Interaction of T-activin with peritoneal macrophages

The action of T-activin on peritoneal macrophages of CBA mice after its introduction into the animals has been studied. In intact mice the phagocytic activity of macrophages and their resistance to the cytopathogenic action of Salmonella typhimurium live cells remains unchanged. The injection of corpuscular pertussis vaccine into mice leads to a decrease in the resistance of macrophages to the action of salmonellae. The simultaneous injection of T-activin into mice in doses of 0.1 and 1.0 microgram per animal abolishes the damaging action of the vaccine. The analysis of the in vitro action of T-activin on macrophages of intact mice revealed that the preliminary incubation of cells with the preparation sharply increases their resistance to the action of salmonellae, while its introduction simultaneously with bacteria or after them rapidly leads to the death of macrophages. The action of T-activin is supposed to be linked with triggering the biosynthetic processes mediating the resistance of macrophages to the cytopathogenic action of salmonellae.     

Effect of T-activin on the production of immune interferon

A study was made of the effect of T-activin on the biosynthesis of immune gamma-interferon. It was shown that in 27% of patients with chronic nonspecific pulmonary diseases, production of gamma-interferon by lymphocytes was substantially reduced during exacerbation of inflammatory process in the lungs. It was discovered that T-activin was not an interferon inductor but enhanced its synthesis in patients with a low capacity of producing immune interferon even at small doses of interferon inductor. The preparation does not produce any effect on this process in normal subjects and in patients showing the normal level of gamma-interferon. Thus T-activin can be used for stimulation of interferonogenesis.     

A critical evaluation of the use of filipin-permeabilized rat hepatocytes to study functions of the endoplasmic reticulum in situ

We have used transmission (TEM) and scanning electron microscopy (SEM) and leakage of lactate dehydrogenase (LDH; EC 1.1.1.27) to evaluate two published procedures which use filipin to render isolated rat hepatocytes permeable to ionic substrates. Cells treated by the procedure of Jorgenson and Nordlie retained less than 10 per cent of their LDH. TEM revealed severe damage to the internal structure of these cells, which included swelling, disintegration and extensive vesicularization of the endoplasmic reticulum (ER). Hepatocytes treated with filipin by the procedure of Gankema et al. retained 65-75 per cent of their LDH and displayed incomplete but highly variable permeability to Trypan blue. SEM revealed the loss of microvilli, other signs of swelling, and the presence of large lesions in the plasma membrane. TEM revealed signs of cell swelling, but the nuclei and the mitochondria were only moderately altered. The rough ER was not swollen, but significant fragmentation was evident and characteristic stacks of lamellar ER were never seen. We conclude that useful information about the functions of the ER in situ cannot be obtained from studies of filipin-treated cells. Our results indicate that retention of LDH is not a sufficient criterion of preservation of cell morphology and that staining with Trypan blue may significantly underestimate the permeability of cells to small ionic metabolites.     

Rapid determination of antimicrobial susceptibility patterns ofListeria monocytogenes isolates by the autobac AIS and MIC procedures

A total of 34 isolates ofListeria monocytogenes were tested against ampicillin, cephalothin, chloramphenicol, erythromycin, tetracycline, and penicillin-G using the Autobac 3-h AIS and the Autobac 5-h MIC procedures. The results were compared to susceptibility category interpretations and MICs determined using the Sceptor system. With the Sceptor System, all isolates were interpreted to be moderately susceptible to ampicillin and penicillin-G, and susceptible to the four other antibiotics. With the Autobac AIS, all isolates were interpreted to be susceptible to all the antibiotics except penicillin-G. All but one of the 34 isolates were interpreted to be resistant to penicillin-G with the Autobac AIS test. The remaining isolate was interpreted to be indeterminant. The Autobac AIS test was unsatisfactory for determining the susceptibility ofL. monocytogenes isolates to penicillin-G. The Autobac MIC results correlated well with the MIC results of the Sceptor system provided that the Autobac was programmed as though it were testing enterococci. The Autobac MIC reported penicillin-G MICs in units per milliliter and required the use of a conversion factor to obtain micrograms per milliliter, and did not allow for the testing of erythromycin. The Autobac MIC susceptibility category interpretations must not be used, as they were derived from an outdated susceptibility standard. The Autobac MIC test may be used if the limitations given above are observed.     

The glucose-6-phosphatase system in rat hepatic microsomes displays hyperbolic kinetics at physiological glucose 6-phosphate concentrations

The kinetics of rat liver glucose-6-phosphatase (EC 3.1.3.9) were studied in intact and detergent-disrupted microsomes from normal and diabetic rats at pH 7.0 using two buffer systems (50 mM Tris-cacodylate and 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and glucose-6-P varied from 20 microM to 10 mM. Identical data were obtained when the phosphohydrolase activity was quantified by a colorimetric determination of Pi or by measuring 32Pi formed during incubations with [32P]glucose-6-P. In every instance the initial rate data displayed excellent concordance with that expected for a reaction obeying Michaelis-Menten kinetics. The present findings agree with recently reported results of Traxinger and Nordlie (Traxinger, R. R., and Nordlie, R. C. 1987) J. Biol. Chem. 262, 10015-10019) that glucose-6-phosphatase activity in intact microsomes exhibits hyperbolic kinetics at concentrations of glucose-6-P above 133 microM, but fail to confirm their finding of sigmoid kinetics at substrate concentrations below 133 microM. We conclude that glucose-6-P hydrolysis conforms to a hyperbolic function at concentrations of glucose-6-P existing in livers of normal and diabetic rats in vivo.     

Synthesis and secretion of mucous glycoprotein by the gill of Mytilus edulis. I. Histochemical and chromatographic analysis of [14C]glucosamine bioincorporation

The ability of the isolated gill epithelium of Mytilus edulis to incorporate [14C]glucosamine as a precursor in the biosynthesis and secretion of mucous glycoproteins was investigated. Localization of mucous cells in the gill filament was achieved using histochemical staining techniques. Mucus cells containing neutral and acidic mucins were found in the lateral region, whereas mucus cells containing primarily neutral or sulfated mucins were found in the abfrontal region. Autoradiographic results showed that in both regions, the mucous cells were rich in content of the incorporated radiolabel. The secreted glycoproteins containing the incorporated radiolabel were analyzed by column chromatography using Bio-Gel P-2 and P-6. Two populations of the glycoproteins differing in molecular size were isolated. Upon alkaline reductive borohydride cleavage of the O-glycosidic linkages of the high molecular weight protein, about 70% of the radiolabel and 85% of the carbohydrate content were removed from the protein. The alkaline borohydride cleavage resulted in the formation of at least six oligosaccharide chains of various lengths of sugar units. Gas chromatographic analysis of the carbohydrate composition shows that the glycoproteins contain N-acetylglucosamine, N-acetylgalactosamine, and galactose, fucose, and mannose as the neutral monosaccharides. The above results indicate that the isolated gill epithelium of M. edulis is capable of incorporating [14C]glucosamine in the synthesis of secretable mucin-type glycoproteins.     

Binding mode transitions of Escherichia coli single strand binding protein-single-stranded DNA complexes. Cation, anion, pH, and binding density effects

We have extended our investigations of the multiple binding modes that form between the Escherichia coli single strand binding (SSB) protein and single-stranded DNA (Lohman, T. M. & Overman, L. B. (1985) J. Biol. Chem. 260, 3594-3603; Bujalowski, W. & Lohman, T. M. (1986) Biochemistry 25, 7799-7802) by examining the effects of anions, pH, BaCl2, and protein binding density on the transitions among these binding modes. "Reverse" titrations that monitor the quenching of the intrinsic tryptophan fluorescence of the SSB protein upon addition of poly(dT) have been used to measure the apparent site size of the complex at 25 degrees C in pH 8.1 and 6.9 as a function of NaF, NaCl, NaBr, and MgCl2 concentrations. Under all conditions in which "reverse" titrations were performed, we observe three distinct binding modes with site sizes of 35 +/- 2, 56 +/- 3, and 65 +/- 3 nucleotides/SSB tetramer; however, the transitions among the three binding modes are strongly dependent upon both the cation and anion valence, type, and concentration as well as the pH. A net uptake of both cations and anions accompanies the transitions from the (SSB)35 to the (SSB)56 binding mode at pH 6.9, whereas at pH 8.1 this transition is anion-independent, and only a net uptake of cations occurs. The transition from the (SSB)56 to the (SSB)65 binding mode is dependent upon both cations and anions at both pH 6.9 and 8.1 (25 degrees C), and a net uptake of both cations and anions accompanies this transition. We have also examined the transitions by monitoring the change in the sedimentation coefficient of the SSB protein-poly(dT) complex as a function of MgCl2 concentration (20 degrees C, pH 8.1) and observe an increase in s20,w, which coincides with the increase in apparent site size of the complex, as measured by fluorescence titrations. The frictional coefficient of the complex decreases by a factor of two in progressing from the (SSB)35 to the (SSB)65 binding mode, indicating a progressive compaction of the complex throughout the transition. The transition between the (SSB)35 and the (SSB)56 complex is dependent on the protein binding density, with the lower site size (SSB)35 complex favored at higher binding density. These results indicate that the transitions among the various SSB protein-single-stranded DNA binding modes are complex processes that depend on a number of solution variables that are thermodynamically linked.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stabilization of glucose-6-phosphatase activity by a 21 000-dalton hepatic microsomal protein.

Hepatic microsomal glucose-6-phosphatase activity was rendered extremely unstable by a variety of techniques: (a) incubation at pH 5.0; (b) extraction of the microsomal fraction in the presence of 1% Lubrol; (c) various purification procedures. These techniques all result in the removal of a 21 kDa polypeptide from the fraction containing glucose-6-phosphatase activity. The 21 kDa protein was purified to apparent homogeneity by solubilization in the detergent Lubrol 12A-9 and chromatography on Fractogel TSK DEAE-650(S) and centrifugation at 105 000 g. The 21 kDa protein stabilizes glucose-6-phosphatase activity, whereas other purified hepatic microsomal proteins do not. The 21 kDa protein appears to be a potential regulator of glucose-6-phosphatase activity.