Sites of metal deposition in the cell wall of Bacillus subtilis

Amine and carboxyl groups of the cell wall of Bacillus subtilis were chemically modified individually to neutralize their electrochemical charge for determination of their contribution to the metal uptake process. Mild alkali treatment removed ca. 94% of the constituent teichoic acid (expressed as inorganic phosphorus) and allowed estimation of metal interaction with phosphodiester bonds. Chemical modifications of amine functions did not reduce the metal uptake values as compared to native walls, whereas extraction of teichoic acid caused a stoichiometric reduction in levels. In contrast, alteration of carboxyl groups severely limited metal deposition of most of the metals tested. X-ray diffraction and electron microscopy suggested, in this case, that the form and structure of the metal deposit could be different from that found in native walls. The observations suggest that carboxyl groups provide the major site of metal deposition in the B. subtilis wall.     

Interaction of mutagenic spermidine-nitrous acid reaction products with uvr- and recA-dependent repair systems in Salmonella.

It has been observed previously that the mutagenic action of nitrous acid may be potentiated by polyamines. We examined the cellular response of two deoxyribonucleic acid repair systems to treatment with spermidine-nitrite reaction products. uvrB- deficient mutants of Salmonella typhimurium LT2 showed enhanced lethal and mutagenic response to the reaction products. Lethal activity was further enhanced in a uvrB recA double mutant, whereas mutagenic activity was not detectable. Dependence of mutagenesis on the recA gene implicates the action of an error-prone repair system in the fixation of a premutagenic lesion as a mutation. From consideration of the substrate characteristics of the two repair systems studied, it is suggested that the deoxyribonucleic acid lesion formed by the reaction products of spermidine and nitrite is an intrastrand cross-link.     

Suitability of L-[35S]methionine for studying the biosynthesis of the polypeptides of mouse liver endoplasmic reticulum membrane fractions in vivo.

The use of L-[35S]methionine (500-700 Ci/mmol (1 Ci = 37 GBq) for labelling the polypeptides of liver rough (R) and smooth (S)endoplasmic reticulum (ER) membrane fractions in vivo was studied. Adult mice were injected intraperitoneally with 400 muCi of the isotope and killed at various times (2'min to 24 h) thereafter. RER and SER fractions were prepared, stripped of ribosomes, and treated with Triton X-100 to remove intravesicular contents. Sufficient radioactivity was present in individual aliquots (75 microgram protein) of the ER membrane fractions to permit their analysis by fluorography after separation by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. By 3 min, although the majority of the labelled components were of intravesicular origin, some 12 membrane polypeptides were labelled in the RER fraction (including one corresponding in migration to cytochrome P-450); some 6 of these latter polypeptides were labelled to a lesser degree in the SER membrane fraction at this time. By 5 min, the patterns of radioactive polypeptides of the RER and SER fractions (including both membrane and intravesicular components) were identical. By 7 min, some 28 labelled membrane polypeptides were detectable in the total microsomal membrane. Analysis of the 24-h samples revealed that all the membrane polypeptides seen by staining with Coomassie blue were visualised by fluorography. Other studies revealed the applicability of the approach used for producing highly labelled cell sap and serum proteins. The overall results demonstrate the suitability of L-[35S]methionine administered in vivo for producing mouse liver ER membrane polypeptides of relatively high radioactivity and are consistent with a rapid conversion of RER to SER by ribosome detachment or membrane flow.     

Neutral glycosphingolipids and gangliosides of human lung and lung tumours.

In order to help determine whether alterations of the profiles of glycosphingolipids occur consistently in human tumours, the neutral glycosphingolipids and gangliosides of nine lung tumours (one adenocarcinoma, four squamous cell, two mixed adeno-squamous cell, one large cell and one oat-cell carcinomata) were analysed. The control tissue consisted of adjacent lung; it contained neutral glycosphingolipids corresponding in properties to glucosyl-, lactosyl-, globotriaosyl- and globotetraosyl-ceramides. All of the tumours also contained these four neutral glycosphingolipids. However, in addition, five of the tumours (two of the squamous, the large cell and the two mixed adeno-squamous cell carcinomata) contained neutral glycosphingolipids corresponding in properties to lactotriaosyl- and neolactotetraosyl-ceramides; these same tumours also exhibited higher amounts of lactosylceramide than the other tumours analysed. Both of the two former neutral glycosphingolipids and very substantial amounts of the latter neutral glycosphingolipid were detected in pneumonic lung and in polymorphonuclear leucocytes; it thus appears possible that these particular compounds were derived from these latter cells rather than from the tumour cells. The ganglioside patterns of the tumours were almost equivalent in complexity to that exhibited by the control lung tissue. This study shows that the profiles of two major classes of glycosphingolipids (neutral glycosphingolipids and gangliosides) occurring in lung tumours are almost as complex as those of the parent tissue, a finding in contrast with the notably simplified patterns of these lipids found in many cancer cells grown in vitro. It also suggests that when lactotriaosyl- and neolactotetraosyl-ceramides and high amounts of lactosylceramide are detected in human tumours, the possibility must be considered that these compounds are derived from polymorphonuclear leucocytes.     

The envelope of Micrococcus radiodurans: isolation, purification, and preliminary analysis of the wall layers

Two methods are presented that separate the complex envelope of Micrococcus radiodurans, strain Sark, into its constituent layers. The first involved treating whole cells with 0.025 M Tris buffer (pH 7.5) containing 2 mM of calcium and 3 mM of magnesium, resulting in the degradation of an intermediate ('compartmentalized') layer and consequent sloughing of the outer subunit and interior layers to form vesicles. This treatment also appears to show that the interior layer may be connected with the peptidoglycan-containing 'holey' layer. The second method involves treating whole cells with benzene followed by sonication; the results suggested that this treatment only released the outer layers from the 'compartmentalized' layer and did not degrade layers. Following benzene treatment, digestion of the 'compartmentalized' layer with cold sodium dodecyl sulfate (SDS) released the 'holey' layer. Electrophoretic analysis of some of the isolated layer preparations suggested that the subunit layer consisted of three major proteins of 90 000, 92 000, and 94 000 molecular weight, one minor protein of 100 000, a small amount of carbohydrate associated with the 94 000 protein, and a small amount of a 55 000 lipoprotein. The interior layer contained at least 10 proteins and may be attached to the peptidoglycan-containing 'holey' layer by means of the 55 000 lipoprotein.     

Effect of polyamines on cyclic AMP-dependent and independent protein kinases from mouse epidermis.

Soluble extracts from mouse epidermis contained both cyclic AMP-dependent and independent protein kinases which could be separated by DEAE-Sephadex chromatography. The cyclic AMP-dependent histone kinase activity was inhibited by millimolar concentrations of the polyamines putrescine, spermidine and spermine. Similar concentrations of polyamines stimulated the cyclic AMP-independent phosphorylation of casein. The polyamines did not inhibit cyclic AMP binding by soluble epidermal extracts.     

Chromosome number and flavonoid synthesis in Briza L. (Gramineae)

Following a recent reidentification of the apigenin C-glycosides in diploid Briza media, the luteolin C-glycosides were reexamined and three acylated derivatives of a luteolin C-glycoside were found. In an attempt to identify the chromosome or group of chromosomes responsible for the change in flavonoid synthesis from 4'-hydroxy- in diploid plants to 3',4'-dihydroxyflavone C-glycosides in autotetraploid plants, leaf flavonoids of artificially produced aneuploids of B. media were examined. Among these plants three different leaf flavonoid profiles were recognized: diploid, tetraploid, and a "modified" tetraploid pattern. All the aneuploids with the normal or "modified" tetraploid pattern were trisomic for one of the small acrocentric chromosomes. Induced polyploids of other Briza species were usually found to have similar flavonoid patterns in the two chromosome races. Flavonoid sulfates were found in three South American species but are absent from all the European species.