Import of proteins into mitochondria. Translatable mRNAs for imported mitochondrial proteins are present in free as well as mitochondria-bound cytoplasmic polysomes

In order to assess the role of different polysomal populations in mitochondrial protein import, yeast spheroplasts were treated with cycloheximide to prevent polysome "run-off" and fractionated into free polysomes, polysomes bound to the mitochondrial outer surface, and polysomes bound to the endoplasmic reticulum. These polysomes were analyzed for translatable mRNAs coding for 8 cytosolic and 12 imported mitochondrial proteins. The mitochondrial proteins included 7 proteins of the inner membrane, 2 proteins of the matrix, 2 proteins of the intermembrane space, and 1 protein of the outer membrane. Of the mRNAs for imported mitochondrial proteins, 8 were enriched in mitochondria-bound polysomes, 3 were enriched in free polysomes, and 1 was enriched in neither. All mRNAs for cytosolic proteins were enriched in free polysomes. Polysomes bound to the endoplasmic reticulum lacked significant levels of translatable mRNAs for either cytosolic or mitochondrial proteins. Even though mRNAs for imported mitochondrial proteins were enriched in mitochondria-bound polysomes, these polysomes represented only 12-18% of the total cytoplasmic polysomes. As a consequence, none of the translatable mRNAs for imported mitochondrial proteins tested was predominantly associated with mitochondria-bound polysomes. While mitochondria-bound polysomes may contribute to mitochondrial protein import, they do not appear to be obligatory for this process.     

Nonenzymatic labeling of proteins by preparations of [35S]methionine

A rapid, simple, and sensitive radiochemical assay for the measurement of purine or pyrimidine nucleoside kinases (EC 2.7.1.-) is described. The substrate (thymidine, deoxyuridine, deoxycytidine, deoxyguanosine, deoxyadenosine, uridine, cytidine, and adenosine) is separated from the product (the respective 5′-nucleotide) on neutral alumina columns which retain the nucleotides but not the nucleosides. The nucleotides are recovered by elution with 0.4 m sodium phosphate buffer, pH 7.6.     

A stable Escherichia coli-Mycobacterium smegmatis plasmid shuttle vector containing the mycobacteriophage D29 origin.

A plasmid shuttle vector for Escherichia coli and mycobacteria was constructed from an E. coli plasmid containing the ColE1 origin, a 2.6-kb PstI fragment from bacteriophage D29 that grows in numerous mycobacterial species, and the kanamycin resistance gene either of Tn903 or of Tn5. The resultant plasmid is 7.63 kb and can be introduced via transformation into Mycobacterium smegmatis with high efficiency. In M. smegmatis the plasmid is stable and apparently present in multiple copies. Bioluminescence (luxA and luxB of Vibrio harveyi and fischeri) has been expressed in M. smegmatis from the aminoglycoside transferase promoter of Tn5. The D29 fragment should carry an origin of replication and some associated genes that act on it since various mutations destroy the ability of this fragment to replicate in M. smegmatis. The fragment was localized on the D29 genome map.     

The extravascular channel system in the rostral pituitary of Mugil cephalus (Teleostei) as revealed by use of horseradish peroxidase

Summary Using the unlabeled antibody peroxidase-antiperoxidase (PAP) technique at the light microscopic level, it was demonstrated that, in the amphibian magnocellular hypothalamo-hypophysial neurosecretory system, vasotocin and mesotocin are synthesized in separate neurons. A tendency to preferential location of the two kinds of neuronal perikarya is described. The neurosecretory perikarya are the origin of separate vasotocinergic and mesotocinergic axons. In the neural lobe, the pattern of distribution of the two types of axons is different. The coarse ventricular dendrites of both kinds of neurons are hormone-containing processes. Staining with anti-bovine neurophysin I serum suggested that the vasotocinergic and the mesotocinergic neurons synthesize different neurophysins.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Effects of temperature and field strength on the NMR relaxation times of Na in frog striated muscle

Pulsed NMR techniques have been applied to the study of the relaxation parameters characterizing ²³Na within frog striated muscle. Experiments were performed at 3°C, 22–24°C and 39°C at a Larmor frequency of 15.7 MHz; at 22–24°C, measurements were obtained both at 15.7 MHz and at 7.85 MHz.As previously reported, only a single spine-lattice relaxation time (T₁) was observed, but both slow (T₂)_I and fast (T₂)_II components of the spin-spin relaxation time were measured. The effect of temperature (θ) upon (1/T₁) was qualitatively similar to that reported for ²³Na in free solution; (θ) did not significantly affect (1/T₂) over the range of temperatures studied. (1/T₂)_I, and to a lesser degreee, (1/T₁) exhibited a modest inverse dependence of doubtful significance on the Larmor frequency.The data are examined within the framework of a simple specific model; a conservative values in assumed for the quadrupolar coupling constant characterizing immobilized intracellular Na⁺. Within this framework, the results suggest that the fraction of bound ions whose molecular tumbling is severely restricted does not exceed some few percent of the total sodium population.     

A theoretical model study of the comparative effectiveness of atropine and scopolamine action in the central nervous system

The molecular structural factors related to the action of atropine and scopolamine at a common biological receptor, with different apparent potencies, are studied from a theoretical viewpoint. The model molecules representing the two cholinergic antagonists are shown to possess the ability to generate an electrostatic potential field which is compatible with the “interaction pharmacophore” of acetylcholine-like molecules. A common site of action for the two types of drugs is rationalized on this basis. The effect of the differences in the molecular structure of the drugs, on their penetration ability and lipid solubility, is analyzed through the proton affinity of model compounds in the gas phase and in solution. These indicate the nature of the stronger solute-solvent interaction of scopolamine. Results from a comparative calculation of the protonation affinity of the solvated drugs are shown to reproduce the correct ratio of protonated versus free base concentrations for the two molecules, from which the reasons for the lower pKa of scopolamine and higher lipophilicity of atropine (free base) can be understood.     

Paraoxonases (PONs) 1, 2, and 3 are expressed in human and mouse gastrointestinal tract and in Caco-2 cell line: selective secretion of PON1 and PON2

The paraoxonase (PON) family contains three genes (PON1/2/3) that are believed to be involved in the protection against oxidative stress. PON1 and PON3 are circulating in serum attached to high-density lipoprotein fraction (HDL), whereas PON2 is ubiquitously expressed. The intestine is the second major organ that synthesizes lipoproteins; therefore, we examined PON mRNA expression and protein levels in gastrointestinal biopsies from humans, from C57BL6 mice, and from Caco-2 cells, a colon carcinoma-derived cell line that exhibits properties of intestinal epithelium at differentiation. PON 1/2/3 mRNA and proteins were present in human biopsies with variable expression among different gastrointestinal segments. Only PON2 and PON3 were present in mice. All PON mRNA, proteins, and enzymatic activities were present in Caco-2 cells. Oxidation of CaCo-2 cells with ferrum ascorbate had no significant effect on PON mRNA expression, but it increased paraoxonase and lactonase activity, whereas statinase activity was decreased. We showed polarized secretion of PON1 (basolateral) and PON2 (apical) into Caco-2 culture medium, raising the possibility that intestine is capable of producing and releasing PON1 and PON3 to the circulation, whereas PON2 is released at the brush-border membrane to intestinal lumen where it may perform another yet unclear function.     

Effect of anions on the photocycle of halorhodopsin. Substitution of chloride with formate anion

Halorhodopsin from Natronomonas pharaonis is a light-driven chloride pump which transports a chloride anion across the plasma membrane following light absorption by a retinal chromophore which initiates a photocycle. It was shown that the chloride anion bound in the vicinity of retinal PSB can be replaced by several inorganic anions, including azide which converts the chloride pump into a proton pump and induces formation of an M-like intermediate detected in the bR photocycle but not in native halorhodopsin. Here we have studied the possibility of replacing the chloride anion with organic anions and have followed the photocycle under several conditions. It is revealed that the chloride can be replaced with a formate anion but not with larger organic anions such as acetate. Flash photolysis experiments detected in the formate pigment an M-like intermediate characterized by a lifetime much longer than that of the O intermediate. The lifetime of the M-like intermediate depends on the pH, and its decay is significantly accelerated at low pH. The decay rate exhibited a titration-like curve, suggesting that the protonation of a protein residue controls the rate of M decay. Similar behavior was detected in N. pharaonis pigments in which the chloride anion was replaced with NO(2)(-) and OCN(-) anions. It is suggested that the formation of the M-like intermediate indicates branching pathways from the L intermediate or basic heterogeneity in the original pigment.     

Studies on acute human infections using FTIR microspectroscopy and cluster analysis

A novel methodology for the diagnosis of acute infections using FTIR microspectroscopy (FTIR-MSP) data on blood components and cluster analysis is presented. Blood samples were collected from 11 patients suffering from various infections and 16 age-matched healthy human controls. Blood components such as white blood cells, red blood cells, and plasma were isolated using standard procedures and FTIR-MSP of these components was utilized. A cluster analysis of the FTIR spectra was performed. The spectra obtained from the three blood components of patients were different from those of controls. The FTIR spectra of white blood cells from patients suffering infections were significantly different from the controls. Cluster analyses of averaged FTIR-MSP spectra of white blood cells provided 100% classification between patients and healthy controls.     

The interaction of water molecules with purple membrane suspension using 2H double-quantum filter, 1H and 2H diffusion nuclear magnetic resonance

Bacteriorhodopsin is a membrane protein of the purple membrane (PM) of Halobacterium salinarum, which is isolated as sheets of highly organized two-dimensional hexagonal microcrystals and for which water molecules play a crucial role that affects its function as a proton pump. In this paper we used single- and double-quantum (2)H NMR as well as (1)H and (2)H diffusion NMR to characterize the interaction of water molecules with the PM in D(2)O suspensions. We found that, under the influence of a strong magnetic field on a concentrated PM sample (0.61 mM), the PM sheets affect the entire water population and a residual quadrupolar splitting (upsilon(q) approximately 5.5 Hz, 298 K, at 11.7 T) is observed for the D(2)O molecules. We found that the residual quadrupolar coupling, the creation time in which a maximal DQF signal was obtained (tau(max)), and the relative intensity of the (2)H DQF spectrum of the water molecules in the PM samples (referred to herein as NMR order parameters) are very sensitive to temperature, dilution, and chemical modifications of the PM. In concentrated PM samples in D(2)O, these NMR parameters seem to reflect the relative organization of the PM. Interestingly, we have observed that some of these parameters are sensitive to the efficiency of the trimer packing, as concluded from the apo-membrane behavior. The data for dionized blue membrane, partially delipidated sample, and detergent-treated PM show that these D(2)O NMR order parameters, which are magnetic field dependent, are sensitive to the structural integrity of the PM. In addition, we revealed that heating the PM sample inside or outside the NMR magnet has, after cooling, a different effect on the NMR characteristics of the water molecules in the concentrated PM suspensions. The difference in the D(2)O NMR order parameters for the PM samples, which were heated and cooled in the presence and in the absence of a strong magnetic field, corroborates the conclusions that the above D(2)O order parameters are indirect reflections of both microscopic and macroscopic order of the PM samples. In addition, (1)H NMR diffusion measurements showed that at least three distinct water populations could be identified, based on their diffusion coefficients. These water populations seem to correlate with different water populations previously reported for the PM system.