Platelet derived growth factor receptor alpha is essential for establishing a microenvironment that supports definitive erythropoiesis

The hematopoietic system undergoes a qualitative change during the embryogenesis of most vertebrates. It is designated as the shift of primitive to definitive hematopoiesis and suitable microenvironment must be established to support this shift. While studying the role of platelet derived growth factor receptor alpha (PDGFR alpha) in embryonic hematopoiesis, we found that it was expressed in a stromal cell component of liver, a major site of this shift, but not in the yolk sac, the site of primitive hematopoiesis. Thus, we considered that development of PDGFRalpha positive stromal cells is an essential requirement for this shift. Without PDFGRalpha positive cell component, erythropoiesis was suppressed in the culture of fetal liver. Moreover, injection of an antagonistic anti-PDGFRalpha monoclonal antibody during embryogenesis suppressed the production of definitive erythrocytes. These indicated that PDGF exerts its effect on a subset of stromal components to prepare a microenvironment that can support the definitive erythropoiesis.     

Evidence that heme d1 is a 1,3-porphyrindione

Heme d1 is the noncovalently associated heme prosthetic group of the bacterial nitrite reductase known as cytochrome cd1. Additional evidence has been obtained in support of a dioxoisobacteriochlorin, or 1,3-porphyrindione, skeleton for this heme. The new data include the natural abundance 13C NMR spectrum of the free base methyl ester derivative of d1, mass spectrometric determinations of the molecular mass of the free base methyl ester and the Cu and the Zn chelates, visible and 1H NMR spectral comparisons between d1 and synthetic porphyrindione model compounds, and the isolation and characterization of several byproducts formed during the purification of the free base methyl ester of d1. The accumulated evidence strongly supports the following structure for the skeleton of d1: 1-oxo-2-methyl-2'-acetyl-3-oxo-4-methyl-4'-acetyl-5-methyl-6-acrylyl+ ++-7- propionyl-8-methylporphyrin.     

Identification of a site on herpes simplex virus type 1 glycoprotein D that is essential for infectivity.

Herpes simplex virus glycoprotein D (gD) plays an essential role during penetration of the virus into cells. There is evidence that it recognizes a specific receptor after initial attachment of virions to cell surface heparan sulfate and also that gD-1, gD-2, and gp50 (the pseudorabies virus gD homolog) bind to the same receptor. Although the antigenic structure of gD has been studied intensively, little is known about functional regions of the protein. Antigenic site I is a major target for neutralizing antibodies and has been partially mapped by using deletion mutants and neutralization-resistant viruses. Working on the assumption that such a site may overlap with a functional region of gD, we showed previously that combining two or more amino acid substitutions within site I prevents gD-1 from functioning and is therefore lethal. We have now used a complementation assay to measure the functional activity of a panel of deletion mutants and compared the results with an antigenic analysis. Several mutations cause gross changes in protein folding and destroy functional activity, whereas deletions at the N and C termini have little or no effect on either. In contrast, deletion of residues 234 to 244 has only localized effects on antigenicity but completely abolishes functional activity. This region, which is part of antigenic site Ib, is therefore essential for gD-1 function. The complementation assay was also used to show that a gD-negative type 1 virus can be rescued by gD-2 and by two gD-1-gD-2 hybrids but not by gp50, providing some support for the existence of a common receptor for herpes simplex virus types 1 and 2 but not pseudorabies virus. Alternatively, gp50 may lack a signal for incorporation into herpes simplex virions.     

DNA primase of human mitochondria is associated with structural RNA that is essential for enzymatic activity

DNA primase isolated from human mitochondria sediments in glycerol density gradients at 30S and 70S. These unusually high sedimentation coefficients are a result of association of the primase activity with RNA. Treatment of primase with nuclease not only affects its sedimentation behavior, but also inactivates the primase activity. The major RNA species that cofractionates with primase activity is shown by direct sequence analysis to be cytosolic 5.8S ribosomal RNA (rRNA). Specific degradation of endogenous 5.8S rRNA using ribonuclease H and oligonucleotides complementary to 5.8S rRNA results in reduction of primase activity. Other small RNAs may play a structural role in the formation of an active DNA primase complex.     

All human granzymes target hnRNP K that is essential for tumor cell viability

Granule exocytosis by cytotoxic lymphocytes is the key mechanism to eliminate virus-infected cells and tumor cells. These lytic granules contain the pore-forming protein perforin and a set of five serine proteases called granzymes. All human granzymes display distinct substrate specificities and induce cell death by cleaving critical intracellular death substrates. In the present study, we show that all human granzymes directly cleaved the DNA/RNA-binding protein heterogeneous nuclear ribonucleoprotein K (hnRNP K), designating hnRNP K as the first known pan-granzyme substrate. Cleavage of hnRNP K was more efficient in the presence of RNA and occurred in two apparent proteolysis-sensitive amino acid regions, thereby dissecting the functional DNA/RNA-binding hnRNP K domains. HnRNP K was cleaved under physiological conditions when purified granzymes were delivered into living tumor cells and during lymphokine-activated killer cell-mediated attack. HnRNP K is essential for tumor cell viability, since knockdown of hnRNP K resulted in spontaneous tumor cell apoptosis with caspase activation and reactive oxygen species production. This apoptosis was more pronounced at low tumor cell density where hnRNP K knockdown also triggered a caspase-independent apoptotic pathway. This suggests that hnRNP K promotes tumor cell survival in the absence of cell-cell contact. Silencing of hnRNP K protein expression rendered tumor cells more susceptible to cellular cytotoxicity. We conclude that hnRNP K is indispensable for tumor cell viability and our data suggest that targeting of hnRNP K by granzymes contributes to or reinforces the cell death mechanisms by which cytotoxic lymphocytes eliminate tumor cells.     

Identification of psl, a locus encoding a potential exopolysaccharide that is essential for Pseudomonas aeruginosa PAO1 biofilm formation

Bacteria inhabiting biofilms usually produce one or more polysaccharides that provide a hydrated scaffolding to stabilize and reinforce the structure of the biofilm, mediate cell-cell and cell-surface interactions, and provide protection from biocides and antimicrobial agents. Historically, alginate has been considered the major exopolysaccharide of the Pseudomonas aeruginosa biofilm matrix, with minimal regard to the different functions polysaccharides execute. Recent chemical and genetic studies have demonstrated that alginate is not involved in the initiation of biofilm formation in P. aeruginosa strains PAO1 and PA14. We hypothesized that there is at least one other polysaccharide gene cluster involved in biofilm development. Two separate clusters of genes with homology to exopolysaccharide biosynthetic functions were identified from the annotated PAO1 genome. Reverse genetics was employed to generate mutations in genes from these clusters. We discovered that one group of genes, designated psl, are important for biofilm initiation. A PAO1 strain with a disruption of the first two genes of the psl cluster (PA2231 and PA2232) was severely compromised in biofilm initiation, as confirmed by static microtiter and continuous culture flow cell and tubing biofilm assays. This impaired biofilm phenotype could be complemented with the wild-type psl sequences and was not due to defects in motility or lipopolysaccharide biosynthesis. These results implicate an as yet unknown exopolysaccharide as being required for the formation of the biofilm matrix. Understanding psl-encoded exopolysaccharide expression and protection in biofilms will provide insight into the pathogenesis of P. aeruginosa in cystic fibrosis and other infections involving biofilms.     

Molecular cloning of a human gene that regulates chromosome condensation and is essential for cell proliferation.

The tsBN2 cell line, a temperature-sensitive (ts) mutant of baby hamster kidney cell line BHK21/13, seems to possess a mutation in the gene that controls initiation of chromosome condensation. At the nonpermissive temperature (39.5 degrees C), the chromatin of tsBN2 cells is prematurely condensed, and the cells die. Using tsBN2 cells as a recipient of DNA-mediated gene transfer, we investigated a human gene that is responsible for regulation of chromosome condensation and cell proliferation. We found that the human gene complementing the tsBN2 mutation resides in the area of the 40- to 50-kilobase HindIII fragment, derived from HeLa cells. Based on this finding, we initiated cloning of a human gene complementing the tsBN2 mutation. From lambda and cosmid libraries carrying partial digests of DNA from the secondary transformants, the 41.8-kilobase HindIII fragment containing the human DNA was isolated. The cloned human DNA was conserved in ts+ transformants through primary and secondary transfections. Two cosmid clones convert the ts- phenotype of tsBN2 cells to ts+ with more than 100 times a higher efficiency, compared with cases of transfection with total human DNA. Thus, the cloned DNA fragments contain an active human gene that complements the tsBN2 mutation.     

Ferrate oxidation of Escherichia coli DNA polymerase-I. Identification of a methionine residue that is essential for DNA binding

Treatment of Escherichia coli DNA polymerase-I with potassium ferrate (K2FeO4), a site-specific oxidizing agent for the phosphate group-binding sites of proteins, results in the irreversible inactivation of enzyme activity as judged by the loss of polymerization as well as 3'-5' exonuclease activity. A significant protection from ferrate-mediated inactivation is observed in the presence of DNA but not by substrate deoxynucleoside triphosphates. Furthermore, ferrate-treated enzyme also exhibits loss of template-primer binding activity, whereas its ability to bind substrate triphosphates is unaffected. In addition, comparative high pressure liquid chromatography tryptic peptide maps obtained before and after ferrate oxidation demonstrated that only five peptides of the more than 60 peptide peaks present in the tryptic digest underwent a major change in either peak position or intensity as a result of ferrate treatment. Amino acid analyses and/or sequencing identified four of these affected peaks as corresponding to peptides that span residues 324-340, 437-455, 456-464, and 512-518, respectively. However, only the last peptide, which has the sequence: Met-Trp-Pro-Asp-Leu-Gln-Lys, was significantly protected in the presence of DNA. This latter peptide was also the only peptide whose degree of oxidation correlated directly with the extent of inactivation of the enzyme. Amino acid analysis indicated that methionine 512 is the target site in this peptide for ferrate oxidation. Methionine 512, therefore, appears to be essential for the DNA-binding function of DNA polymerase-I from E. coli.     

Identification of a Drosophila gene encoding xylosylprotein beta4-galactosyltransferase that is essential for the synthesis of glycosaminoglycans and for morphogenesis

In mammals, the xylosylprotein beta4-galactosyltransferase termed beta4GalT7 (XgalT-1, EC ) participates in proteoglycan biosynthesis through the transfer of galactose to the xylose that initiates each glycosaminoglycan chain. A Drosophila cDNA homologous to mammalian beta4-galactosyltransferases was identified using a human beta4GalT7 cDNA as a probe in a BLAST analysis of expressed sequence tags. The Drosophila cDNA encodes a type II membrane protein with 322 amino acids and shows 49% identity to human beta4GalT7. Extracts from L cells transfected with the cDNA exhibited marked galactosyltransferase activity specific for a xylopyranoside acceptor. Moreover, transfection with the cloned cDNA restored glycosaminoglycan synthesis in beta4GalT7-deficient Chinese hamster ovary cells. In transfectant lysates the properties of Drosophila and human beta4GalT7 resembled each other, except that Drosophila beta4GalT7 showed a less restricted specificity and was active at a wider range of temperatures. Drosophila beta4GalT7 is expressed throughout development, with higher expression levels in adults. Reduction of Drosophila beta4GalT7 levels using expressed RNA interference (RNAi) in imaginal discs resulted in an abnormal wing and leg morphology similar to that of flies with defective Hedgehog and Decapentaplegic signaling, which are known to depend on intact proteoglycan biosynthesis. Immunohistochemical analysis of tissues confirmed that both heparan sulfate and chondroitin sulfate biosynthesis were impaired. Our results demonstrate that Drosophila beta4GalT7 has the in vitro and in vivo properties predicted for an ortholog of human beta4GalT7 and is essential for normal animal development through its role in proteoglycan biosynthesis.     

Identification of a human SCARB2 region that is important for enterovirus 71 binding and infection

We previously identified human scavenger receptor class B, member 2 (SCARB2), as a cellular receptor for enterovirus 71 (EV71). Expression of human SCARB2 (hSCARB2) permitted mouse L929 cells to efficiently bind to virions and to produce both viral proteins and progeny viruses upon EV71 infection. Mouse Scarb2 (mScarb2) exhibited 85.8% amino acid identity and 99.9% similarity to hSCARB2. The expression of mScarb2 in L929 cells conferred partial susceptibility. Very few virions bound to mScarb2-expressing cells. The viral titer in L929 cells expressing mScarb2 was approximately 40- to 100-fold lower than that in L929 cells expressing hSCARB2. Using hSCARB2-mScarb2 chimeric mutants, we attempted to map the region that was important for efficient EV71 infection. L929 cells expressing chimeras that carried amino acids 142 to 204 from the human sequence were susceptible to EV71, while chimeras that carried the mouse sequence in this region were not. Moreover, this region was also critical for binding to virions. The determination of this region in hSCARB2 that is important for EV71 binding and infection greatly contributes to the understanding of virus-receptor interactions. Further studies will clarify the early steps of EV71 infection.     

Yeast LEU5 is a PET-like gene that is not essential for leucine biosynthesis

Summary Alpha-IPM synthase catalyzes the first committed step in leucine biosynthesis in the yeast S. cerevisiae. LEU4 is known to encode this enzyme activity. A second gene, LEU5, has been proposed to encode a second enzyme with this activity.We cloned LEU5 and genetically defined the locus. LEU5 maps to chromosome VIII and is tightly linked to CEN8.Five different mutations in LEU5 were analyzed: a sitedirected deletion and a disruption, as well as three distinct mutations produced by chemical mutagenesis. In a leu4 background, each leu5 mutation causes a Leu — phenotype; in a LEU4 background, none of the mutations alters the Leu+ phenotype. This shows that LEU5 is not essential for leucine biosynthesis. In either a leu4 or LEU4 background, each leu5 mutation causes a glycerol — phenotype. This operationally defines LEU5 as a PET gene.Two distinct suppressors of the Pet — phenotype of leu5 strains have been isolated. These suppressors revert the Pet — phenotype of each of four mutant leu5 alleles that were tested. Suppression occurs regardless of the allele at LEU4. Moreover, the suppressors co-revert the Leu — phenotype for each of the four leu5 mutations that is combined with a leu4 allele. This establishes the presence of a gene other than LEU5 that encodes a second alpha-IPM synthase. Further analysis provided no evidence for synthase activity that is encoded by LEU5.Abbreviation EMS ethylmethane sulfonate - IPM isopropylmalate - NPD nonparental ditype - PD parental ditype - TT tetratype     

Granzyme M targets host cell hnRNP K that is essential for human cytomegalovirus replication

Human cytomegalovirus (HCMV) is the most frequent viral cause of congenital defects and HCMV infection in immunocompromised patients may trigger devastating disease. Cytotoxic lymphocytes control HCMV by releasing granzymes towards virus-infected cells. In mice, granzyme M (GrM) has a physiological role in controlling murine CMV infection. However, the underlying mechanism remains poorly understood. In this study, we showed that human GrM was expressed by HCMV-specific CD8⁺ T cells both in latently infected healthy individuals and in transplant patients during primary HCMV infection. We identified host cell heterogeneous nuclear ribonucleoprotein K (hnRNP K) as a physiological GrM substrate. GrM most efficiently cleaved hnRNP K in the presence of RNA at multiple sites, thereby likely destroying hnRNP K function. Host cell hnRNP K was essential for HCMV replication not only by promoting viability of HCMV-infected cells but predominantly by regulating viral immediate-early 2 (IE2) protein levels. Furthermore, hnRNP K interacted with IE2 mRNA. Finally, GrM decreased IE2 protein expression in HCMV-infected cells. Our data suggest that targeting of hnRNP K by GrM contributes to the mechanism by which cytotoxic lymphocytes inhibit HCMV replication. This is the first evidence that cytotoxic lymphocytes target host cell proteins to control HCMV infections.     

SOCS3 is essential in the regulation of fetal liver erythropoiesis.

SOCS3 (CIS3/JAB2) is an SH2-containing protein that binds to the activation loop of Janus kinases, inhibiting kinase activity, and thereby suppressing cytokine signaling. During embryonic development, SOCS3 is highly expressed in erythroid lineage cells and is Epo independent. Transgene-mediated expression blocks fetal erythropoiesis, resulting in embryonic lethality. SOCS3 deletion results in an embryonic lethality at 12-16 days associated with marked erythrocytosis. Moreover, the in vitro proliferative capacity of progenitors is greatly increased. SOCS3-deficient fetal liver stem cells can reconstitute hematopoiesis in lethally irradiated adults, indicating that its absence does not disturb bone marrow erythropoiesis. Reconstitution of lymphoid lineages in JAK3-deficient mice also occurs normally. The results demonstrate that SOCS3 is critical in negatively regulating fetal liver hematopoiesis.     

Heme catabolism in cultured hepatocytes: evidence that heme oxygenase is the predominant pathway and that a proportion of synthesized heme is converted rapidly to biliverdin

Heme oxygenase has been considered to be involved in the predominant pathway of heme degradation in vivo. However, alternative pathways involving cytochrome P-450 reductase, and lipid peroxidation, have previously been demonstrated in vitro, and studies with cultured rat hepatocytes were interpreted to show a majority of endogenous hepatic heme breakdown by non-heme oxygenase pathways. To clarify the pathway of heme breakdown in hepatocytes and the role of heme oxygenase in this process, cultured hepatocytes were pre-labelled with 5-[5-14C]aminolevulinate [( 14C]ALA). Radioactivity in heme, carbon monoxide, and bile pigments was measured for 8-24 h after the removal of [14C]ALA. In cultured chick embryo hepatocytes, which lack biliverdin reductase, the rate of production of biliverdin IXa was closely similar to the rate of catabolism of exogenous heme and radioactivity in carbon monoxide and biliverdin IXa was similar to the loss of radioactivity from endogenous heme. These results support the conclusion that heme breakdown occurred predominantly, if not solely, by heme oxygenase. Also, no evidence of non-heme oxygenase pathways was found in the presence of tin protoporphyrin, an inhibitor of heme oxygenase or mephenytoin, an inducer of both cytochrome P-450 and heme oxygenase. Similarly, in untreated cultured rat hepatocytes, radioactivity in carbon monoxide corresponded with loss of radioactivity in endogenous heme. In other experiments with chick hepatocyte cultures, rates of heme synthesis and breakdown were measured, and data were fitted to various models of hepatic heme metabolism. The results observed were consistent only with models in which an appreciable fraction (control cells, 17%, mephenytoin treated cells, 41%) of the newly synthesized heme was degraded rapidly to biliverdin.     

Identification of a subdomain of CENP-B that is necessary and sufficient for localization to the human centromere

We have combined in vivo and in vitro approaches to investigate the function of CENP-B, a major protein of human centromeric heterochromatin. Expression of epitope-tagged deletion derivatives of CENP-B in HeLa cells revealed that a single domain less than 158 residues from the amino terminus of the protein is sufficient to localize CENP-B to centromeres. Centromere localization was abolished if as few as 28 amino acids were removed from the amino terminus of CENP-B. The centromere localization signal of CENP-B can function in an autonomous fashion, relocating a fused bacterial enzyme to centromeres. The centromere localization domain of CENP-B specifically binds in vitro to a subset of alpha-satellite DNA monomers. These results suggest that the primary mechanism for localization of CENP-B to centromeres involves the recognition of a DNA sequence found at centromeres. Analysis of the distribution of this sequence in alpha-satellite DNA suggests that CENP-B binding may have profound effects on chromatin structure at centromeres.