Targeting and fusion in vesicular transport

Vesicular transport of proteins and lipids between distinct subcellular compartments is directly responsible for generating and maintaining the structure of the organelles of the secretory and endocytic pathways in eukaryotic cells. Rapid advances in a variety of experimental systems have resulted in the identification of molecules involved in late steps of the transport process. This article presents a general paradigm for vesicular fusion and reviews the available experimental evidence.     

Construction of a dihydrofolate reductase-deficient mutant of Escherichia coli by gene replacement.

The dihydrofolate reductase (fol) gene in Escherichia coli has been deleted and replaced by a selectable marker. Verification of the delta fol::kan strain has been accomplished using genetic and biochemical criteria, including Southern analysis of the chromosomal DNA. The delta fol::kan mutation is stable in E. coli K549 [thyA polA12 (Ts)] and can be successfully transduced to other E. coli strains providing they have mutations in their thymidylate synthetase (thyA) genes. A preliminary investigation of the relationship between fol and thyA gene expression suggests that a Fol- cell (i.e., a dihydrofolate reductase deficiency phenotype) is not viable unless thymidylate synthetase activity is concurrently eliminated. This observation indicates that either the nonproductive accumulation of dihydrofolate or the depletion of tetrahydrofolate cofactor pools is lethal in a Fol- ThyA+ strain. Strains containing the thyA delta fol::kan lesions require the presence of Fol end products for growth, and these lesions typically increase the doubling time of the strain by a factor of 2.5 in rich medium.     

Superficial and deeper layers of dog normal articular cartilage. Role of hyaluronate and link protein in determining the sedimentation coefficients distribution of the nondissociatively extracted proteoglycans

Proteoglycans were extracted under nondissociative conditions from superficial and deeper layers of dog normal articular cartilage. The purified a-A1 preparations were characterized by velocity gradient centrifugation. Superficial specimens exhibited an abundant population of slow sedimenting aggregates whereas the aggregates of deeper preparations sedimented as two well-defined families of molecules. These dissimilarities in the size distribution of the aggregates observed between superficial and deeper a-A1 preparations derived most of all from differences in their content of hyaluronate and link proteins: (a) superficial preparations contained twice as much hyaluronate as deeper specimens; (b) superficial aggregates were link-free and unstable at pH 5.0 whereas deeper preparations contained link-proteins and their faster sedimenting aggregates were stabilized against dissociation at pH 5.0. In these proteoglycan preparations from different cartilage layers, the monomers exhibited an identical capacity for aggregation and the hyaluronate molecules displayed quite similar molecular weight (Mr = 5 x 10(5] and aggregating capacity. These observations as well as aggregating studies conducted with highly purified link protein and purified hyaluronate specimens of different molecular weights support the following conclusions: (a) link protein not only stabilizes proteoglycan aggregates but also enhances the aggregating capacity of hyaluronate; (b) for all practical purposes, the slow sedimenting aggregates represent a secondary complex of hyaluronate and proteoglycan monomers whereas the fast sedimenting aggregates may be considered as a ternary complex wherein link protein stabilizes the hyaluronate-proteoglycans interaction; (c) the distinctive heterogeneity of articular cartilage can be related to structurally different proteoglycan aggregates. The structural dissimilarities observed between superficial and deeper aggregates could reflect the different macromolecular organization of the proteoglycan molecules in the territorial and interterritorial matrices, respectively.     

Cytodiagnosis of in situ and early carcinoma of the upper gastrointestinal tract

Three cases of adenocarcinoma of the stomach, two in situ and one superficially invasive, and one of superficially invasive squamous-cell carcinoma of the esophagus are presented to illustrate the problems encountered in the diagnosis of early lesions of the upper gastrointestinal (GI) tract and the contribution that cytodiagnosis can make. The symptomology and roentgenographic findings in these cases were largely nonspecific. While endoscopic biopsies were repeatedly negative in three of the four cases, endoscopic brushing cytology consistently indicated the presence of a malignancy. Surgery was finally performed on the basis of the cytologic findings, confirming the presence of early malignancy. The cytologic findings, with histologic correlations, are presented in an effort to define some specific criteria for the diagnosis of early malignancy of the upper GI tract.     

Hemodynamic effects of anti-G suit inflation in a 1-G environment

This study evaluated effects of various anti-G inflation pressures on cardiac volumes and the relationship of these volume changes to mean arterial pressure changes. Ventricular volumes were calculated using two-dimensional echocardiography. An anti-G suit was inflated to 2, 4, and 6 psi in the standing and supine positions for 10 male subjects. In the supine position, mean arterial pressure increased from base line for all three inflation pressures (P = 0.05). The end-diastolic volume increased after 2-psi inflation (P = 0.03). Cardiac output or stroke volume did not change. After standing, mean arterial pressure (P = 0.002), end-diastolic volume (P = 0.002), and stroke volume (P = 0.05) fell after suit deflation. Peripheral vascular resistance fell in the 2- and 4-psi inflation profiles. In the standing protocol, mean arterial pressure, end-diastolic volume, stroke volume, and cardiac output rose with all three inflation pressures (P less than 0.05). After reclining, heart rate increased (P = 0.02) and mean arterial pressure fell (P less than 0.05) in the 4- and 6-psi inflation profiles after suit deflation. Increases in mean arterial pressure are caused by increases in cardiac preload and cardiac output after inflation of the anti-G suit while subjects were standing. Increased cardiac preload was not consistently seen after inflation while subjects were supine. Changes in end-diastolic volume and mean arterial pressure were dependent on the pressure used to inflate the anti-G suit.     

Role of the Colorless Polypeptides in Phycobilisome Assembly in Nostoc sp

We have identified the function of the `extra' polypeptides involved in phycobilisome assembly in Nostoc sp. These phycobilisomes, as those of other cyanobacteria, are composed of an allophycocyanin core, phycoerythrin- and phycocyanin-containing rods, and five additional polypeptides of 95, 34.5, 34, 32, and 29 kilodaltons. The 95 kilodalton polypeptide anchors the phycobilisome to the thylakoid membrane (Rusckowski, Zilinskas 1982 Plant Physiol 70: 1055-1059); the 29 kilodalton polypeptide attaches the phycoerythrin- and phycocyanin-containing rods to the allophycocyanin core (Glick, Zilinskas 1982 Plant Physiol 69: 991-997). Two populations of rods can exist simultaneously or separately in phycobilisomes, depending upon illumination conditions. In white light, only one type of rod with phycoerythrin and phycocyanin in a 2:1 molar ratio is synthesized. Associated with this rod are the 29, 32, and 34 kilodalton colorless polypeptides; the 32 kilodalton polypeptide links the two phycoerythrin hexamers, and the 34 kilodalton polypeptide attaches a phycoerythrin hexamer to a phycocyanin hexamer. The second rod, containing predominantly phycocyanin, and the 34.5 and 29 kilodalton polypeptides, is synthesized by redlight-adapted cells; the 34.5 kilodalton polypeptide links two phycocyanin hexamers. These assignments are based on isolation of rods, dissociation of these rods into their component biliproteins, and analysis of colorless polypeptide composition, followed by investigation of complexes formed or not formed upon their recombination.     

Arabidopsis mutants with increased sensitivity to aluminum.

Al-sensitive (als) mutants of Arabidopsis were isolated and characterized with the aim of defining mechanisms of Al toxicity and resistance. Most als mutants selected on the basis of root growth sensitivity to Al were recessive, and together the mutants constituted eight complementation groups. Also, in most als mutants, Al sensitivity appeared to be specific for Al relative to La (another trivalent cation), except als2, which was more sensitive to La than wild type. The tendency of roots on mutant seedlings to accumulate Al was examined by staining with morin and hematoxylin, dyes used to indicate the presence of Al. A significant increase in morin staining was observed in als5, consistent with its increased sensitivity to Al. Unexpectedly, als7 and als4 showed less morin staining, suggesting that the roots on these mutants accumulate less Al than wild type seedlings after exposure to Al-containing solutions. Roots of wild-type seedlings produce callose in response to AlCl3 concentrations that inhibit root growth. Only als5 accumulated more callose than wild type in response to low levels (25 mu M) of AICI3 However, als4 and als7 did not accumulate callose at this AlCl3 concentration even though root growth was significantly inhibited. The lack of callose accumulation in als4 and als7 suggests that there is not an obligatory relationship between callose deposition and Al-induced inhibition of root growth.     

Heme Inhibition of [delta]-Aminolevulinic Acid Synthesis Is Enhanced by Glutathione in Cell-Free Extracts of Chlorella

In plants, algae, and many bacteria, the heme and chlorophyll precursor, [delta]-aminolevulinic acid (ALA), is synthesized from glutamate in a reaction involving a glutamyl-tRNA intermediate and requiring ATP and NADPH as cofactors. In particulate-free extracts of algae and chloroplasts, ALA synthesis is inhibited by heme. Inclusion of 1.0 mM glutathione (GSH) in an enzyme and tRNA extract, derived from the green alga Chlorella vulgaris, lowered the concentration of heme required for 50% inhibition approximately 10-fold. The effect of GSH could not be duplicated with other reduced sulfhydryl compounds, including mercaptoethanol, dithiothreitol, and cysteine, or with imidazole or bovine serum albumin, which bind to heme and dissociate heme dimers. Absorption spectroscopy indicated that heme was fully reduced in incubation medium containing dithiothreitol, and addition of GSH did not alter the heme reduction state. Oxidized GSH was as effective in enhancing heme inhibition as the reduced form. Co-protoporphyrin IX inhibited ALA synthesis nearly as effectively as heme, and 1.0 mM GSH lowered the concentration required for 50% inhibition approximately 10-fold. Because GSH did not influence the reduction state of heme in the incubation medium, and because GSH could not be replaced by other reduced sulfhydryl compounds or ascorbate, the effect of GSH cannot be explained by action as a sulfhydryl protectant or heme reductant. Preincubation of enzyme extract with GSH, followed by rapid gel filtration, could not substitute for inclusion of GSH with heme during the reaction. The results suggest that GSH must specifically interact with the enzyme extract in the presence of the inhibitor to enhance the inhibition.