A small molecule inhibitor partitions two distinct pathways for trafficking of tonoplast intrinsic proteins in Arabidopsis

Tonoplast intrinsic proteins (TIPs) facilitate the membrane transport of water and other small molecules across the plant vacuolar membrane, and members of this family are expressed in specific developmental stages and tissue types. Delivery of TIP proteins to the tonoplast is thought to occur by vesicle-mediated traffic from the endoplasmic reticulum to the vacuole, and at least two pathways have been proposed, one that is Golgi-dependent and another that is Golgi-independent. However, the mechanisms for trafficking of vacuolar membrane proteins to the tonoplast remain poorly understood. Here we describe a chemical genetic approach to unravel the mechanisms of TIP protein targeting to the vacuole in Arabidopsis seedlings. We show that members of the TIP family are targeted to the vacuole via at least two distinct pathways, and we characterize the bioactivity of a novel inhibitor that can differentiate between them. We demonstrate that, unlike for TIP1;1, trafficking of markers for TIP3;1 and TIP2;1 is insensitive to Brefeldin A in Arabidopsis hypocotyls. Using a chemical inhibitor that may target this BFA-insensitive pathway for membrane proteins, we show that inhibition of this pathway results in impaired root hair growth and enhanced vacuolar targeting of the auxin efflux carrier PIN2 in the dark. Our results indicate that the vacuolar targeting of PIN2 and the BFA-insensitive pathway for tonoplast proteins may be mediated in part by common mechanisms.     

CROSS-SECTIONAL ECHOCARDIOGRAPHIC FINDINGS IN VEGETATIVE AORTIC VALVE ENDOCARDITIS

M-mode echocardiograms of two patients with bacterial endocarditis of approximately 4 months' duration showed dense echoes in the area of the aortic valve. In one patient, who had no prior abnormal cardiac findings, the echoes were clearly suggestive of valvular vegetations. The second patient, however, was known to have had aortic valve disease and a systolic murmur for more than a decade; therefore, dense echoes arising from the aortic valve also could have resulted from valvular calcification. In both patients, cross-sectional echocardiography provided important information. In the first patient, retrograde cardiac catheterization was prevented by large and highly mobile masses attached to the aortic cusps that prolapsed into the left ventricular outflow tract during diastole. Aortic valve replacement without further hemodynamic evaluation was recommended. In the second patient, whose blood cultures remained negative after the acute phase of his illness had been treated, cross-sectional echocardiography showed large vegetations on the aortic valve. Intraoperative findings confirmed the echocardiographic interpretation in each case.     

Rapid neurotrophic actions of an ACTH/MSH(4–9) analogue after nigrostriatal 6-OHDA lesioning

ACTH peptide fragments demonstrate potent neurotrophic effects on peripheral nerves in situ, central neurons in culture, and have been implicated to have effects on central neurons in vivo. Neurotoxic lesioning of the nigrostriatal system, which depletes the striatum of dopamine, provides a feasible model of central regeneration in which to test these peptides. Male Sprague-Dawley rats were lesioned unilaterally with 6-hydroxydopamine (8 μg/4 μl), infused into the substantia nigra. They were subsequently treated with 10 μg/kg IP of Org 2766 [ACTH/MSH(4–9) analogue] or saline every 24 h starting immediately after the infusion and were observed for 2 weeks. Rotational behavior data indicate that Org 2766 significantly decreases ipsiversive turning (p < 0.05), induced by amphetamine (2 mg/kg), as well as accelerating the onset of denervation supersensitivity induced by apomorphine (0.05 mg/kg). Evaluation of dopamine immunohistochemistry, using an anti-tyrosine hydroxylase antibody, demonstrates an enhanced intensity of staining in the ORG 2766-treated tissue compared to its saline counterpart. This difference is confirmed and quantified through specific high-affinity dopamine uptake. Dopamine uptake is about 17% higher in the striata of animals treated with Org 2766. Higher dopamine uptake levels in these ACTH-treated animals correlate with greater fiber density in this group. Therefore, it appears that treatment with the ACTH/MSH(4–9) analogue Org 2766 (10 μg/kg/24 h) offers a protective effect from 6-OHDA lesions in the substantia nigra as well as accelerating various compensatory mechanisms involved in functional recovery.     

Genomic location and characterisation of MIC genes in cattle

Major histocompatibility complex (MHC) class I chain-related (MIC) genes have been previously identified and characterised in human. They encode polymorphic class I-like molecules that are stress-inducible, and constitute one of the ligands of the activating natural killer cell receptor NKG2D. We have identified three MIC genes within the cattle genome, located close to three non-classical MHC class I genes. The genomic position relative to other genes is very similar to the arrangement reported in the pig MHC region. Analysis of MIC cDNA sequences derived from a range of cattle cell lines suggest there may be four MIC genes in total. We have investigated the presence of the genes in distinct and well-defined MHC haplotypes, and show that one gene is consistently present, while configuration of the other three genes appears variable.     

Role of tryptophan residues of Erv1: Trp95 and Trp183 are important for its folding and oxidase function

Erv1 is an FAD-dependent thiol oxidase of the ERV (essential for respiration and viability)/ALR (augmenter of liver regeneration) sub-family and an essential component of the mitochondrial import and assembly pathway. Erv1 contains six tryptophan residues, which are all located in the highly conserved C-terminal FAD-binding domain. Though important structural roles were predicted for the invariable Trp⁹⁵, no experimental study has been reported. In the present study, we investigated the structural and functional roles of individual tryptophan residues of Erv1. Six single tryptophan-to-phenylalanine yeast mutant strains were generated and their effects on cell viability were tested at various temperatures. Then, the mutants were purified from Escherichia coli. Their effects on folding, FAD-binding and Erv1 activity were characterized. Our results showed that Erv1^W95F has the strongest effect on the stability and function of Erv1 and followed by Erv1^W183F. Erv1^W95F results in a decrease in the T_m of Erv1 by 23°C, a significant loss of the oxidase activity and thus causing cell growth defects at both 30°C and 37°C. Erv1^W183F induces changes in the oligomerization state of Erv1, along with a pronounced effect on the stability of Erv1 and its function at 37°C, whereas the other mutants had no clear effect on the function of Erv1 including the highly conserved Trp¹⁵⁷ mutant. Finally, computational analysis indicates that Trp⁹⁵ plays a key role in stabilizing the isoalloxazine ring to interact with Cys¹³³. Taken together, the present study provided important insights into the molecular mechanism of how thiol oxidases use FAD in catalysing disulfide bond formation.     

Morphometrics and character state recognition for cladistic analyses in theBletia reflexa complex (Orchidaceae)

Different criteria have been suggested for decisions on character state recognition for morphological characters showing continuous variation from taxon to taxon. A method proposed byMishler andDe Luna seeks to recognize as many character states as the groups of taxon means that are revealed by an analysis of variance (ANOVA) followed by multiple range tests. We apply this method as a guide in decisions of selecting taxonomic characters in species of theBletia reflexa complex. Seven species of the complex, represented by 156 individuals belonging to 54 populations, were the terminal taxa of the study. Character state codes were assigned to 20 characters. These characters together with 12 non-quantitative characters were incorporated into cladistic analyses. Results indicate that the complex is monophyletic only under one outgroup combination.     

[3H]5-Hydroxytryptamine Binding to Brain Astroglial Cells: Differences Between Intact and Homogenized Preparations and Mature and Immature Cultures

[3H]5-hydroxytryptamine ([3H]serotonin) binds with high affinity (KD 2-12 nM) to a finite number of sites on brain astroglial cells. The number of binding sites in the C6 glioma line is decreased significantly (Bmax = 315 versus 30 fmol/mg) by homogenization. In intact primary cultures, derived from newborn rat brain, the number of binding sites is far greater in cultures of immature astrocytes than in cultures treated with dibutyryl cyclic AMP (Bmax = 1,520 versus 580 fmol/mg). A role for these receptors in development is suggested.     

5-HT1A Receptor Agonist Reverses Adrenalectomy-Induced Loss of Granule Neuronal Morphology in the Rat Dentate Gyrus

Adrenal steroids are important for maintaining neuronal maturation in the adult rats. Two weeks after bilateral adrenalectomy (ADX), hippocampal MAP-2 (microtubule associated protein-2) and calbindin immunoreactivity (IR) decreased in the molecular layer of the superior blade of the dentate gyrus. The molecular and granular cell layer at the lateral tip of the superior blade decreased in width by 32% and 50%, respectively. The granule neurons showed reduced staining with Nissl and an anti-calbindin antibody. These changes suggested a loss of the mature neuronal morphology. In this same localized regions, two glial proteins, glial fibrillary acidic protein (GFAP) and S-100 showed dramatically reduced immunoreactivity. These effects induced by ADX were reduced within 72 hrs by ipsapirone (1 mg/kg), a 5HT_1A receptor agonist. Loss of adult neuronal morphology by ADX, and reversal by the 5HT_1A agonist, may be evidence of the trophic importance of the 5HT_1A receptor in granule neurons of hippocampus.