Interaction of poly(L-lysine) and copolymers of lysine with immobilized DNA.

Sonicated DNA has been covalently attached to Sepharose 4B by a carbodiimide method [Rickwood, P. (1972) Biochim. Biophys. Acta269, 47–50] which minimizes modification of the DNA and matrix. Columns of this material have been used to study the interaction between cationic polypeptides and DNA. When poly(l-lysine) is bound to such columns at low ionic strength and then eluted with a linear salt gradient the polypeptide elutes over a broad range of salt concentration, presumably reflecting different strengths of interaction with various sites on the DNA. The broadness of the elution profile cannot be attributed to heterogeneity in the poly(l-lysine) sample but rather to the $\text{AT}\text{GC}$ content of various DNA sites. Studies with other polypeptides, poly(l-Lys⁷⁹, l-Leu²¹) and poly-(l-Lys-l-Ala-Gly), as well as studies at different temperatures, have helped to clarify the possible roles of conformational mobility, polypeptide hydrophobicity, and the presence of contiguous lysines in determining the strength of interaction of polypeptides and proteins with DNA sites.     

A cytochemical approach to the wound repair mechanism inUdotea petiolata (Siphonales)

Summary When injured, the thalli of the coenocytic algaUdotea petiolata undergo a rapid sealing process mainly due to the extrusion of two successive plugs. In the first, external and transitory plug, sulphated polysaccharides are the predominant components. In the second, permanent and internal plug, roundish bodies having a complex polysaccharidic composition are embedded in a fibrillar matrix of still unknown nature. The sulphated sugars were identified and located by means of Alcian Blue staining and X-ray microanalysis. A periodic acid-thiocarbohydrazide-silver proteinate technique proved useful especially in the study of the roundish bodies and in the compositional and structural comparison of the siphon wall with the wound wall. Phosphotungstic acid at low pH was used to evidentiate an extensive plasma membrane activity in the repairing cytoplasm.Supported by a grant of C.N.R.     

Antifungal properties of some pyrazolyl-alkyl-sulfides

Four 5-amino-4-alkylthio-pyrazoles were synthesized and their antifungal activity was evaluated in vitro in Trichophyton mentagrophytes, Microsporum cookei and Candida albicans. The compounds slightly influenced the growth kinetics of the yeast, but at concentrations ranging from 20 to 40 g/ml completely prevented the mycelial growth of the two dermatophytes cultivated on Sabouraud's agar medium. An electron microscopic study, undertaken by using the most active compound, showed that in C. albicans mitochondria were the only cell targets affected whereas in the dermatophytes cell wall, plasmalemma and the main cytoplasmic organelles were damaged in various degrees. Since the most remarkable alterations were connected with membrane abnormalities, the cytological changes observed were tentatively interpreted as a consequence of the compound intrusion into the lipid bilayer of the membranes, since the drug is lipophilic in nature.Investigations supported by a grant from Consiglio Nazionale delle Ricerche of Italy (Contract No. 79.01871.04)     

Activities in vitro and in vivo of enzymes of benzodiazepine alkaloid biosynthesis during development of Penicillium cyclopium

In the hyphae of Penicillium cyclopium the in vitro measurable activities of 3 enzymes of alkaloid biosynthesis are induced endogenously during     

Location in the yeast hexokinase structure of residues related to the enzyme activity

Seven residues implicated as acting directly in substrate binding in yeast hexokinase B have been identified in the crystallographic structure by chemical sequencing. The cysteine which is regarded as a residue critically maintaining the active conformation of yeast hexokinase has been selectively labelled and likewise located in the structure. In some parts of the amino acid sequence predicted from the high-resolution electron density map it is found that alignments of chemically sequenced peptides can be made unambiguously; however, the extent of matching to the predicted sequence varies considerably along the chain.     

Clearance and fate of leukemia-inhibitory factor (LIF) after injection into mice

Leukemia-inhibitory factor (LIF) elicits effects on a broad range of cell types, including cells of the monocytic and megakaryocytic series, embryonal stem cells, hepatocytes, adipocytes, and osteoblasts. Native and recombinant LIF, injected intravenously into adult mice, had an initial half-life of 6-8 min and a more prolonged second clearance phase. Clearance of 125I-LIF from the circulation was paralleled by a rapid accumulation in the kidneys, liver, lungs, and spleen and a more gradual accumulation in the thyroid gland. Labeling of the renal glomerular tufts, parenchymal hepatocytes, splenic red pulp, alveolar pneumocytes, and thyroid follicular cells as well as of megakaryocytes and osteoblasts in the bone cavities, placental trophoblasts, and cells of the choroid plexus was demonstrable autoradiographically. The appearance of a large amount of nonprecipitable 125I in the urine suggested that the kidneys were the major route of LIF clearance from the body.     

Metabolism of nitroxide spin labels in subcellular fraction of rat liver. I. Reduction by microsomes

As part of an ongoing study of the role of subcellular fractions on the metabolism of nitroxides, we studied the metabolism of a set of seven nitroxides in microsomes obtained from rat liver. The nitroxides were chosen to provide information on the effects of the type of charge, lipophilicity and the ring on which the nitroxide group is located. Important variables that were studied included adding NADH, adding NADPH, induction of enzymes by intake of phenobarbital and the effects of oxygen. Reduction to nonparamagnetic derivatives and oxidation back to paramagnetic derivatives were measured by electron-spin resonance spectroscopy. In general, the relative rates of reduction of nitroxides were similar to those observed with intact cells, but the effects of the various variables that were studied often differed from those observed in intact cells. The rates of reduction were very slow in the absence of added NADH or NADPH. The relative effect of these two nucleotides changed when animals were fed phenobarbital, and paralleled the levels of NADPH cytochrome c reductase, cytochrome P-450, cytochrome b5 and NADH cytochrome c reductase; results with purified NADPH-cytochrome c reductase were consistent with these results. In microsomes from uninduced animals the rate of reduction was about 10-fold higher in the absence of oxygen. The products of reduction of nitroxides by microsomes were the corresponding hydroxylamines. We conclude that there are significant NADH- and NADPH-dependent paths for reduction of nitroxides by hepatic microsomes, probably involving cytochrome c reductases and not directly involving cytochrome P-450. From this, and from parallel studies now in progress in our laboratory, it seems likely that metabolism by microsomes is an important site of reduction of nitroxides. However, mitochondrial metabolism seems to play an even more important role in intact cells.     

Two actin variants in developing axolotl heart

Heart development in the Mexican salamander, Ambystoma mexicanum has been studied using two-dimensional gel electrophoresis and electron microscopy. At the preheart beat stage two forms of action, α and β, are present in the heart in approximately equal quantity, however, very few definitive thin filaments can be distinguished at this stage. Once the heart beat is initiated and heart development progresses α-actin increases relative to β-actin. This increase in muscle-specific actin is coincidental with the appearance of numerous thin filaments in the myocyte.     

A Substrate for Direct Measurement of L-iduronic Acid 2-sulfate sulfatase

Commercially available sodium heparinate has been sequentially treated with methanolic 0.06M hydrogen chloride and nitrous acid. The nondegraded material was separated by gel filtration from the nonsulfated and monosulfated disaccharides produced. The latter ones, obtained in 10% yield, have been used as a substrate for the direct measurement of the enzyme L-iduronic acid 2-sulfate sulfatase present in human plasma and fibroblast homogenates. Studies of the kinetics and pH optimum of the enzyme, by use of plasma of a patient with mucolipidosis II, indicated an apparent K_m of 2.5mM and a pH optimum of 4.6-4.8. The levels of activity in normal plasma and plasma of a patient with Hunter's disease were found to be 20.4 ± 1.22 units (μmol sulfate/24 h/g protein) and 3.25 ± 0.35 units, respectively. In homogenates of cultured skin fibroblasts, the levels were 137.6 ± 10.7 units for normal controls and 6.4 ± 5.1 for patients with Hunter's disease. The plasma of two obligated heterozygotes gave intermediate levels of activity, whereas the plasma of two possible heterozygotes gave either intermediate levels or entirely normal levels of activity.     

Secretion of granule enzymes from alveolar macrophages : Regulation by intracellular Ca-buffering capacity

Rabbit alveolar macrophages exposed to the ionophore A23187 in the presence of extracellular Ca²⁺ take up about 12 nmoles of Ca²⁺/1 × 10⁶ cells. This uptake induces a slight, but significant, extracellular release of granule enzymes, β-glucuronidase and lysozyme, but not of the cytoplasmic marker, lactate dehydrogenase. If either EGTA is added to the medium or Mg²⁺ replaces Ca²⁺ no stimulation of secretion is observed. If the energy supply is decreased by treating the macrophages with mitochondrial inhibitors such as oligomycin, cyanide, or rotenone and antimycin, Ca²⁺-dependent secretion is potentiated several fold. Selective release of granule enzymes from macrophages exposed to A23187 and Ca²⁺ is also stimulated by cytochalasin B (CB).