Enzymes catalyzing steps from ethanol to acetyl-coenzyme A, from malate to pyruvate, and from pyruvate to glucose 6-phosphate were identified in ethanol-grown Pseudomonas indigofera. Enzymes catalyzing the catabolism of glucose to pyruvate via the Entner-Doudoroff pathway were identified in glucose-grown cells. Phosphofructokinase could not be detected in Pseudomonas indigofera. Itaconate, a potent inhibitor of isocitrate lyase, abolished growth of P. indigofera on ethanol at concentrations that had little effect upon growth on glucose. The date obtained through enzyme analyses and studies of itaconate inhibition with both extracts and toluene-treated cells suggest that itaconate selectively inhibits and reduces the specific activity of isocitrate lyase.

Four cases of patients in whom graft vs. host disease developed after liver transplantation are described. The clinical course of each patient was similar with fever, pancytopenia, diarrhea and a skin rash developing 1 or 2 mo after liver transplantation. The clinical diagnosis was made from skin or colon biopsy specimens. Liver dysfunction did not occur in the patients at the time of diagnosis. Extrahepatic donor DNA was identified in the three patients it was tested for. Three patients died from the complications of the disease primarily related to sepsis. The other patient recovered from the graft vs. host disease but died from lymphoproliferative disease.

Analogs of human choriogonadotropin (hCG) lacking N-glycosyl chains at alpha52Asn and alpha78Asn were purified from the culture media of insect cells by immunoaffinity chromatography using a monoclonal antibody column. As previously reported, while analogs lacking carbohydrate at alpha52Asn and alpha78Asn had similar receptor binding activities compared with the wild type recombinant hCG (hCGwt), they differed in their signal transduction properties. The mutant lacking carbohydrate at alpha78Asn had 20% less cAMP-stimulating activity than hCGwt, but the absence of glycosylation at alpha52Asn resulted in the reduction of cAMP accumulation by 90-95%. A similar effect of the mutations was observed on the stimulation of steroidogenesis. Circular dichroism spectra of the two mutants showed significant differences. The mutant lacking carbohydrate at alpha52Asn had a much higher negative mean residue ellipticity (MRE) at 200 nm and a lower negative MRE at 220 nm than that lacking carbohydrate at alpha78Asn and hCGwt. The dissociation rates of the alpha52Asn and alpha78Asn carbohydrate deficient mutants at pH 3 and room temperature, measured by using 1-anilino-8-naphthalenesulfonate, were 9.4 x 10(-5) and 3.8 x 10(-5) s-1, respectively, as compared with 1.5 x 10(-5) s-1 for hCGwt. The results of both CD measurements and dissociation studies strongly suggest that the absence of carbohydrate at alpha52Asn results in conformational changes in the mutant which might explain the loss in its signal transduction function. This is further supported by indirect evidence from two other lines of experimentation. Unlike the mutant lacking carbohydrate at alpha78Asn, the one lacking carbohydrate at alpha52Asn cross-reacted with the two subunit specific monoclonal antibodies, anti-hCGalpha and anti-hCGbeta, which normally did not cross-react with the native or the hCGwt. Also, polyclonal anti-hCGbeta but not anti-hCGalpha was able to restore the cAMP-producing activity of the alpha52Asn carbohydrate deficient mutant. From all the data taken together, it appears that the loss of second messenger-producing activity of hCG with the absence of the glycosyl chain at alpha52Asn was probably due to a conformational change in the heterodimer rather than due to the loss of the alpha52Asn-carbohydrate-receptor interaction.

Apurinic/apyrimidinic endonuclease (APE), an essential DNA repair enzyme, initiates the base excision repair pathway by creating a nick 5′ to an abasic site in double‐stranded DNA. Although the Chinese hamster ovary cells remain an important model for DNA repair studies, the Chinese hamster APE (chAPE1) has not been studied in vitro in respect to its kinetic characteristics. Here we report the results of a kinetic study performed on cloned and overexpressed enzyme in sf9 cells. The kinetic parameters were fully compatible with the broad range of kinetic parameters reported for the human enzyme. However, the activity measures depended on the time point of the culture. We applied inductivity coupled plasma spectrometry to measure the phosphorylation level of chAPE1. Our data showed that a higher phosphorylation of chAPE1 in the expression host was correlated to a lower endonuclease activity. The phosphorylation of a higher activity batch of chAPE1 by casein kinase II decreased the endonuclease activity, and the dephosphorylation of chAPE1 by lambda phosphatase increased the endonuclease activity. The exonuclease activity of chAPE1 was not observed in our kinetic analysis. The results suggest that noticeable divergence in reported activity levels for the human APE1 endonuclease might be caused by unaccounted phosphorylation. Our data also demonstrate that only selected kinases and phosphatases exert regulatory effects on chAPE1 endonuclease activity, suggesting further that this regulatory mechanism may function in vivo to turn on and off the function of this important enzyme in different organisms.

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