Purification and characterization of the phosphoenolpyruvate carboxylase from the facultative chemolithotroph Thiobacillus novellus (ATCC 8093).

Phosphoenolpyruvate carboxylase (EC4.1.1.31), which catalyzes the carboxylation of phosphoenolpyruvate to produce oxaloacetate was purified 465-fold from extracts of organotrophically grown Thiobacillus novellus. Nondenaturing polyacrylamide gel electrophoresis (PAGE) of the purified enzyme revealed the presence of two bands after staining with Buffalo Black. Gels stained with Fast Violet B after incubation with PEP, HCO3-, Mg2+ and acetyl CoA also showed two bands of activity with the faster moving the more active of the two. Sodium dodecylsulfate (SDS)-PAGE of the enzyme heated at 100 degrees C for 5 min revealed the presence of three intensely stained bands of M(r) 95 K, 51 K, and 28 K. However, electrophoresis of the enzyme heated for 2 min showed a single band of about 100 K, indicating that the preparation was likely homogeneous. The 51 K and 28 K subunits are thus products of the 95 K subunit. Gel filtration studies of the native enzyme yielded a M(r) of 360 K. Therefore, the enzyme is a tetramer. The optimum pH in Tris buffer was 8.0, with Km for PEP 0.64 mM, HCO3- 0.11 mM, and acetyl CoA a potent activator, 1.3 microM. A divalent cation best served by Mg2+ gave sigmoidal initial velocity plots. Hill plots of the data gave coefficients (nH) of 2.6. None of the metabolites tested, nucleotide triophosphates excepted, significantly affected enzyme activity. Binding studies with 14C-labelled PEP revealed the binding of about 20 moles PEP per mole (360,000 g) of PEPC. Initial velocity studies suggest that the reaction is catalyzed by a random Bi Bi mechanism. Despite the lack of inhibition by certain metabolites, the enzyme's function is probably anaplerotic.

Effect of storage time with different lens care systems on in-office hydrogel trial lens disinfection efficacy: a multi-center study.

A combined prospective and retrospective study was conducted to evaluate the efficacy of in-office disinfection methods for hydrogel trial contact lenses. Two hundred and twenty-one trial contact lenses, disinfected by four different disinfection methods, were collected from seven Study Centers and cultured for microbial contamination after various storage periods. Negative and positive control lenses were included as an additional Center in this double-masked study. There was a significant difference in the incidence of microbial contamination among the Centers for all storage times (chi 2 p < 0.001). Contamination of trial lenses in Centers using thermal disinfection with preserved saline (SoftWear Saline) was negative and thermal disinfection with nonpreserved saline (LensPlus Saline) was 8.7%. Lens contamination in Centers using chemical disinfection was 13.6% with ReNu and 40.7% with OptiFree. The degree of contamination ranged from 90 colony forming units (CFU)/ml to over 10 million CFU/ml. Among the microorganisms isolated after the different disinfection methods were Alcaligenes xylosoxidans, Serratia marcescens, Moraxella phenylpyruvica, Enterobacter agglomerans, Pseudomonas stutzeri, and various gram-positive organisms. This study suggests that practitioners should redisinfect all inventory trial lenses at least once a month to minimize the risk of patient infection.

Antimicrobial effectiveness of some soft contact lens care systems.

A study of the antimicrobial effectiveness of three soft contact lens care systems, In-A-Wink, Oxysept, and Hydrocare revealed that viable microorganisms are less likely to be present in the storage solution of the Hydrocare system than in either the Oxysept or In-A-Wink systems after lenses are removed by patients. The limited antimicrobial activity of sorbic acid in the In-A-Wink neutralizing solution could be attributed to the pH of the formulation. It is recommended that the neutralizing solution be discarded after the lenses are removed from the case, as microorganisms transferred by the hands to the solution in the case could remain viable, thus increasing the risk of eye contamination.

Natural killer cell and islet killer cell activities in human type 1 diabetes.

Peripheral blood mononuclear cells from 14 type 1 diabetic patients were examined for natural killer cell activity using the K562 cell line as 51Cr labeled targets. Mean cytotoxicity of K562 cells by unseparated mononuclear cells derived from new onset type 1 patients (12 +/- 1.6%) was lower (P less than .01) than that observed in non diabetic controls, (25 +/- 4.2%). Mean natural killer cell cytotoxicity mediated by enriched non-T cells from patients (41 +/- 5.8%) was also lower (P less than 0.03) than in the control group (56 +/- 3.7%). Specificity of these findings was evaluated by also examining other diabetic patient subgroups. Mean non T cell mediated natural killer cell activity in type 2 diabetic patients and type 1 patients with long term disease was 65 +/- 5.4% and 62 +/- 4.8% respectively (p less than 0.001 vs new onset type 1 patients). Longitudinal studies of new onset type 1 patients during the remission (honeymoon) phase revealed no improvement of impaired natural killer cell activity. In 30 new onset and 11 remission diabetic patients, mean non-T cell-mediated cytotoxicity was also measured using dispersed 51Cr labeled pancreatic islet target cells. Mean islet cytotoxicity mediated by cells from new onset patients was 34 +/- 2.4%, whereas in nondiabetic control subjects mean cytotoxicity was 25 +/- 1.8% (p less than 0.005). During remission, islet cytotoxicity returned to normal values in over half of the patients. There was no correlation between K562 and islet cell cytotoxicity in either of the latter two patient groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial flora of the eye and contact lens. Cases during hydrogel lens wear.

Bacteriological comparisons between the tear fluids of soft contact lens wearers and noncontact lens wearers indicate that there is an increase in the bacterial population in contact lens wearers but not a significant change in the varieties present. Differences between groups of contact lens wearers appear to depend on the method of disinfection used.

Autologous mixed lymphocyte reaction in man: XV. Cellular and molecular basis of deficient autologous mixed lymphocyte response in insulin-dependent diabetes mellitus.

The autologous mixed lymphocyte response (AMLR) and the allogeneic mixed lymphocyte response were deficient in a subset of patients with newly diagnosed insulin-dependent diabetes mellitus (IDDM). Using a single set of HLA-identical twins, the cellular and molecular basis of deficient AMLR was investigated and appears to be due to a defect in both responder T cells and stimulator non-T cells. Interleukin-2 production was diminished in the patient but not in the healthy twin. The in vitro addition of purified interleukin-2 enhanced the depressed AMLR in the diseased twin. This suggests that the deficient AMLR in IDDM may be in part due to a deficiency in the production of interleukin-2.

Unique antigens on neoplastic cells of the soft shell clam Mya arenaria.

Soft shell clams (Mya arenaria) are commonly found in coastal waters of both the eastern and western United States. These invertebrates, which have an open circulatory system, may develop neoplasms of the haemolymph which ultimately kill the host. In this study we have 1) recorded the prevalence of hematopoietic neoplasms (HN) in Mya arenaria within a 50 mile radius of Woods Hole, Massachusetts and 2) utilized cells from one HN bearing clam to generate a series of monoclonal antibodies. Our data show that determinants are expressed on HN cells which are not detected on normal clam haemocytes, suggesting separate ontogenetic pathways of cell differentiation.

Physical properties and metabolite regulation of ribulose bisphosphate carboxylase from Thiobacillus A2.

Purified ribulose-bisphosphate carboxylase (EC 4.1.1.39) was strongly and equally inhibited either by ADP or GDP and to a lesser extent by IDP. AMP or ATP exerted little effect on activity. Inhibition by the nucleotide diphosphates was competitive with respect to RuBP and non-competitive with respect to "CO2" and Mg2+, respectively. Treatment of the enzyme with urea or guanidine-HCl resulted in rapid loss of activity that was not restored by dialysis even in the presence of Mg2+ and cysteine. Sodium dodecyl sulfate electrophoresis of 8.0 M urea treated enzyme revealed the presence of a fast-moving (small) sub-unit with molecular weight 14150 and a slower moving (large) sub-unit with molecular weight 68000. Examination of native enzyme by sodium dodecyl sulfate electrophoresis gave sub-units of 13700 and 55500 respectively. The amino acid content standardized to phenylalanine was essentially similar to that from other sources. Arrhenius plots showed a "break" at 29 degrees C with an Ea of 12.34 kcal per mole for the steeper part of the curve and a deltaH of 11.43 kcal per mole while for the less steep region, the Ea was 1.04 kcal per mole and the deltaH 1.92 kcal per mole.

Ribulose biophosphate carboxylase from Thiobacillus A2. Its purification and properties.

Ribulose bisphosphate carboxylase (EC 4.1.1.39) from Thiobacillus A2 has been purified to homogeneity on the basis of polyacrylamide gel electrophoresis and U.V. analysis during sedimentation velocity studies. The enzyme had an optimum pH of about 8.2 with Tris-HCl buffers. The molecular weight was about 521000 with an Srel. of 16.9. Km for RuBP was 122 muM, for total "CO2" it was 4.17 mM, and for Mg2+ 20.0 muM. The absolute requirement for a divalent cation was satisfied by Mg2+ which was replaceable to a certain extent by Mn2+. Activity was not significantly affected by SO(2-4), SO(2-3), or S(2)O(2-3) at 1.0 mM. At this concentration S(2-) caused a 27% stimulation. All mercurials tested were inhibitory. pHMB was the most potent causing about 60% inhibition at 0.04 mM. This inhibition was reversible by low concentrations of cysteine. Cyanide was also inhibitory. Its mode of inhibition with respect to RuBP was un-competitive and with a Ki of 20 muM. Lost activity could be restored partially by GSH or Cu2+. Although azide at the concentration tested had no significant effect on enzyme activity, 2, 4-dinitrophenol at 1.0 mM caused 91% inhibition. Finally, activity was also affected by energy charge.

Properties and regulation of ribulose diphosphate carboxylase from Thiobacillus novellus.

Ribulose-diphosphate carboxylase from Thiobacillus novellus has been purified to hemogeneity as observed by polyacrylamide gel electrophoresis and U.V. light observation during sedimentation velocity analysis. The optimum pH for the enzyme with Tris-HCl buffers was about 8.2. Concentrations of this buffer in excess of 80 mM were inhibitory. The apparent Km for RuDP was about 14.8 muM with a Hill value of 1.5, for HCO3- the apparent Km was about 11.7 mM with an n value of 1.18 and for Mg2+ about 0.61 mM. The enzyme was specific for this cation. Relatively high concentrations of either Hg2+ or pCMB were required before significant inhibition was observed. Activity declined slowly during a 4-hr incubation period in either 3.0 M or 8.0 M urea. Incubation for 12 hrs resulted in complete loss of activity which was not prevented by 10 mM Mg2+ and was not reversed by dialysis and subsequent addition of 10 mM cysteine. Polyacrylamide gel electrophoresis revealed a loss of the major band and the appearance of 2 new bands. SDS polyacrylamide gel electrophoresis gave an average M.W. of 73500 +/- 2500 for the slower moving band and 12250 +/- 2500 for the faster moving. However, incubation in urea for up to 40 hrs revealed a decrease in the M.W. of the slower moving band to about 60000. The Ea for the enzyme was calculated to be about 18.85 kcal mole-1, with the possibility of a "break" between 40 and 50 degrees C. The Q10 was 3.07 between 20 and 30 degrees C whereas between 30 to 40 degrees C it was 3.31. Only phosphorylated compounds caused significant inhibition of enzyme activity. They included ADP, FDP, F6P, G6P, PEP, 6PG, 2-PGA, R1P, R5P, and Ru5p.