Bacterial mesosomes: Real structures of artifacts?

The ultrastructural study of membrane organization in gram-positive bacteria related to the OsO₄ fixation conditions revealed that large, complex mesosomes are observed only when the bacteria are subjected to an initial fixation with 0.1% OsO₄ in the culture broth, as in the prefixation step of the Ryter-Kellenberger procedure. Evidence was obtained suggesting that the large mesosomes are produced by this prefixation. The kinetic study of the membrane morphological alterations occurring during the prefixation of Bacillus cereus with 0.1% OsO₄ in the culture broth showed that the amount of mesosome material increases linearly from zero to a maximum observed at 1.7 min of prefixation and that at about this time a maximum is reached for the number of mesosomes per unity of cell area and for the average individual mesosome area. The large mesosomes observed in gram-positives fixed by the complete Ryter-Kellenberger procedure would be the result of the membrane-damaging action of 0.1% OsO₄. Such damaging action was deduced from the observation that 0.1% OsO₄ quickly lyses protoplasts and induces a quick and extensive leakage of intracellular K⁺ from B. cereus and Streptococcus faeculis. In support of that interpretation is the observation that in bacteria subjected to several membrane-damaging treatments, mesosome-like structures are seen after three different fixation procedures. In bacteria initially fixed with 1% OsO₄, 4% OsO₄ or 2.5% glutaraldehyde, no large, complex mesosomes are observed, small and simple invaginations of the cytoplasmic membrane being present. The size of these minute mesosomes is inversely proportional that causes of fixation. Uranyl acetate was found among the studied fixatives the one to the rate the least damage to bacterial membranes. This fixative satisfactorily preserves protoplasts. In bacteria initially fixed with uranyl acetate no mesosomes were found. The results of the present work throw serious doubts on the existence of mesosomes, both large and small, as real structures of bacterial cells. It is proposed that a continuous cytoplasmic membrane without infoldings (mesosomes) would be the real pattern of membrane organization in gram-positives.     

Evidence for mobility of immunoglobulin domains obtained by spin-label method.

It is found that EPR spectra of immunoglobulins and their subunits spin-labeled by iminoxyl radical 2,2,6,6-tetramethyl-4-amino (N-dichlorotriazine) at pH 7.5 are similar in form and reflect the capability of spin-label to be in two states. Formation of specific complexes of spin-labeled antibodies with antigens is accompanied by increased correlation time of labels as well as by increased fraction of the labels in a more immobilized state. It is shown that splitting spin-labeled light chains to halves results in the label losing its capacity of being in the more rigid microenvironment. EPR spectra are interpreted as due to the relative motion of domains.     

Fluorescence histochemistry of peptide hormone-producing cells: Observations on the nitroso-naphthol method for the demonstration of tyrosine residues

Summary Nitroso-naphthol reacts with tyrosine residues of peptides (and probably also proteins) to yield intensely fluorescent condensation products. This reaction forms the basis of a fluorescence histochemical procedure designed to demonstrate cells that are rich in tyrosine-containing peptides or proteins. In models the method was found to be specific for p-hydroxylated phenolic compounds. Fluorescence was induced also following formaldehyde vapour fixation. With the nitroso-naphthol technique the zymogen granules of gastric chief cells, intestinal Paneth cells, pancreatic acinar cells and certain peptide hormone-secreting cells such as the GH cells in the adenohypophysis, the insulin cells of the pancreatic islets and the calcitonin cells of the thyroid gave intense fluorescence with spectral characteristics indistinguishable from those of the fluorophores of tyrosine-containing peptides. In addition, a population of endocrine-like cells in the antral and intestinal mucosa of certain mammals displayed fluorescence.Grant support from the Swedish Medical Research Council (04X-04499)     

Trgrps — an interactive algorithm for group recogniton with an example from spartinetea

Summary TRGRPS can detect groups or signal if discrete groups cannot be found in a sample. The present paper elaborates on the concepts, describes the algorithm and provides illustrations from syntaxonomy. A computer program (TRGRPS.BAS) is available from the author upon request.Contribution from the Working Group for Data Processing in Phytosociology, International Society for Vegetation Science. For nomenclature of species see Lausi, Beeftink & Kortekaas (1975).The research project, from which this paper summarizes partial results, is supported by a National Research Council of Canada grant. Computer time was provided by the University of Western Ontario.     

A heuristic test for homogeneity in species populations

Summary This paper elaborates on the notion of homogeneity in plant populations and its quantification. It describes a method for testing homogeneity without the need to make assumptions about generalized distributions. The implementation of the method is illustrated through actual numerical examples.This paper presents results from a study of plant populations for which a National Research Council of Canade grant has been received (L. Orlóci).     

Leukocyte migration inhibitory factor (LMIF) production in unidirectional mixed lymphocyte cultures.

Puromycin treatment of lymphocytes was used to develop a one-way test for leukocyte migration inhibitory factor (LMIF) production in the mixed lymphocyte culture (MLC) reaction. Lymphocytes incubated with this protein synthesis inhibitor induced a vigorous mediator production by nontreated allogeneic cells, being themselves unable to respond to stimulator cells. When puromycin-treated cells were stimulated with the mitogens PHA, ConA, or PWM, overall protein and DNA synthesis were significantly decreased with concomitant abolishment of LMIF production. Viability of stimulator lymphocytes was found to be essential for generation of the mediator in MLC reaction.     

Effect of epinephrine on the oxidative desaturation of fatty acids in the rat.

The effect of epinephrine on the oxidative desaturation of fatty acids by liver microsomal preparations of rats has been studied. Administration of epinephrine (1 mg/kg body weight) produced a significant decrease in desaturation of [l-14C]=linoleic acid to gamma-linolenic acid and of [L-14C]alpha-linolenic acid to actadeca-6,9,12,15-tetraenoic acid 12 hr after the infection. Lower doses produced a lesser effect on the delta6-desaturation activity. Epinephrine administration modified the V max of linoleic acid desaturation but not the K m. There was also a slight increase in palmityl desaturation activity. The effect of epinephrine on delta6-desaturation activity was postulated to be mediated through an enhancement of the intracellular cyclic AMP levels that lead to an increase of a glucose metabolite. This metabolite would inhibit delta6-desaturation activity.     

Charge transfer mediated by nigericin in black lipid membranes.

Nigericin, in the concentration range (10(-6) M or higher) at which it uncouples intact mitochondria, was found to increase the conductance of black lipid membranes (BLM) by several orders of magnitude. The dependence of the membrane conductance on pH and K+ concentration suggests a mechanism for the transfer of charge mediated by this ionophore based on a mobile dimer with both nigericin molecules protonated and complexed with one K+. This charged complex accounts for the uncoupling effect observed in intact mitochondria.     

Ion-pair formation as a source of enhanced reactivity of the essential thiol group of D-glyceraldehyde-3-phosphate dehydrogenase.

The reactivity and the mode of activation of the essential--SH group (Cys-149) of D-glyceraldehyde-3-phosphate dehydrogenase have been studied by means of a spectrophotometric method [Polgár, L., FEBS Lett. 38, 187-190 (1974)], capable of detecting the dissociated form of the thiol group in proteins. Alkylations of Cys-149 of NAD-free D-glyceraldehyde-3-phosphate dehydrogenase with iodoacetamide and iodoacetate were investigated. The corrected absorbance change on alkylation at 250 nm (which is a direct parameter of the dissociation of the thiol group) and the alkylation rate were determined as a function of pH. The pH profiles of both dissociation and alkylation rate of Cys-149 conform to doubly sigmoid curves. All these curves implicate two ionizing groups (pK1 equals 5.5, pK2 equals 8.2). It is concluded that there are two reactive forms of the--SH group in the apoenzyme between pH 5 and 10. One reactive form corresponds to the free mercaptide ion. The other can be identified with an ion-pair composed of a mercaptide ion and some base, possibly the imidazolium group of His-176. The ion-pair has lower molar absorption coefficient and nucleophilicity than the free mercaptide ion. The two reactive forms are transformed into each other with pK2 equals 8.2. The ion-pair decomposes to a nondissociated thiol group and a protonated base with pK1 equals 5.5. In the presence of NAD, only the pH-rate profile of alkylation of D-glyceraldehyde-3-phosphate dehydrogenase was measured (at 370 nm). Using iodoacetamide as alkylating agent we also obtained a doubly sigmoid curve. A slight downward shift on pK1 and an upward shift in pK2 indicate that the ion-pair exists in a somewhat wider pH-range in the enzyme-coenzyme complex. An increase in the ionic strength of the reaction mixture from 0.09 to 0.45 M does not abolish the doubly sigmoid character of the curves determined either in the presence or in the absence of NAD.     

Diadenosine triphosphate splitting by rat liver extracts.

A splitting activity on diadenosine triphosphate has been found in rat liver. One of the products of the cleavage is ADP. A Km of 10 μM has been found. This activity on diadenosine triphosphate seems to be specific as diadenosine tetraphosphate, a nucleotide previously described by others to occur in rat liver at very low concentration, is not a substrate of the reaction. The occurrence of diadenosine triphosphate in rat liver has not been so far reported, but a dinucleoside triphosphate structure has been described at the 5′ end of certain mRNAs. The possibility that this enzymatic activity may be involved in the hydrolysis of diadenosine triphosphate or in the processing of mRNAs is suggested.