Ernest D. Gray

EFFECT OF EXTRACELLULAR SLIME SUBSTANCE FROM STAPHYLOCOCCUS EPIDERMIDIS ON THE HUMAN CELLULAR IMMUNE RESPONSE

Staphylococcus epidermidis infection of plastic catheters is often associated with heavy deposits of slime. To test whether this slime affects the human cellular immune response, its effect on the lympho-proliferative response of mononuclear cells to polyclonal stimulators was measured. Slime drastically reduces this response. Its inhibitory action was not immediate but took place over a few days and resulted in destruction of affected cells. The effect is dose related. This inhibition of cellular response may contribute to S epidermidis infection of implanted prostheses.

A sensitive method for the assay of hyaluronidase activity.

Abstract A spectrophotometric method for the assay of hyaluronidase activity, based on the binding of a carbocyanine dye (1-ethyl-2-[3-(1-ethyl-naphtho[1,2 d ]thiazolin-2-ylidene)-2-methyl-propenyl]naphtho[1,2d]thiazolium bromide) to undegraded substrate, is described. The binding results in a spectral shift with an absorbance maximum at 640 nm which is proportional to the amount of hyaluronic acid within a prescribed range. The end products of hyaluronidase activity do not cause a shift in the dye spectrum. The method allows the determination of an amount of hyaluronidase equivalent to 0.00005 NF unit.

Cellular Immune Responses to Extracellular Streptococcal Products in Rheumatic Heart Disease

The lymphocyte transformation responses to purified preparations of two extracellular products of group A streptococci (blastogen A and nuclease B), to phytohemagglutinin, and to Candida albicans antigen were measured in tonsillar and peripheral blood lymphocytes from patients with rheumatic heart disease (RHD) and suitably matched nonrheumatic (control) subjects. The mean phytohemagglutinin dose responses of tonsillar and peripheral lymphocytes from RHD patients were essentially indistinguishable from those of controls. In contrast, the responses of tonsillar and peripheral blood lymphocytes to the two extracellular products of group A streptococci were significantly lower in RHD patients than in nonrheumatic control subjects. Candida antigen produced very little stimulation of lymphocytes in any of the subjects. The geometric means of antibody levels against streptolysin O, nuclease B, and nicotinamide adenine dinucleotidase showed no consistent differences between the control group and the group of RHD subjects. Group A streptococci were isolated from the tonsils of approximately 25% of both groups of subjects. The RHD patients clearly had a depressed cellular immune response to the two purified streptococcal extracellular antigens. The equal frequency in recovery of group A streptococci from tonsils and the absence of consistent difference in titers of humoral antibodies to streptococcal extracellular antigens, particularly nuclease B, suggest that this differential response is not due to a lower level of stimulation by repeated exposure to group A streptococcal products.

Studies on the adaptive formation of photosynthetic structures in Rhodopseudomonas spheroides: I. Synthesis of macromolecules

Abstract 1. 1. Dark aerobically growing cells of Rhodopseudomonas spheroides in adapting to anaerobic-light conditions undergo a lag phase, during which bacteriochlorophyll is synthesized, followed by a resumption of growth. 2. 2. The cellular levels of RNA, protein and DNA are unaltered during this adaptive period. 3. 3. Considerable fluctuations occur during this period in rate of uptake of RNA, DNA and protein precursors. These fluctuations have been ascribed to the restricted energy available to the adapting cells. 4. 4. Fractionation of cell components demonstrates that a preferential synthesis of protein occurs at the membrane site during adaption although rapidly formed RNA is not associated with these membrane fractions. 5. 5. The proportion of rapidly formed RNA which forms ribonuclease-resistant hybrids with DNA increases when aerobically grown cells are transferred to anaerobic conditions.

Variations in ppGpp levels in Rhodopseudomonas spheroides during adaptation to decreased light intensity

The guanosine nucleotides ppGpp and pppGpp have been found in the photosynthetic bacterium Rhodopseudomonas spheroides supporting a suggestion that these are ubiquitous compounds in bacteria. ppGpp levels undergo an abrupt rise coincident with the cessation of RNA accumulation which occurs in this bacterium following a down-shift in incident light intensity. An abrupt decrease in the rate of [3H] uridine uptake into acid-precipitable material characteristic of light shift-down is also coincident with the rise in ppGpp levels. A regulatory role for this nucleotide in R. spheroides similar to that proposed in other bacteria is suggested.

Isoaccepting transfer rna species in differing morphogenetic states of Rhodopseudomonas spheroides

Abstract 1. Benzoylated DEAE-cellulose chromatography was employed to compare isoaccepting tRNA species from aerobic and anaerobic photosynthetic cultures of Rhodopseudomonas spheroides . 2. Variation in isoaccepting species were observed with phenylalanine and tryptophan tRNAs which were independent of the source of aminoacyl synthetase. 3. Tryptophanyl-tRNA formed in vivo was chromatographed and found to show similar variations to those observed with in vitro charged tRNA.

Structural studies of the ribosomes of Rhodopseudomonas spheroides

Abstract Ribosomes isolated from Rhodopseudomonas spheroides have a sedimentation coefficient at infinite dilution of 66s. Values of 45s and 29s were obtained for the ribosomal subunits. The two ribosomal subunits of this organism have different proportions of RNA and protein. The 45s has 62% RNA, 38% protein, and the 29s has 51% RNA, and 49% protein. It was found that the 29s subunit is structurally labile to both T 1 and pancreatic RNase whereas the 45s particle is relatively stable. 66s ribosomes exhibit diverse responses to RNase depending on both the mode of isolation and the presence of potassium chloride. Evidence is presented to show that different states of intraribosomal binding are responsible for the apparent stability of some and lability of other 66s ribosomes to RNase action. Polyuridylic acid binds both to the 29s subunit and to the 66s ribosome and protects these particles from the action of T 1 RNase. It is suggested that polyuridylic acid binds to 66s and 29s particles in a similar manner. Since the binding of polyuridylic acid protects against nuclease action, it is proposed that the structurally important RNA attacked by the nuclease is either associated with or part of the messenger binding site.

Studies on the adaptive formation of photosynthetic structures in Rhodopseudomonas spheroides: II. Nucleotide compositions during adaptation

Abstract 1. 1. Nucleotide compositions of total RNA isolated from Rhodopseudomonas spheroides were identical for cells grown under both aerobic and anaerobic conditions, and during the adaptation period from aerobic-dark to anaerobic-light conditions. 2. 2. Studies employing proflavine showed that with increasing times of [14C]-uracil incorporation a greater proportion of radioactivity was in the form of stable RNA, while at short time periods of incorporation (6 min), over 80 % of the RNA formed was unstable. 3. 3. Nucleotide compositions of rapidly labelled RNA are different from total RNA but showed no variation during the adaptation period from aerobic-dark to anaerobic-light conditions. 4. 4. It is concluded that if specific changes in the kind or amount of RNA species are occurring as R. spheroides responds to adaptive changes, they must be relatively minor in character and undetectable by the methods employed in the present studies.

Requirement of protein synthesis for maturation of ribosomal RNA in Rhodopseudomonas spheroides

Abstract 1. The role of protein synthesis in the maturation of ribosomal RNA was studied in the photosynthetic bacterium Rhodopseudomonas spheroides . 2. Methionine starvation and chloramphenicol both caused the accumulation of the 1.07 and 0.65 Mdalton ribosomal RNA precursors. 3. In methionine starved cultures accumulated precursor RNAs were processed upon addition of methionine. 4. The processing of these precursors upon methionine addition was inhibited in the presence of chloramphenicol. 5. It was concluded that protein synthesis is necessary for the cleavage of precursor ribosomal RNA.

The effect of CO2 on the biosynthesis of carotenoids in Rhodopseudomonas spheroides

Abstract In anaerobic photosynthetic cultures of Rhodopseudomonas spheroides , the light-induced induced synthesis of carotenoid pigments is greatly affected by the presence or absence of CO 2 . Cultures gassed with N 2 alone synthesize predominantly the yellow carotenoid(s). Gassing with a mixture of N 2 CO 2 results in the preferential synthesis of red carotenoids. CO 2 exerts similar effects in cultures maintained at consultant light intensity. The pigments formed in the presence of CO 2 are similar in their spectra to those normally found in Rhodopseudomonas spheroides . The addition of chloramphenicol to growing cultures prevents the CO 2 -induced preferential synthesis of red carotenoid, as well as general pigment formation. The results indicate that the effect of CO 2 is not mediated through a conversion of preformed yellow carotenoid to red carotenoid.