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J M Novak

Publications and source records attributed to J M Novak.

8 recordsLinked to original sources
PubMed1992

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.

Animals↗
PubMed1990

Effect of osmotic pressure on uptake of chemotherapeutic agents by carcinoma cells.

Intraperitoneal chemotherapy has been used to treat cancers which are confined to the abdominal cavity. Several variables which affect drug delivery into tumor cells have been identified, but the effect of osmotic pressure has not been studied. Tumor cell lines were used to evaluate the effect of fluid tonicity on drug uptake. HeLa cells and a murine teratoma cell line were suspended in solutions of tonicities 154, 308, and 616 mosM, each containing the same quantity of 5-fluorouracil, and uptake of the drug was measured at different intervals over 30 min. At all time points the amount of 5-fluorouracil taken up by cells in solutions of 154 mosM was greater than that in 310 mosM solutions, which was greater than the uptake in 616 mosM solutions, each by an average of 40-50%. Incorporation of drug into tumor cells was also assayed in vivo using a teratoma cell line propagated i.p. in mice. Tumor cell uptake of doxorubicin was increased to a similar extent when this drug was administered in hypotonic solutions of 154 mosM and was decreased by administration in hypertonic solutions of 465 mosM, as compared to solutions of 310 mosM. These results demonstrate that the uptake of chemotherapeutic agents into tumor cells is increased significantly when these drugs are infused in solutions of lower osmolalities, a finding which may be exploited in clinical situations.

Animals↗
PubMed1989

Effect of volume and pH on surface receptor number in macrophages.

Incubation of rabbit alveolar macrophages in hypo-osmotic solutions transiently increases cell volume and inhibits membrane internalization, resulting in an increase in surface receptor number. Since recent reports suggest that hypo-osmotic treatment decreases intracellular pH, and that reduced pH inhibits receptor internalization, pH was measured in hypo-osmotically treated macrophages. We found that cells incubated in iso-osmotic solutions of pH less than 7.2 exhibited a decrease in intracellular pH upon exposure to hypo-osmotic solutions, while cells in iso-osmotic solutions of pH greater than 7.2 had an increase in pH upon exposure to hypo-osmotic solutions. The relative increase in surface receptor number was unaffected by the initial pH or by the direction of change in pH. Incubation of cells in high K+/low Na+ hypotonic buffers induced a persistent increase in cell volume and surface receptor number. Cell volume and surface receptor number fell to baseline values after restoration of isotonicity by the addition of hypertonic sucrose. These manipulations had little effect on intracellular pH. We conclude that the inhibition of membrane internalization observed in cells exposed to hypo-osmotic solutions is independent of changes in intracellular pH. The inhibition of internalization observed in this system may be due directly to forces produced as a consequence of cell swelling.

Animals↗
PubMed1988

Effect of hypo-osmotic incubation on membrane recycling.

Incubation of alveolar macrophages in hypo-osmotic media causes a time-and temperature-dependent increase in the number of surface receptors for three different ligands. Exposure of cells to solutions of 210 mOsM or less, at 37 degrees C but not at 0 degree C, resulted in an increase in the number of surface receptors for diferric transferrin, alpha-macroglobulin-protease complexes, and mannose-terminated glycoproteins. Upon media dilution at 37 degrees C, surface receptor number reached a maximum within 5 min and returned to near-normal values by 30 min. The increase in surface receptor number was the result of a decrease in the rate of internalization of receptors, either occupied or unoccupied. The rate of receptor exteriorization was unaltered by hypo-osmotic incubation of cells. The rate of fluid-phase pinocytosis was also inhibited upon incubation in hypo-osmotic solution. In experiments in which both receptor-mediated endocytosis and fluid phase pinocytosis were measured on the same samples, inhibition of both processes occurred with the same kinetics and to a similar extent. The rate of receptor-mediated endocytosis recovered to normal rates after 60 min in hypo-osmotic solutions, whereas the rate of fluid phase pinocytosis did not recover to the same extent.

Animals↗
PubMed1988

Regulatory volume decrease in alveolar macrophages: cation loss is not correlated with changes in membrane recycling.

Alveolar macrophages regain their normal volume after swelling in hypo-osmotic solutions. This process, termed regulatory volume decrease (RVD), is initiated 3-5 minutes after exposure of cells to hypo-osmotic solutions, and by 30 min, near-normal volumes are attained. Volume decrease does not occur at 0 degrees C or in solutions in which Na+ has been replaced by K+, or Cl- by the impermeant anion gluconate. These results, as well as direct measurement of intracellular cations, indicate that decreases in cell volume result primarily from the loss of K+ and Cl- and are similar to RVD in lymphocytes. Kinetic analysis of cation loss, both by directly measuring changes in intracellular cation content and by assaying rubidium efflux, showed that cation loss occurred immediately upon media dilution. The rate of cation loss fit first-order kinetics and preceded both the initiation of volume decrease and the maximum increase in surface receptor number. These results suggest that the cation transporters responsible for RVD are located at the cell surface and that regulation of activity is not dependent on alterations in membrane movement.

Animals↗
PubMed1987

Optic nerve hypoplasia.

Optic nerve hypoplasia is now recognized as a common developmental anomaly of the optic nerve. It has a large clinical spectrum of findings, but is most often associated with impaired vision and a small optic disc. Additional findings which aid in the diagnosis include a Marcus Gunn pupil, a double peripapillary ring, and pathological thinness of the retinal nerve fiber layer. Bilateral optic nerve hypoplasia may be a clue to midline developmental anomalies of the brain. Our case of optic nerve hypoplasia illustrates its variability of presentation and underscores the importance in investigating every case of unexplained decreased vision.

Diagnosis, Differential↗
PubMed1979

Effects of radiation on the human gastrointestinal tract.

Radiation therapy directed at the abdomen may damage the digestive tract, the type and extent of injury depending on the dose of the radiation and the radiation sensitivity of the gut. Characteristic early changes are manifest in the mucosa of the gut: for later ulceration, changes in the collagen tissues and particularly in the vascular channels occur. This paper describes and characterizes injuries to the esophagus, stomach, small intestine and colon. It emphasizes the importance of recognizing radiation-induced damage to the gut which may occur early or late after radiation.

Adult↗