Surface loop dynamics in adeno-associated virus capsid assembly

The HI loop is a prominent domain on the adeno-associated virus (AAV) capsid surface that extends from each viral protein (VP) subunit overlapping the neighboring fivefold VP. Despite the highly conserved nature of the residues at the fivefold pore, the HI loops surrounding this critical region vary significantly in amino acid sequence between the AAV serotypes. In order to understand the role of this unique capsid domain, we ablated side chain interactions between the HI loop and the underlying EF loop in the neighboring VP subunit by generating a collection of deletion, insertion, and substitution mutants. A mutant lacking the HI loop was unable to assemble particles, while a substitution mutant (10 glycine residues) assembled particles but was unable to package viral genomes. Substitution mutants carrying corresponding regions from AAV1, AAV4, AAV5, and AAV8 yielded (i) particles with titers and infectivity identical to those of AAV2 (AAV2 HI1 and HI8), (ii) particles with a decreased virus titer (1 log) but normal infectivity (HI4), and (iii) particles that synthesized VPs but were unable to assemble into intact capsids (HI5). AAV5 HI is shorter than all other HI loops by one amino acid. Replacing the missing residue (threonine) in AAV2 HI5 resulted in a moderate particle assembly rescue. In addition, we replaced the HI loop with peptides varying in length and amino acid sequence. This region tolerated seven-amino-acid peptide substitutions unless they spanned a conserved phenylalanine at amino acid position 661. Mutation of this highly conserved phenylalanine to a glycine resulted in a modest decrease in virus titer but a substantial decrease (1 log order) in infectivity. Subsequently, confocal studies revealed that AAV2 F661G is incapable of efficiently completing a key step in the infectious pathway nuclear entry, hinting at a possible perturbation of VP1 phospholipase activity. Molecular modeling studies with the F661G mutant suggest that disruption of interactions between F661 and an underlying P373 residue in the EF loop of the neighboring subunit might adversely affect incorporation of the VP1 subunit at the fivefold axis. Western blot analysis confirmed inefficient incorporation of VP1, as well as a proteolytically processed VP1 subunit that could account for the markedly reduced infectivity. In summary, our studies show that the HI loop, while flexible in amino acid sequence, is critical for AAV capsid assembly, proper VP1 subunit incorporation, and viral genome packaging, all of which implies a potential role for this unique surface domain in viral infectivity.     

Fission and selective fusion govern mitochondrial segregation and elimination by autophagy

Accumulation of depolarized mitochondria within beta-cells has been associated with oxidative damage and development of diabetes. To determine the source and fate of depolarized mitochondria, individual mitochondria were photolabeled and tracked through fusion and fission. Mitochondria were found to go through frequent cycles of fusion and fission in a 'kiss and run' pattern. Fission events often generated uneven daughter units: one daughter exhibited increased membrane potential (delta psi(m)) and a high probability of subsequent fusion, while the other had decreased membrane potential and a reduced probability for a fusion event. Together, this pattern generated a subpopulation of non-fusing mitochondria that were found to have reduced delta psi(m) and decreased levels of the fusion protein OPA1. Inhibition of the fission machinery through DRP1(K38A) or FIS1 RNAi decreased mitochondrial autophagy and resulted in the accumulation of oxidized mitochondrial proteins, reduced respiration and impaired insulin secretion. Pulse chase and arrest of autophagy at the pre-proteolysis stage reveal that before autophagy mitochondria lose delta psi(m) and OPA1, and that overexpression of OPA1 decreases mitochondrial autophagy. Together, these findings suggest that fission followed by selective fusion segregates dysfunctional mitochondria and permits their removal by autophagy.     

Tetranor PGDM, an abundant urinary metabolite reflects biosynthesis of prostaglandin D2 in mice and humans

Prostaglandin D(2) (PGD(2)) is a cyclooxygenase (COX) product of arachidonic acid that activates D prostanoid receptors to modulate vascular, platelet, and leukocyte function in vitro. However, little is known about its enzymatic origin or its formation in vivo in cardiovascular or inflammatory disease. 11,15-dioxo-9alpha-hydroxy-2,3,4,5-tetranorprostan-1,20-dioic acid (tetranor PGDM) was identified by mass spectrometry as a metabolite of infused PGD(2) that is detectable in mouse and human urine. Using liquid chromatography-tandem mass spectrometry, tetranor PGDM was much more abundant than the PGD(2) metabolites, 11beta-PGF(2alpha) and 2,3-dinor-11beta-PGF(2alpha), in human urine and was the only endogenous metabolite detectable in mouse urine. Infusion of PGD(2) dose dependently increased urinary tetranor PGDM > 2,3-dinor-11beta-PGF(2alpha) > 11beta-PGF(2alpha) in mice. Deletion of either lipocalin-type or hemopoietic PGD synthase enzymes decreased urinary tetranor PGDM. Deletion or knockdown of COX-1, but not deletion of COX-2, decreased urinary tetranor PGDM in mice. Correspondingly, both PGDM and 2,3-dinor-11beta-PGF(2alpha) were suppressed by inhibition of COX-1 and COX-2, but not by selective inhibition of COX-2 in humans. PGD(2) has been implicated in both the development and resolution of inflammation. Administration of bacterial lipopolysaccharide coordinately elevated tetranor PGDM and 2,3-dinor-11beta-PGF(2alpha) in volunteers, coincident with a pyrexial and systemic inflammatory response, but both metabolites fell during the resolution phase. Niacin increased tetranor PGDM and 2,3-dinor-11beta-PGF(2alpha) in humans coincident with facial flushing. Tetranor PGDM is an abundant metabolite in urine that reflects modulated biosynthesis of PGD(2) in humans and mice.     

E2F-1 is essential for normal epidermal wound repair.

E2F factors are involved in proliferation and apoptosis. To understand the role of E2F-1 in the epidermis, we screened wild type and E2F-1(-/-) keratinocyte mRNA for genes differentially expressed in the two cell populations. We demonstrate the reduced expression of integrins alpha(5), alpha(6), beta(1), and beta(4) in E2F-1(-/-) keratinocytes associated with reduced activation of Jun terminal kinase and Erk upon integrin stimulation. As a consequence of altered integrin expression and function, E2F-1(-/-) keratinocytes also show impaired migration, adhesion to extracellular matrix proteins, and a blunted chemotactic response to transforming growth factor-gamma1. E2F-1(-/-) keratinocytes, but not dermal fibroblasts, exhibit altered patterns of proliferation, including significant delays in transit through both G(1) and S phases of the cell cycle. Recognizing that proliferation and migration are key for proper wound healing in vivo, we postulated that E2F-1(-/-) mice may exhibit abnormal epidermal repair upon injury. Consistent with our hypothesis, E2F-1(-/-) mice exhibited impaired cutaneous wound healing. This defect is associated with substantially reduced local inflammatory responses and rates of re-epithelialization. Thus, we demonstrate that E2F-1 is indispensable for a hitherto unidentified cell type-specific and unique role in keratinocyte proliferation, adhesion, and migration as well as in proper wound repair and epidermal regeneration in vivo.     

Non-random inheritance of mitochondrial genomes in Citrus hybrids produced by protoplast fusion

Somatic hybridization offers the possibility of manipulating chloroplast and mitochondrial genomes and evaluating their role on cultivar qualities in citrus. Numerous associations between Willow-leaf mandarin (Citrus deliciosa Ten.), as embryogenic parent, and sweet orange cv. Valencia (Citrus sinensis (L.) Osb.), as mesophyll parent, and between Willow-leaf mandarin (embryogenic parent) and grapefruit cv. Duncan (Citrus paradisi Macf.) (mesophyll parent) were obtained by the fusion of protoplasts induced by polyethylene glycol. Regenerated plants were characterized by flow cytometry and nuclear and mitochondrial DNA restriction fragment length polymorphism (RFLP). All plants were diploid. Diploid plants with the nuclear RFLP patterns of mandarin or sweet orange were identified in the progeny between these two parents, while only grapefruit nuclear types were found in the mandarin + grapefruit progeny. The diploid plants with the nuclear profile of the mesophyll parent originated systematically from cells formed through spontaneous association of the nuclear genome of the mesophyll parent and the mitochondrial genome of the embryogenic parent. These plants are assumed to be alloplasmic hybrids or cybrids. They were viable and have been propagated for field testing.     

Dehydroepiandrosterone regulation of the hepatic glucocorticoid receptor in the zucker rat. The obesity research program

Dehydroepiandrosterone (DHEA) decreases the activity of hepatic tyrosine aminotransferase (TAT), a glucocorticoid-inducible enzyme, in the obese, hypercorticosteronemic Zucker rat. To investigate the mechanism of this antiglucocorticoid action, the effect of exogenous DHEA on hepatic glucocorticoid receptor (GC) number and affinity was quantitated. Food supplementation with DHEA (0.6% w/w) for 1 or 7 days had no effect on either receptor number or affinity in obese Zucker rats. After 28 days, however, DHEA treatment resulted in a nearly 40% decrease in cytosolic hepatic receptor content (B_max; fmol/mg cytosolic protein) without any change in affinity (K_d) in both lean and obese rats. DHEA treatment for 28 days also resulted in an increased liver size and cytosolic protein content. When the hepatic GC receptor content was normalized based on the change in liver size and protein content, the apparent number of GC binding sites per liver was not affected by DHEA treatment. This observation suggests that DHEA's effect on GC receptor content may not be a specific action and that downregulation of the GC receptor is not the mechanism of DHEA action on GC induced TAT activity. This is supported by the effect of DHEA on obese rat TAT activity in the same experiment where the greatest inhibition occurred after only 1 day of treatment. From these experiments it is concluded that although long-term DHEA treatment may decrease the relative concentration of GC receptors in rat liver, this change is not the mechanism through which DHEA mediates its acute antiglucocorticoid action.     

Structural basis for IL-12 and IL-23 receptor sharing reveals a gateway for shaping actions on T versus NK cells

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Enhanced resistance to citrus canker in transgenic mandarin expressing Xa21 from rice

Genetic engineering approaches offer an alternative method to the conventional breeding of Citrus sp. ‘W. Murcott’ mandarin (a hybrid of ‘Murcott’ and an unknown pollen parent) is one of the most commercially important cultivars grown in many regions around the world. Transformation of ‘W. Murcott’ mandarin was achieved by direct DNA uptake using a protoplast transformation system. DNA construct (pAO3), encoding Green Fluorescent Protein (GFP) and the cDNA of Xa21, a Xanthomonas resistance gene from rice, was used to transform protoplasts of ‘W. Murcott’ mandarin. Following citrus protoplast culture and regeneration, transformed micro calli were microscopically designated via GFP expression, physically isolated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. More than 150 transgenic embryos were recovered and from them, ten transgenic lines were regenerated and cultured on rooting medium for shoot elongation. Transgenic shoots were micrografted and established in the greenhouse with 3–5 replicates per line. The insertion of Xa21 and GFP was confirmed by PCR and southern blot analysis. GFP expression was verified by fluorescence microscopy and western blot analysis revealed expression of Xa21 although it was variable among transgenic lines, as shown by RT-qPCR. Transgenic plants challenged with the citrus canker pathogen by syringe inoculation showed a reduction in lesion number and bacterial populations within lesions compared to non-transgenic control plants. Transgenic ‘W. Murcott’ mandarin lines with improved canker resistance via protoplast transformation from embryogenic callus with the Xa21 gene from rice are being evaluated under field conditions to validate the level of resistance.     

Über den Einfluß der Kultivierungsbedingungen auf die Farbstoffbildung durch Penicillium-Arten

Zusammenfassung Es wird gezeigt, daß sich die Farbstoffbildung der Penicillium-arten in recht erheblichem Ausmaß durch die Züchtungsbedingungen beeinflussen läßt. Bereits bei der vergleichenden Kultivierung der Pilze in der Oberflächen- und Submerskultur können auffallende Unterschiede beobachtet werden. Ferner konnte durch den Zusatz von Asparagin, Glutaminsäure und Zinksulfat besonders in der Submerskultur die Farbstoffbildung recht erheblich geändert werden. Dabei verhielten sich allerdings die einzelnen Pilze individuell außerordentlich verschieden, so daß keine allgemein gültigen Gesetzmäßigkeiten über die Beeinflußbarkeit des Prozesses abgeleitet werden können. Wohl aber können bestimmte Typen von Fällen aufgestellt werden, denen jeweils eine Reihe von Penicillien zuzuordnen ist.Aus dem vormaligen Institut für Biochemie u. Nahrungsmittelchemie der Deutschen Technischen Hochschule in Prag. — Vgl. auch Diplom-Arbeit H. Kundtner, Prag, März 1945.