmental factors, the effects due to their interactions are less understood. The two main problems now facing the food microbiologist are optimization of environmental parameters and the selection of strains with specific properties. A better understanding of the mechanisms of action and interactions between the various environmental factors, coupled with the application of modern techniques to produce strains with particular properties, will lead to optimum use of food supplies and improvements in quality. There is also potential for the development of new and novel foods.     

Effects of oligomycin on the partial reactions of the sodium plus potassium-stimulated adenosine triphosphatase

The effects on phosphoenzyme (E-P) formation of ligands which activate Electrophorus (Na,K)-ATPase were investigated in the presence of oligomycin. When the enzyme was allowed to bind oligomycin in the presence of NaCl and MgCl2, subsequent addition of ATP plus KCl produced a monoexponential time course of E-P formation with a rate of 56 s-1, similar to the rate obtained in the uninhibited enzyme phosphorylated by ATP in the absence of KCl. Pi liberation under these conditions was slow and showed no initial burst phase, consistent with the inhibitory effect oligomycin has on the E1-P to E2-P conformational transition. Addition to KCl to a preincubation medium containing oligomycin, NaCl, and MgCl2 had no further effect on E-P formation. However, equilibration with oligomycin, KCl, and MgCl2 prior to the addition of NaCl plus ATP gave a much slower rate of E-P formation (5 s-1) and resulted in an initial rapid release of Pi similar to that found in the uninhibited enzyme. The slow increase in E-P level observed after incubation with oligomycin, KCl, and MgCl2 may be due to secondary formation of an inhibition complex following rapid binding of oligomycin. In contrast to the monophasic behavior which resulted from pre-exposure to NaCl or KCl, preincubation with oligomycin in the presence of MgCl2 plus Tris or Tris alone gave a biphasic pattern of E-P formation in which about 50% of the intermediate accumulated at a rate of 56 s-1 and the remainder at a rate of 5 s-1. In addition, the Pi burst amplitude was reduced, indicating partial inhibition of the enzyme. These results suggest that in the absence of Na+ and K+ only half of the enzyme is inhibited by oligomycin while the remainder undergoes inhibition subsequent to initiation of phosphorylation. Since the oligomycin concentration was saturating, the partial inhibition reflected in the biphasic pattern of E-P formation may be due to half-of-the-sites reactivity in which only half of the subunits bind oligomycin in the absence of monovalent cations.     

The Baermann technique for estimating Protostrongylus infection in bighorn sheep: effect of laboratory procedures

The modified Baermann funnel technique was evaluated to determine the effects of time of baermannization, fecal preparation, type and size of funnel, and type of filter on the number of first stage larvae of Protostrongylus spp. recovered from feces of Rocky Mountain bighorn sheep (Ovis canadensis). More larvae were recovered when fecal pellets were baermannized for 24 hr compared to 8 hr, and when feces were crushed than when left intact. Use of small funnels resulted in the recovery of more larvae per gram of feces than larger funnels, and glass funnels more than plastic ones. There was no difference in recovery of larvae between cheesecloth filters and cellulose filters.     

Conjugal Transfer of Plasmid-Borne Multiple Antibiotic Resistance in Streptococcus faecalis var. zymogenes

A strain of Streptococcus faecalis var. zymogenes, designated JH1, had high-level resistance to the antibiotics streptomycin, kanamycin, neomycin, erythromycin, and tetracycline. These resistances were lost en bloc from approximately 0.1% of cells grown in nutrient broth at 45 C. The frequency of resistance loss was not increased by growth in the presence of the "curing" agents acriflavine or acridine orange, but after prolonged storage in nutrient agar 17% of cells became antibiotic sensitive. Covalently closed circular deoxyribonucleic acid (DNA) molecules were isolated from the parental strain and from antibiotic-sensitive segregants by using cesium chloride-ethidium bromide gradients. DNA molecular species were identified by using neutral sucrose gradients. Strain JH1 contained two covalently closed circular DNA species of molecular weights 50 x 10(6) and 38 x 10(6). An antibiotic-sensitive segregant, strain JH1-9, had lost the larger molecular species. A second sensitive segregant, strain JH1-5, had also lost the larger molecular species but a new molecular species of approximate molecular weight 6 x 10(6) was present. The antibiotic resistances that were curable from the parental strain were transferred to antibiotic-sensitive strains of S. faecalis and to strain JH1-9, during mixed incubation in nutrient broth at 37 C. Data to be described are interpreted to suggest that the transfer is by a conjugal mechanism. Analysis of the plasmid species in recipient clones showed that all had received the plasmid of molecular weight 50 x 10(6). Strain JH1-5 was not a good recipient. Analysis of one successful recipient clone of JH1-5 revealed that it had gained the 50 x 10(6) molecular weight plasmid but lost the 6 x 10(6) molecular weight species. These data are interpreted to mean that the multiple antibiotic resistance is borne by a transferable plasmid of 50 x 10(6) molecular weight, and that in clone JH1-5 this plasmid suffered a large deletion leaving only a 6 x 10(6) remnant which was incompatible with the complete replicon.     

Fixation of Microscopic Fresh-Water Green Algae by 31% OsO4 in CCI4 in an Unbuffered, Two-Phase Fixative System

The fixative was prepared by dissolving 0.1 gm of OsO₄ in 0.2 ml of CCI₄. It was applied to microscopic green algae, concentrated by centrifugation, in a volume equal to that of the residual culture medium and allowed to act for 45 min at either 20 or 4 C. Such fixation has proved of value in producing an electron micrographic picture of particular use in the study of cytoplasmic microtubules. The fixative acts by diffusion of OsO₄, from the nonaqueous to the aqueous phase and thus provides a condition similar to that of vapour fixation but with a much higher concentration of OsO₄