Comparison of anti-inflammatory compounds in the carrageenan induced paw edema model and the reversed passive Arthus model utilizing the same animal.

In order to better define antiinflammatory activity in new agents, a test was devised utilizing both carrageenan induced paw edema and the reversed passive Arthus reaction in the same animal. The model of carrageenan induced rat paw edema is a standard laboratory assay used to predict classical "aspirin-like" antiinflammatory molecules. The reversed passive cutaneous Arthus reaction involves precipitating antigen-antibody complexes, complement and infiltrating polymorphonuclear leukocytes (PMN's) and can be used to identify agents that affect one or more of these factors specifically. Antiinflammatory compounds were given orally one hour prior to the administration of carrageenan and goat anti-rat serum. Comparisons were made between several non-steroidal compounds and the steroid hydrocortisone. All of the compounds tested gave good carrageenan activity, but only hydrocortisone produced significant Arthus lesion inhibition in this assay.

Lymphocyte reactivity contributes to protection conferred by specific antibody passively transferred to herpes simplex virus-infected mice.

Passively acquired immunity to herpes simplex virus (HSV) was studied in antithymocyte serum (ATS)-treated mice and athymic nude mice to determine whether immunocompetent lymphocytes contribute to the protection observed after transfer of HSV-specific antibody to infected animals. Mice were given three intraperitoneal injections of 0.1 ml of ATS at 24-h intervals. This treatment reduced concanavalin A and lipopolysaccharide stimulation of lymphocytes harvested from these animals by 90% when compared with the stimulation of lymphocytes harvested from untreated animals. It was found that intraperitoneal injection of 0.5 ml of specific antibody 8 h after corneal HSV type 1 infection or subcutaneous HSV type 2 infection did not protect ATS-treated animals from virus infection. Specific antibody passively transferred to ATS-treated animals 8 and 120 h postinfection also failed to protect lymphocyte-depleted animals from HSV. However, ATS-treated animals were protected from HSV infection by passively acquired antibody when lymphocytes harvested from these animals regained 80% of their ability to be stimulated with concanavalin A and lipopolysaccharide. It was also found that specific antibody conferred protection to nude mice infected with HSV only if they were first reconstituted with syngeneic thymus cells 48 h before infection. The results suggest that both antiviral antibody and thymus-derived lymphocytes contribute to the recovery of HSV-infected hosts after passive immunization.

Virus-induced cellular immunity to simian virus 40 tumor-specific surface antigen(s) in adult LVG:LAK hamsters.

Cell-mediated immunity directed against simian virus 40 (SV40) tumor-specific surface antigen(s) (TSSA) was detected in SV40-immunized noninbred LVG:LAK hamsters with the use of a lymphocyte transformation assay. Sensitized spleen cells underwent a proliferative response in the presence of cells of an SV40-induced hamster sarcoma cell line (WF5-1) or the plasma membranes from these cells. In addition, 3-M KCl extracts of the plasma membranes of WF5-1 cells elicited such a lymphocyte proliferative response. Neither the cells of a chemically induced sarcoma cell line (OBT), their plasma membranes, nor 3-M KCl extracts of these membranes caused a similar proliferative response. These results established that immunization of adult hamsters with SV40 resulted in a specific autochthonous cell-mediated immune response against SV40 TSSA. The in vitro demonstration of this virus-induced immunity provided a unique method for the investigation of the SV40 TSSA involved in the establishment or rejection of tumors. The described lymphocyte transformation assay was also used to follow the solubilization and purification of the SV40 TSSA.

Initial membrane reaction in peptidoglycan synthesis: perturbation of lipid-phospho-N-acetylmuramyl-pentapeptide translocase interactions by n-butanol.

Phospho-N-acetylmuramyl-pentapeptide translocase, the initial membrane enzyme in the biosynthesis of peptidoglycan, requires a lipid microenvironment for function. n-Butanol was reversibly intercalated into membranes to perturb the hydrophobic interactions in this microenvironment in order to define further the role of lipid. In the concentration range for maximal stimulation of enzymic activity (0.12-0.18 M), n-butanol causes a 40% decrease in the fluorescence emission of the dansylated product, undecaprenyl diphosphate-(N epsilon-dansyl)pentapeptide. Since no change in emission maximum occurs below 22 degrees C in the presence of 0.12 M n-butanol, it is concluded that intercalation of this alkanol causes an increase in fluidity. Above 22 degrees C this concentration of n-butanol causes both a decrease in the fluorescence emission and a red shift in the emission maximum. It is concluded that a polarity change as well as fluidity change occurs above 22 degrees C. n-Butanol also causes a significant change in the phase transition experienced by the dansylated lipid product. Thus, it is possible with n-alkanols, e.g. n-butanol, to perturb lipid-translocase interactions resulting in an increase in fluidity in the microenvironment of the enzyme. This change in fluidity correlates with a stimulation of enzymic activity.

Preparative fractionation of T and B lymphocytes of the Syrian golden hamster with soybean agglutinin.

T and B lymphocytes of the Syrian golden hamster were separated from spleen cell preparations on the basis of their differential agglutinability with the lectin soybean agglutinin. Only B lymphocytes were agglutinated by this lectin, and they could be separated from the unagglutinated T lymphocytes by sedimentation through 50% heat inactivated calf serum at unit gravity. The B lymphocyte aggregates could be dissociated into single cells that were viable and functional after treatment with 0.5 M galactose. The isolated cell fractions were characterized by their blastogenic response to various T cell and B cell specific mitogens and by the presence or absence of cell surface IgG.

Initial membrane reaction in peptidoglycan synthesis. Interaction of lipid with phospho-N-acetylmuramyl-pentapeptide translocase.

The initial membrane reaction in the biosynthesis of peptidoglycan is catalyzed by phospho-N-acetylmuramyl (MurN Ac)-pentapeptide translocase (UDP-MurNAc-Ala-gamma DGlu-Lys-DAla-DAla undecaprenyl phosphate phospho-MurNAc-pentapeptide transferase). In addition to the transfer reaction, the enzyme catalyzes the exchange of [3H]uridine monophosphate with the uridine monophosphate moiety of UDP-MurN Ac-pentapeptide. Two distinct discontinuities are observed in the slopes of the Arrhenius plots of the exchange and transfer activities at 22 and 30 degrees C for the enzyme from Staphylococcus aureus Copenhagen. Anisotropy measurements of perylene fluorescence and electron spin resonance measurements of N-oxyl-4',4'-dimethyloxazolidine derivatives of 12- and 16-ketostearic acid intercalated into membranes from this organism define the lower (T1 = 16--22 degrees C) and upper (Th = 30 degrees C) boundaries of a phase transition. These values correlate with the discontinuities observed for the activity measurements. Thus, it is proposed that the physical state of the lipid micro-environment of phospho-MurNAc-penetapeptide translocase has a significant effect on the catalytic activity of this enzyme.

The initial attachment of transforming DNA to competent Bacillus subtilis.

The initial attachment of transforming DNA to competent Bacillus subtilis is temperature independent between 25 degrees and 45 degrees. However, below 15 degrees there is a significant reduction in the amount of DNA attached to compentent cells. The DNA that is attached at 4 degrees can lead to transformation or interfere effectively with the subsequent attachment of a distinctive DNA when the cells are shifted to a permissive temperature (37 degrees). These data suggest that the attachment of DNA at 4 degrees is to sites normally involved in the transformation process. The amount of DNA that is initially attached to the bacteria at 4 degrees or 37 degrees after perturbation of the cells by ionic strength changes, repetitive washings, or periodate oxidation varies with the temperature at which the treatment occurs. These results are consistent with a reorientation of the DNA attachment sites upon lowering the temperature to 4 degrees, such that their affinity for DNA and susceptibility inhibitory treatments are reduced.

The effect of Waring Blendor treatment on transformation in Bacillus subtilis.

Treatment of competent Bacillus subtilis in a Waring Blendor for 10 s increases transformability of the culture about twofold while reducing the attachment of DNA to competent cells by 80%. The effectiveness of attached DNA in producing transformants is increased 10-fold by this treatment. The uptake of transforming DNA into a DNase-resistant state is progressively reduced by 70% during a 120-s blending treatment. Blending for 30-45 s diminishes transformability to about 10% of the original nonblended value without affecting the viable cell titer. No effect is produced by 30 s of blending on transformability if the irreversible uptake of DNA has been completed. Thus, the inhibition occurs at an early step in the transformation sequence. Treatment of the competent culture for 60 s or longer in the Waring Blendor reduces both the number of transformants obtained and the total number of viable cells.

Fluorescent substrate for nascent peptidoglycan synthesis. Uridine diphosphate-N-acetylmuramyl-(Nepsilon-5-dimethylaminonaphthalene-1-sulfonyl)pentapeptide.

The synthesis of UDP-MurNAc-Ala-DGlu-Lys[Nepsilon-dimethylaminonaphthalene sulfonyl (Dns)]-DAla-DAla provides a method for the specific introduction of a fluorescent reporter group into the membrane environment of nascent peptidoglycan synthesis. To assess the degree of perturbation of this environment caused by the introduction of the dansyl substituent, this nucleotide was compared with UDP-MurNAc-Ala-DGlu-Lys-DAla-DAla in the reaction catalyzed by phospho-MurNAc-pentapeptide translocase (UDP-MurNAc-Ala-gammaDGlu-Lys-DAla-DAla:undecaprenyl phosphate phospho-MurNA-C-pentapeptide transferase) and in the membrane-associated synthesis of nascent peptidoglycan. Phospho-MurNAc-pentapeptide translocase in membrane fragments from Staphylococcus aureus Copenhagen catalyzed the transfer of phospho-MurNAc-Ala-DGlu-Lys(Nepsilon-Dns)-DAla-DAla to undecaprenyl phosphate with a Vmax/Km of 3.8 and a Vmax of 1.6 times the values for UDP-MurNAc-pentapeptide. In the exchange reaction catalyzed by the translocase, the Rmax/Km and Rmax for the dansylated substrate were 1.8 and 0.78 times the respective values for the reference nucleotide. The equilibrium constant for the transfer reaction utilizing UDP-MurNAc-(Nepsilon-Dns)pentapeptide was 5.9 +/- 0.13 compared to 1.1 +/- 0.02 for UDP-MurNAc-pentapeptide. With respect to the proposed reaction model (Pless, D. D., and Neuhaus, F. C. (1973) J. Biol. Chem. 248, 1568-1576), the increase in Keq is consistent with a decrease in the affinity of undecaprenyl diphosphate-MurNAc-Ala-DGlu-Lys(Nepsilon-Dns)-DAla-DAla for the translocase. The fluorescence emission maximum of the phospho-MurNAc-(Nepsilon-Dns)pentapeptide moiety of UDP-MurNAc-(Nepsilon-Dns)pentapeptide was blue-shifted from 525 to 495 nm upon transfer from UMP to undecaprenyl phosphate with a 6-fold increase in quantum yield. These spectral changes provided a sensitive and continuous assay for the formation of undecaprenyl diphosphate-MurNAc-Ala-DGlu-Lys(Nepsilon-Dans)-DAla-DAla. The nascent peptidoglycan synthesizing system from Gaffkya homari utilized the dansylated nucleotide with a Vmax/Km of 0.05 and a Vmax of 0.10 times the values for UDP-MurNAc-pentapeptide. These results demonstrate that phospho-MurNAc-Ala-DGlu-Lys(Nepsilon-Dns)-DAla-DAla linked to the undecaprenyl phosphate will serve as a precursor for the synthesis of nascent peptidoglycan and that the dansyl moiety will report on the membrane environment it experiences during this synthesis.