A mutually inhibitory feedback loop between the 20S proteasome and its regulator, NQO1

NAD(P)H:quinone-oxidoreductase-1 (NQO1) is a cytosolic enzyme that catalyzes the reduction of various quinones using flavin adenine dinucleotide (FAD) as a cofactor. NQO1 has been also shown to rescue proteins containing intrinsically unstructured domains, such as p53 and p73, from degradation by the 20S proteasome through an unknown mechanism. Here, we studied the nature of interaction between NQO1 and the 20S proteasome. Our study revealed a double negative feedback loop between NQO1 and the 20S proteasome, whereby NQO1 prevents the proteolytic activity of the 20S proteasome and the 20S proteasome degrades the apo form of NQO1. Furthermore, we demonstrate, both in vivo and in vitro, that NQO1 levels are highly dependent on FAD concentration. These observations suggest a link between 20S proteolysis and the metabolic cellular state. More generally, the results may represent a regulatory mechanism by which associated cofactors dictate the stability of proteins, thus coordinating protein levels with the metabolic status.     

Identification of DNA-binding Proteins Using Structural, Electrostatic and Evolutionary Features

DNA-binding proteins (DBPs) participate in various crucial processes in the life-cycle of the cells, and the identification and characterization of these proteins is of great importance. We present here a random forests classifier for identifying DBPs among proteins with known 3D structures. First, clusters of evolutionarily conserved regions (patches) on the surface of proteins were detected using the PatchFinder algorithm; earlier studies showed that these regions are typically the functionally important regions of proteins. Next, we trained a classifier using features like the electrostatic potential, cluster-based amino acid conservation patterns and the secondary structure content of the patches, as well as features of the whole protein, including its dipole moment. Using 10-fold cross-validation on a dataset of 138 DBPs and 110 proteins that do not bind DNA, the classifier achieved a sensitivity and a specificity of 0.90, which is overall better than the performance of published methods. Furthermore, when we tested five different methods on 11 new DBPs that did not appear in the original dataset, only our method annotated all correctly.The resulting classifier was applied to a collection of 757 proteins of known structure and unknown function. Of these proteins, 218 were predicted to bind DNA, and we anticipate that some of them interact with DNA using new structural motifs. The use of complementary computational tools supports the notion that at least some of them do bind DNA.     

The herpes simplex virus type 1 vhs-UL41 gene secures viral replication by temporarily evading apoptotic cellular response to infection: Vhs-UL41 activity might require interactions with elements of cellular mRNA degradation machinery

We have previously shown that herpes simplex virus type 1 (HSV-1) infection is associated with early destabilization/degradation of infected cell mRNAs and consequent shutoff of host protein synthesis by the activity of the virion-associated host shutoff (vhs) UL41 protein. Wild-type (wt) virus destabilized/degraded the housekeeping beta-actin and alpha-tubulin mRNAs as well host stress functions, like the heat shock 70 protein induced postinfection. vhs mutants did not degrade the mRNAs. Elaborate studies by others have been concerned with the mode of mRNA degradation and the mRNAs affected. We now describe vhs activity in primary cultures of mouse cerebellar granule neurons (CGNs). Specifically, (i) upon infection in the presence of actinomycin D to test activity of input viral particles, there was a generalized inhibition of protein synthesis, which depended on the input multiplicity of infection (MOI). (ii) Low-MOI infection with vhs-1 mutant virus was associated with increased synthesis of all apparent proteins. Higher MOIs caused some shutoff, albeit significantly lower than that of wt virus. This pattern could reflect an interaction(s) of vhs-1 protein with host machinery involved in cellular mRNA destabilization/degradation, sequestering this activity. (iii) wt virus infection was associated with cell survival, at least for a while, whereas mutant virus induced apoptotic cell death at earlier times. (iv) wt virus replicated well in the CGNs, whereas there was no apparent replication of the vhs-1 mutant virus. (v) The vhs-1 mutant could serve as helper virus for composite amplicon vectors carrying marker genes and the human p53 gene. Ongoing studies test the use of vhs-1-based composite oncolytic vectors towards cancer gene therapy.     

Glutathione S-transferase Ya subunit is coded by a multigene family located on a single mouse chromosome.

A cloned DNA probe of Ya, the major glutathione S-transferase subunit in rat liver, was used to study the organization of Ya genes in the mouse genome. Southern blot analysis of mouse genomic DNA indicates that the Ya subunit is encoded by a multigene family. The chromosomal distribution of Ya genes was determined by analysis of DNA from a panel of mouse-Chinese hamster somatic cell hybrids. All detectable Ya genes were found to be located on chromosome 9. At least some of the Ya-specific DNA sequences are clustered since, by screening a mouse genomic library, two recombinant phages, each containing two different Ya DNA sequences in the same insert, have been isolated. The finding that Ya is encoded by a cluster of different genes raises the question of the specificity of the different Ya DNA sequences.     

Carp growth hormone: molecular cloning and sequencing of cDNA

cDNA clones of the fish Cyprinus carpio growth hormone (GH) mRNA have been isolated from a cDNA library prepared from carp pituitary gland poly(A)+RNA. The nucleotide sequence of one of the carp GH cDNA clones containing an insert of 1164 nucleotides (nt) was determined. The cDNA sequence was found to encode a polypeptide of 210 amino acids (aa) including a signal peptide of 22 aa and to contain 5' and 3' untranslated regions of the mRNA of 36 and 498 nt, respectively. The carp GH presents a 63% amino acid sequence homology with the salmon GH, has structural features common with other GH polypeptides of mammalian or avian origin and contains domains of conserved sequence near the N- and C-terminal regions. Southern blot hybridization of carp genomic DNA with GH cDNA probes shows the presence of at least two GH-coding sequences in the fish genome.     

Isolation and characterization of a plasmid involved with enterotoxin B production in Staphylococcus aureus.

Genetic analysis and molecular characterization of plasmid deoxyribonucleic acid (DNA) was performed in a toxigenic isolate of Staphylococcus aureus strain DU4916. Elimination, transduction, and transformation experiments provided us with a series of derivatives similar except for the presence or absence of genes mediating resistance to penicillin (penr), methicillin (mecr), and tetracycline (tetr) and enterotoxin type B (SEB) production (entB+). The derivatives were examined for the presence of a plasmid species which encodes for SEB production. Two distinct species of covalently closed circular DNA of about 2.8 X 10(6) and 0.75 X 10(6) daltons were identified in an ethidium bromide-cured, penicillinase-negative (pens) isolate, SN109 (mecr tetr emtB+). Further segregation of either methicillin resistance or tetracycline resistance or of both together resulted in the loss of SEB production and the disappearance of both plasmids. Transduction from strain SN109 showed that determinants for tetracycline resistance are carried by the 2.8 X 10(6) dalton plasmid. Transformation with covalently closed circular DNA from strain SN109 yielded mecs tetr entB- transformants harboring the tetracycline resistance plasmid alone and mecr tetr entB+ transformants harboring both the tetracycline resistance and the 0.75 X 10(6)-dalton plasmid. Further segregation of methicillin resistance in transformants was not associated with any change in plasmid DNA. The results indicate that a genetic determinant for SEB production is carried by the 0.75 X 10(6)-dalton plasmid. It is possible, however, that this plasmid cannot be maintained in the host independently from the tetracycline resistance plasmid. Methicillin resistance in the strains examined could not be ascribed to any of the covalently closed circular DNA components resolved in strain DU4916.     

Enantioselective synthesis and complement inhibitory assay of A/B-ring partial analogues of oleanolic acid.

A series of oleanolic acid A/B-ring partial analogues was synthesized and tested for their complement inhibitory activity as well as cytotoxic properties. All target compounds and one intermediate exhibited moderate complement inhibitory potency. These compounds also showed cytotoxicity on malignant melanoma cell line, SK-MEL.     

Post-transplant Kaposi sarcoma originates from the seeding of donor-derived progenitors

Kaposi sarcoma (KS) is a vascular tumor that can develop in recipients of solid tissue transplants as a result of either primary infection or reactivation of a gammaherpesvirus, the KS- associated herpesvirus, also known as human herpesvirus-8 (HHV-8). We studied whether HHV-8 and the elusive KS progenitor cells could be transmitted from the donor through the grafts. We used a variety of molecular, cytogenetic, immunohistochemical and immunofluorescence methods to show that the HHV-8-infected neoplastic cells in post-transplant KS from five of eight renal transplant patients harbored either genetic or antigenic markers of their matched donors. These data suggest the use of donor-derived HHV-8-specific T cells for the control of post-transplant KS.     

Characterization of midface maxillary membranous bone formation during distraction osteogenesis

The purpose of the study was to follow the early events in bone formation and neovascularization during maxillary distraction and after the consolidation period and to define the characterization of the new bone in the distracted area. Maxillary osteotomy was performed in seven sheep. In five animals, an external distraction device was used for maxillary lengthening of 20 mm at a rate of 1 mm/day for 20 days. Another two animals served as controls without distraction. Sequential biopsies were performed. The methods used for analysis were histologic, immunohistochemical, and ultrastructural by transmission electron microscopy. During the 5 days of latency, a fibrin clot was formed that after 5 days of distraction was replaced by granulation tissue, proliferating mesenchyme-like cells, and capillaries. After 10 days of distraction, the regenerated tissue could be divided into three main zones and two transitional areas: a central zone occupied by many polygonal mesenchyme-like cells and spindle-shaped cells that proliferated intensively; two paracentral zones on both sides of the central zone in which many cells showed morphologic signs of apoptosis leading to a decreased number of fibroblast-like cells embedded in wavy collagen fibers; a transitional area from the central to the paracentral zone in which concentric cellular colonies were believed to represent a novel form of vasculogenesis; distal-proximal zones, located on both sides of the paracentral zones and in continuation with the old bone, showed delicate new woven bone trabeculae that grew continuously in the direction of lengthening and gradually became mineralized; and a transitional area from the paracentral to the distal-proximal zones in which there was recruitment of preosteoblasts from the distracted tissue to the trabecular tips. These further differentiated into osteoblasts that contributed to the trabecular growth. The histologic feature pattern was similar after 15 and 20 days of continuous distraction. At the end of lengthening, after 20 days, delicate longitudinally oriented trabeculae continued to grow by recruiting preosteogenic cells from the central distracted tissue, became mineralized, and were rimmed by osteoblasts. After 6 weeks of retention, the trabeculae thickened and consisted of a mixture of lamellar and woven bone. In conclusion, the distraction force creates a pool of undifferentiated mesenchyme-like cells with osteogenic potential and triggers capillary formation, a clear zonation can be observed during active lengthening, and new bone trabeculae begin to form between 5 and 10 days after distraction, soon become aligned with osteoblasts, and continue to grow as long as distraction force is applied. This characterization may help in any exogenous involvement with growth factors to improve bone quality.