Deep transcriptome sequencing reveals differences in global gene expression between normal and pale, soft, and exudative turkey meat.

Previous studies from our laboratory suggested that differential expression of genes between normal and pale, soft, and exudative (PSE) turkey is associated with development of the PSE syndrome. However, a detailed understanding of molecular mechanisms responsible for the development of this meat defect remains unclear. The objective of this study was to extend and complement our previous work by using deep transcriptome RNA sequence analysis to compare the respective transcriptome profiles and identify molecular mechanisms responsible for the etiology of PSE turkey meat. Turkey breasts (n = 43) were previously classified as normal or PSE using marinade uptake as an indicator of quality (high = normal; low = PSE). Total RNA from breast muscle samples with the highest (n = 4) and lowest (n = 4) marinade uptake were isolated and sequenced using the Illumina GA(IIX) platform. The results indicated differential expression of 494 loci (false discovery rate < 0.05). Changes in gene expression were confirmed using quantitative real-time PCR. Pathway analysis of differentially expressed genes suggested abnormalities of calcium homeostasis and signaling pathways regulating actin cytoskeleton structure as well as carbohydrate metabolism and energy production in PSE samples. Dysregulation of postmortem glucose oxidation in PSE turkey was suggested by both dramatic downregulation of pyruvate dehydrogenase kinase, isozyme 4 (PDK4) mRNA, the most downregulated gene, and a decrease in the protein product (P = 0.0007) as determined by immunoblot analysis. These results support the hypothesis that differential expression of several genes and their protein products contribute to development of PSE turkey.

Osteogenic and adipogenic cell fractions isolated from postnatal mouse calvaria.

The use of stem/progenitor cells represents a promising approach to treat craniofacial bone defects, but successful treatments will rely on the availability of cells that can be expanded in vitro and which will differentiate appropriately in vivo. The calvaria may represent a source of autologous cells for such purposes. We demonstrate expression of stem cell antigen-1 (Sca-1) in mouse calvaria. We isolated Sca-1(+) and Sca-1(-) cells at high purity and tested the ability of these cells to differentiate into adipose and bone. We show that the Sca-1(+) cell fraction has adipogenic differentiation potential and that the cell Sca-1(-) fraction has osteogenic differentiation potential. The Sca-1(+) cell fraction partially retains its adipogenic differentiation potential and the Sca-1(-) cell fraction partially retains its osteogenic differentiation potential after in vitro expansion. These data suggest that the calvaria may be used as a source of stem/progenitor cells that can be expanded in vitro and transplanted in vivo for craniofacial tissue regeneration.

Stoichiometry of DnaA and DnaB protein in initiation at the Escherichia coli chromosomal origin.

Initiation of DNA replication at the Escherichia coli chromosomal origin, oriC, occurs through an ordered series of events that depend first on the binding of DnaA protein, the replication initiator, to DnaA box sequences within oriC followed by unwinding of an AT-rich region near the left border. The prepriming complex then forms, involving the binding of DnaB helicase at oriC so that it is properly positioned at each replication fork. We assembled and isolated the prepriming complexes on an oriC plasmid, then determined the stoichiometries of proteins in these complexes by quantitative immunoblot analysis. DnaA protein alone binds to oriC with a stoichiometry of 4-5 monomers per oriC DNA. In the prepriming complex, the stoichiometries are 10 DnaA monomers and 2 DnaB hexamers per oriC plasmid. That only two DnaB hexamers are bound, one for each replication fork, suggests that the binding of additional molecules of DnaA in forming the prepriming complex restricts the loading of additional DnaB hexamers that can bind at oriC.

Essential amino acids of Escherichia coli DnaC protein in an N-terminal domain interact with DnaB helicase.

Escherichia coli DnaC protein bound to ATP forms a complex with DnaB protein. To identify the domain of DnaC that interacts with DnaB, a genetic selection was used based on the lethal effect of induced dnaC expression and a model that inviability arises by the binding of DnaC to DnaB to inhibit replication fork movement. The analysis of dnaC alleles that preserved viability under elevated expression revealed an N-terminal domain of DnaC involved in binding to DnaB. Mutant proteins bearing single amino acid substitutions (R10P, L11Q, L29Q, S41P, W32G, and L44P) that reside in regions of predicted secondary structure were inert in DNA replication activity because of their inability to bind to DnaB, but they retained ATP binding activity, as indicated by UV cross-linking to [alpha-(32)P]ATP. These alleles also failed to complement a dnaC28 mutant. Other selected mutations that map to regions carrying Walker A and B boxes are expected to be defective in ATP binding, a required step in DnaB-DnaC complex formation. Lastly, we found that the sixth codon from the N terminus encodes aspartate, resolving a reported discrepancy between the predicted amino acid sequence based on DNA sequencing data and the results from N-terminal amino acid sequencing (Nakayama, N., Bond, M. W., Miyajima, A., Kobori, J., and Arai, K. (1987) J. Biol. Chem. 262, 10475-10480).

Physician compliance with warfarin prophylaxis for central venous catheters in patients with solid tumors.

PURPOSE: There is an established benefit of prophylactic warfarin in cancer patients with central venous catheters. This study assessed the compliance rate of prophylactic low-dose warfarin prescription in cancer patients with central venous catheters at a single institution. PATIENTS AND METHODS: Oncology patients with central venous catheters were identified by a retrospective chart review. Information retrieved included whether prophylactic warfarin had been prescribed and whether the patient had suffered a thrombotic or bleeding event. After the initial chart review, physicians were notified of the benefits of warfarin prophylaxis, and subsequently, a physician-independent mechanism of prescribing prophylactic warfarin was instituted. After each of these interventions, we retrospectively reviewed a further two cohorts of patients to assess compliance with warfarin prophylaxis. RESULTS: During the baseline study, only 10% of patients were prescribed prophylactic warfarin. After physician notification, the compliance rate increased to only 20% (P =.3). After instituting the physician-independent mechanism of prescribing prophylactic warfarin, the compliance rate increased to 95% (P <.001). The rate of catheter-related thrombosis was 11% for patients who were prescribed warfarin compared with 21% in those who were not anticoagulated (P =.2). CONCLUSION: At our institution, the rate of prescribing prophylactic warfarin was low in this patient population, and there was a reluctance of treating physicians to change their prescribing practice. Mechanisms exist to improve the rate of anticoagulant prophylaxis in this clinical setting. We recommend that institutions review their rate of compliance with prophylactic anticoagulation for patients with central venous catheters and solid tumors.

Factitious methemoglobinemia.

We report the case of a 26 year-old female who was treated on numerous occasions for methemoglobinemia believed secondary to surreptitious abuse of dapsone as part of a factitious disorder.

Escherichia coli DnaA protein. The N-terminal domain and loading of DnaB helicase at the E. coli chromosomal origin.

Initiation of DNA replication at the Escherichia coli chromosomal origin occurs through an ordered series of events that depends first on the binding of DnaA protein, the replication initiator, to DnaA box sequences followed by unwinding of an AT-rich region. A step that follows is the binding of DnaB helicase at oriC so that it is properly positioned at each replication fork. We show that DnaA protein actively mediates the entry of DnaB at oriC. One region (amino acids 111-148) transiently binds to DnaB as determined by surface plasmon resonance. A second functional domain, possibly involving formation of a unique nucleoprotein structure, promotes the stable binding of DnaB during the initiation process and is inactivated in forming an intermediate termed the prepriming complex by removal of the N-terminal 62 residues. Based on similarities in the replication process between prokaryotes and eukaryotes, these results suggest that a similar mechanism may load the eukaryotic replicative helicase.

Molecular characterization of the murine Hif-1 alpha locus.

Hypoxia inducible factor 1 alpha (HIF-1 alpha) is a basic helix-loop-helix-PAS (bHLH-PAS) transcription factor that mediates certain cellular responses to low oxygen tension, iron chelators, Co2+, Ni2+, Mg2+, and low intracellular glucose concentration. Upon exposure to the above conditions, HIF-1 alpha is upregulated and heterodimerizes with the Ah receptor nuclear translocator (ARNT, also known as HIF-1 beta), the heterodimeric complex binds TACGTG-containing genomic enhancer elements, and activates transcription of target genes. As a first step in developing genetic models to study the biology related to cellular hypoxia, we have cloned the murine HIF-1 alpha cDNA, determined the tissue-specific expression of its mRNA, functionally analyzed its protein product, and characterized its promoter and its genomic structure. A comparison between the murine and human HIF-1 alpha protein sequence reveals 95%, 99%, and 83% identity in the bHLH, PAS, and variable domains, respectively. RNAse protection assays demonstrate that in adult mice, the mHIF-1 alpha mRNA is expressed at high levels in kidney, heart, brain, thymus, and placenta, with moderate expression in liver, spleen, testis, and lung and much lower expression in skeletal muscle testis. Northern blot analysis indicates that the mRNA of the murine HIF-1 alpha is transcribed in two forms, a major 4-kb species and a minor 5-kb species; both are present in all tissues examined. The Hif-1 alpha promoter is GC rich, does not have a TATA element near its transcriptional start site, and does not respond to hypoxia or Co2+. The mHIF-1 alpha structural gene is composed of 15 exons. The splice junction sites within the bHLH and the PAS domains of HIF-1 alpha gene are highly conserved with respect to a number of previously characterized members of the bHLH-PAS superfamily. However, unlike other bHLH-PAS genes, where the variable domain is encoded by 2 exons, the variable region of the mHIF-1 alpha gene is encoded by 7 exons. Furthermore, most of these splice junction sites in the variable region are conserved with that of HIF-2 alpha, a recently cloned hypoxia-responsive bHLH-PAS protein (also known as MOP2, EPAS1, and HLF). These data suggest that HIF-1 alpha, along with HIF-2 alpha, represents a new subclass of the bHLH-PAS superfamily.

Identification of palmitoylation sites within the L-type calcium channel beta2a subunit and effects on channel function.

The hydrophilic beta2a subunit of the L-type calcium channel was recently shown to be a membrane-localized, post-translationally modified protein (Chien, A. J., Zhao, X. L., Shirokov, R. E., Puri, T. S., Chang, C. F., Sun, D. D., Rios, E., and Hosey, M. M. (1995) J. Biol. Chem. 270, 30036-30044). In this study, we demonstrate that the rat beta2a subunit was palmitoylated through a hydroxylamine-sensitive thioester linkage. Palmitoylation required a pair of cysteines in the N terminus, Cys3 and Cys4; mutation of these residues to serines resulted in mutant beta2a subunits that were unable to incorporate palmitic acid. Interestingly, a palmitoylation-deficient beta2a mutant still localized to membrane particulate fractions and was still able to target functional channel complexes to the plasma membrane similar to wild-type beta2a. However, channels formed with a palmitoylation-deficient beta2a subunit exhibited a dramatic decrease in ionic current per channel, indicating that although mutations eliminating palmitoylation did not affect channel targeting by the beta2a subunit, they were important determinants of channel modulation by the beta2a subunit. Three other known beta subunits that were analyzed were not palmitoylated, suggesting that palmitoylation could provide a basis for the regulation of L-type channels through modification of a specific beta isoform.

Domains of DnaA protein involved in interaction with DnaB protein, and in unwinding the Escherichia coli chromosomal origin.

DnaA protein of Escherichia coli is a sequence-specific DNA-binding protein required for the initiation of DNA replication from the chromosomal origin, oriC. It is also required for replication of several plasmids including pSC101, F, P-1, and R6K. A collection of monoclonal antibodies to DnaA protein has been produced and the primary epitopes recognized by them have been determined. These antibodies have also been examined for the ability to inhibit activities of DNA binding, ATP binding, unwinding of oriC, and replication of both an oriC plasmid, and an M13 single-stranded DNA with a proposed hairpin structure containing a DnaA protein-binding site. Replication of the latter DNA is dependent on DnaA protein by a mechanism termed ABC priming. These studies suggest regions of DnaA protein involved in interaction with DnaB protein, and in unwinding of oriC, or low-affinity binding of ATP.

The A184V missense mutation of the dnaA5 and dnaA46 alleles confers a defect in ATP binding and thermolability in initiation of Escherichia coli DNA replication.

The temperature-sensitive dnaA5 and dnaA46 alleles each contain two missense mutations. These mutations have been separated and the resulting mutant proteins studied with regard to their role in initiation of DNA replication in vitro. Whereas the His-252 to tyrosine substitution (H252Y) unique to the dnaA46 allele did not affect the activities of DnaA protein, the unique substitution of the dnaA5 allele, Gly-426 to serine (G426S), was reduced in its DNA-binding affinity for oriC, the chromosomal origin. This suggests that the C-terminal region of the DnaA protein is involved in DNA binding. The alanine-to-valine substitution at amino acid 184 (A184V) that is common to both of the alleles is responsible for the thermolabile defect and lag in DNA synthesis of these mutants. Mutant proteins bearing the common substitution were defective in ATP binding and were inactive in a replication system reconstituted with purified proteins. DnaK and GrpE protein activated these mutant proteins for replication and ATP binding; the latter was measured indirectly by the ATP-dependent formation of a trypsin-resistant peptide. However, with this assay, the ATP-binding affinity appeared to be reduced relative to wild-type DnaA protein. Activation was by conversion of a self-aggregate to the monomer, and also by a conformational alteration that correlated with ATP binding.

Digitate intravascular appendages and aneurysm of the vein of Galen.

Peculiar intravascular appendages were found in a case of aneurysm of the vein of Galen. Such appendages consisted of finger-like polypoid growths which protruded within the lumen of ectatic cerebral veins and arteries. They were solid, relatively large, and non-branching. Histologically, their structure was comparable to that of a vessel wall, but was concentrically laminated around the longitudinal axis. Some appendages were associated with thrombi. It is suggested that appendages of this kind probably represent secondary vascular malformations, that they are likely to develop in reaction to abnormal hemodynamic strains upon the vessel walls, and that they might be thrombogenic and, therefore, potentially beneficial for patients with arteriovenous shunts.

Open-complex formation by the host initiator, DnaA, at the origin of P1 plasmid replication.

Replication of P1 plasmid requires both the plasmid-specific initiator, RepA, and the host initiator, DnaA. Here we show that DnaA can make the P1 origin reactive to the single-strand specific reagents KMnO4 and mung bean nuclease. Addition of RepA further increased the KMnO4 reactivity of the origin, although RepA alone did not influence the reaction. The increased reactivity implies that the two initiators interact in some way to alter the origin conformation. The KMnO4 reactivity was restricted to one strand of the origin. We suggest that the roles of DnaA in P1 plasmid and bacterial replication are similar: origin opening and loading of the DnaB helicase. The strand-bias in chemical reactivity at the P1 origin most likely indicates that only one of the strands is used for the loading of DnaB, a scenario consistent with the unidirectional replication of the plasmid.