Functional characterization of protease-treated Bacillus anthracis protective antigen.

Characterization of the functional domains of Bacillus anthracis protective antigen (PA, 83-kDa), the common cellular binding molecule for both anthrax edema toxin and anthrax lethal toxin, is important for understanding the mechanism of entry and action of the anthrax toxins. In this study, we generated both biologically active (facilitates killing of J774A.1 cells in combination with lethal factor, LF) and inactive preparations of PA by protease treatment. Limited proteolytic digestion of PA in vitro with trypsin generated a 20-kDa fragment and a biologically active 63-kDa fragment. In contrast, limited digestion of PA with chymotrypsin yielded a preparation containing 37- and 47-kDa fragments defective for biological activity. Treatment with both chymotrypsin and trypsin generated three major fragments, 20, "17," and 47 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This PA preparation was also biologically inactive. To investigate the nature of the defect resulting from chymotrypsin treatment, we assayed PA preparations for the ability to bind to the cellular receptor and to bind and internalize 125I-LF. All radiolabeled PA preparations bound with specificity to J774A.1 cells and exhibited affinities similar to native 83-kDa PA. Once bound to the cell surface receptor, both trypsin-treated PA and chymotrypsin/trypsin-treated PA specifically bound 125I-LF with high affinity. Finally, these PA preparations delivered 125I-LF to a Pronase-resistant cellular compartment in a time- and temperature-dependent fashion. Thus, the biological defect exhibited by chymotrypsin-treated PA is not at the level of cell binding or internalization but at a step later, such as toxin routing or processing by J774A.1 cells. These protease-treated preparations of PA should prove useful in both elucidating the intracellular processing of anthrax lethal toxin and determining the structure-function relationship of PA and LF.     

Inhibitory effects of orotate on precursor incorporation into nucleic acids.

The influence of orotic acid on the incorporation of precursors into nucleic acids was studied in mice and rats and in isolated cells. In vivo, orotate levels were modified by two diets which are known to increase the rate of pyrimidine nucleotide synthesis in rat liver. Of these diets, a 1% orotate diet had greater inhibitory effects than an arginine-deficient diet on the incorporation of [3H]orotate into RNA of mouse kidney than mouse liver. This contrasted with the situation in the rat where there was a greater effect in the liver than the kidney. The situation in the rat was more readily interpreted than in the mouse in terms of previously established effects of these diets on ribonucleotide pool sizes. However, studies using [3H]adenosine as a precursor for incorporation into RNA suggested that even in the mouse the effects of orotate were on pool sizes rather than an inhibitory effect on RNA synthesis. The incorporation of [3H]thymidine into DNA was inhibited by orotate to a similar degree in cultured HTC hepatoma cells and a line of rat liver epithelial cells. An effect on DNA synthesis rather than solely on pool sizes was suggested by the observation that the pool size of dTTP was not increased by 5 mM orotate under conditions in which there was a four-fold increase in the level of UTP in HTC cells. An inhibitory effect of orotate on DNA synthesis was further supported by an observation of decreased incorporation of [3H]deoxyadenosine into DNA and a lower rate of cellular proliferation.     

Role of agitation conditions in nitrification

The objective of this study was to determine the role of agitation conditions in the oxidation of nitrite ions by Nitrobacter. Batch reaction kinetic experiments were conducted in baffled stirred tanks. The range of agitation conditions studied was 6200 ? 95700 ergs/cm³ sec. This power input corresponds to 3.2 ? 45.6 hp/ 1000 gal, or a “hem Scale” of 3 ? 9. After a lag phase, the reaction kinetics were found to be zero order with respect to nitrite over a concentration range of 590 to 10 mg/liter nitrite nitrogen (NO₂^?-N). The zero-order rate constants were found to significantly decrease with increasing impeller power input per volume of liquid (P / V).     

Liver transplantation in a patient with cholangiocarcinoma and ulcerative colitis.

A 39 year-old patient with cholangiocarcinoma and pre-existing ulcerative colitis was successfully treated by orthotopic liver transplantation. He was given low doses of prednisone and azathioprine and survived for more than 9 months, dying with tumour metastases, thrombosis of the inferior vena cava and an intra-abdominal abscess. At autopsy the homograft showed little evidence of rejection. Preoperatively the patient had septicemia. Removal of his liver was difficult. The discrepancy between donor and recipient in size of blood vessels and the presence of two hepatic arteries in the donor caused problems during the vascular anastomoses. During the operation cardiac arrest occurred. Postoperatively there were several medical and surgical problems, including intraperitoneal and gastrointestinal hemorrhage, paralysis of the right dome of the diaphragm, sinus bradycardia, massive diuresis, peroneal nerve palsy, and one major and three minor episodes of rejection, which were reversed by giving pulse doses of methylprednisolone intravenously.     

The occurrence of glutamate synthase in algae.

The presence of glutamate synthase in the green algae $\text{Chlorella fusca}$ var. $\text{vacuolata}$ has been demonstrated using a whole cell assay as well as cell free extracts. The assay is complicated by the presence of glutamine (amino): α-oxoglutarate transaminase, but this enzyme can be inhibited by amino oxyacetate. The rates of glutamate synthase activity are sufficient to account for the known rates of nitrate assimilation to occur via the glutamine synthetase/glutamate synthase pathway.     

Actions of exogenous histones and other proteins on [3H]-thymidine incorporation into DNA of Novikoff hepatoma cells.

The effects of exogenous proteins on the incorporation of [3H]-thymidine into DNA was studied in Novikoff hepatoma ascites cells incubated in Eagle's minimal essential medium. A liver cytosol fraction (8 mg protein/ml) caused approximately 80% inhibition of isotope incorporation. The inhibitory activity of cytosol fractions from Morris hepatomas 9618A2, 5123C, and 20 were inversely related to their growth rate. Under conditions in which there appeared to be a density dependent inhibition of growth, a mean 10-20% stimulation of isotope incorporation was observed after addition of total calf thymus histones and individual fractions in the concentration range of 100-400 microgram/ml. In experiments with lower cell concentrations, a 60% or greater increase in [3H]-thymidine incorporation could be obtained with total calf thymus histone and with F1 and arginine-rich histones from rat liver. At concentrations of 1-2 mg/ml, histones inhibited DNA synthesis. Bovine serum albumin had little effect on DNA synthesis. Polylysine caused an 80-90% inhibition at a concentration of 1 mg/ml, but stimulatory effects were detected under certain conditions at 10 microgram/ml. The results suggest critical dependence on the ratio of cell and exogenous protein concentration in the action of proteins on DNA synthesis.