Reduced hepatic global hydroxymethylation in mice treated with non-genotoxic carcinogens is transiently reversible with a methyl supplemented diet.

Non-genotoxic carcinogens (NGCs) are known to cause perturbations in DNA methylation, which can be an early event leading to changes in gene expression and the onset of carcinogenicity. Phenobarbital (PB) has been shown to alter liver DNA methylation and hydroxymethylation patterns in mice in a time dependent manner. The goals of this study were to assess if clofibrate (CFB), a well-studied rodent NGC, would produce epigenetic changes in mice similar to PB, and if a methyl donor supplementation (MDS) would modulate epigenetic and gene expression changes induced by phenobarbital. CByB6F1 mice were treated with 0.5% clofibrate or 0.14% phenobarbital for 7 and 28 days. A subgroup of PB treated and control mice were also fed MDS diet. Liquid Chromatography-Ionization Mass Spectrometry (LC-MS) was used to quantify global liver 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) levels. Gene expression analysis was conducted using Affymetrix microarrays. A decrease in liver 5hmC but not 5mC levels was observed upon treatment with both CFB and PB with varying time of onset. We observed moderate increases in 5hmC levels in PB-treated mice when exposed to MDS diet and lower expression levels of several phenobarbital induced genes involved in cell proliferation, growth, and invasion, suggesting an early modulating effect of methyl donor supplementation. Overall, epigenetic profiling can aid in identifying early mechanism-based biomarkers of non-genotoxic carcinogenicity and increases the quality of cancer risk assessment for candidate drugs. Global DNA methylation assessment by LC-MS is an informative first step toward understanding the risk of carcinogenicity.

Response to Comment on "Sensitivity of seafloor bathymetry to climate-driven fluctuations in mid-ocean ridge magma supply".

Tolstoy reports the existence of a characteristic 100 thousand year (ky) period in the bathymetry of fast-spreading seafloor but does not argue that sea level change is a first-order control on seafloor morphology worldwide. Upon evaluating the overlap between tectonic and Milankovitch periodicities across spreading rates, we reemphasize that fast-spreading ridges are the best potential recorders of a sea level signature in seafloor bathymetry.

Response to Comment on "Sensitivity of seafloor bathymetry to climate-driven fluctuations in mid-ocean ridge magma supply".

Huybers et al present new bathymetric spectra from an intermediate-spreading ridge as evidence for a primary contribution of sea level cycles to the morphology of the seafloor. Although we acknowledge the possibility that sea level-modulated magmatic constructions may be superimposed on a first-order tectonic fabric, we emphasize the difficulty of deciphering these different contributions in the frequency domain alone.

Sensitivity of seafloor bathymetry to climate-driven fluctuations in mid-ocean ridge magma supply.

Recent studies have proposed that the bathymetric fabric of the seafloor formed at mid-ocean ridges records rapid (23,000 to 100,000 years) fluctuations in ridge magma supply caused by sealevel changes that modulate melt production in the underlying mantle. Using quantitative models of faulting and magma emplacement, we demonstrate that, in fact, seafloor-shaping processes act as a low-pass filter on variations in magma supply, strongly damping fluctuations shorter than about 100,000 years. We show that the systematic decrease in dominant seafloor wavelengths with increasing spreading rate is best explained by a model of fault growth and abandonment under a steady magma input. This provides a robust framework for deciphering the footprint of mantle melting in the fabric of abyssal hills, the most common topographic feature on Earth.

Vascular anomalies in a case of situs inversus.

Situs inversus is a developmental condition in which the thoracic and abdominal organs fail to negotiate their normal migration patterns and the result is a mirror-image arrangement of these viscera. The literature provides evidence that individuals with this condition have a higher incidence of other congenital malformations (e.g. heart anomalies). Here we describe the dissection of a 71 year-old female cadaver with situs inversus, in which we discovered multiple anomalous vessels associated with the coeliac trunk directed toward the liver. In addition, we identified the inferior vena cava on the left side and a persistent supracardinal vein on the right, constituting a double inferior vena cava. Finally, we identified multiple abnormal venous channels associated with the sub-renal inferior vena cava. These vascular patterns are indeed a rare finding and have surgical implications but may indicate a higher incidence of vascular anomalies in cases of situs inversus.

Phylogenetic evidence of a rapid radiation of pleurocarpous mosses (Bryophyta).

Pleurocarpous mosses, characterized by lateral female gametangia and highly branched, interwoven stems, comprise three orders and some 5000 species, or almost half of all moss diversity. Recent phylogenetic analyses resolve the Ptychomniales as sister to the Hypnales plus Hookeriales. Species richness is highly asymmetric with approximately 100 Ptychomniales, 750 Hookeriales, and 4400 Hypnales. Chloroplast DNA (cpDNA) sequences were obtained to compare partitioning of molecular diversity among the orders with estimates of species richness, and to test the hypothesis that either the Hookeriales or Hypnales underwent a period (or periods) of exceptionally rapid diversification. Levels of biodiversity were quantified using explicitly historical "phylogenetic diversity" and non-historical estimates of standing sequence diversity. Diversification rates were visualized using lineage-through-time (LTT) plots, and statistical tests of alternative diversification models were performed using the methods of Paradis (1997). The effects of incomplete sampling on the shape of LTT plots and performance of statistical tests were investigated using simulated phylogenies with incomplete sampling. Despite a much larger number of accepted species, the Hypnales contain lower levels of (cpDNA) biodiversity than their sister group, the Hookeriales, based on all molecular measures. Simulations confirm previous results that incomplete sampling yields diversification patterns that appear to reflect a decreasing rate through time, even when the true phylogenies were simulated with constant rates. Comparisons between simulated results and empirical data indicate that a constant rate of diversification cannot be rejected for the Hookeriales. The Hypnales, however, appear to have undergone a period of exceptionally rapid diversification for the earliest 20% of their history.

The feline model of neuroAIDS: understanding the progression towards AIDS dementia.

Feline immunodeficiency virus (FIV) is a neurotropic lentivirus that produces a protracted state of immunodeficiency and encephalopathy in the cat. Recent evidence has shown several similarities to the natural progression of human immunodeficiency virus infection (HIV-1) associated degenerative effects on the central and peripheral nervous systems. Similar to HIV-1, FIV-induced encephalopathy neurovirulence is strain dependent, results in progressive immunodeficiency and increasing early peripheral but not brain viral load, preferentially affects the developing nervous system, produces quantifiable behavioural and neurophysiological impairment that is not directly linked to neuronal infectivity, and induces neuronal injury and loss both in vivo and in vitro. This paper highlights the cumulative scientific body of evidence supporting the use of the feline model of neuroAIDS.

Testing morphological concepts of orders of pleurocarpous mosses (Bryophyta) using phylogenetic reconstructions based on TRNL-TRNF and RPS4 sequences.

The ordinal classification of pleurocarpous mosses rests on characters such as branching mode and architecture of the peristome teeth that line the mouth of the capsule. The Leucodontales comprise mainly epiphytic taxa, characterized by sympodial branching and reduced peristomes, whereas the Hypnales are primarily terricolous and monopodially branching. The third order, the Hookeriales, is defined by a unique architecture of the endostome. We sampled 78 exemplar taxa representing most families of these orders and sequenced two chloroplast loci, the trnL-trnF region and the rps4 gene, to test the monophyly and relationships of these orders of pleurocarpous mosses. Estimates of levels of saturation suggest that the trnL-trnF spacer and the third codon position of the rps4 gene have reached saturation, in at least the transitions. Analyses of the combined data set were performed under three optimality criteria with different sets of assumptions, such as excluding hypervariable positions, downweighting the most likely transformations, and indirect weighting of rps4 codon positions by including amino acid translations. Multiple parallelism in nonsynonymous mutations led to little or no improvement in various indices upon inclusion of amino acid sequences. Trees obtained under likelihood were significantly better under likelihood than the trees derived from the same matrix under parsimony. Our phylogenetic analyses suggest that (1) the pleurocarpous mosses, with the exception of the Cyrtopodaceae, form a monophyletic group which is here given formal recognition as the Hypnidae; (2) the Leucodontales are at least paraphyletic; and (3) the Hypnales form, with most members of the Leucodontalean grade, a monophyletic group sister to a Hookerialean lineage. The Hypopterygiaceae, Hookeriales, and a clade composed of Neorutenbergia, Pseudocryphaea, and Trachyloma likely represent a basal clade or grade within the Hypnidae. These results suggest that mode of branching and reduced peristomes are homoplastic at the ordinal level in pleurocarpous mosses.

Neurophysiologic and immunologic abnormalities associated with feline immunodeficiency virus molecular clone FIV-PPR DNA inoculation.

Although direct feline immunodeficiency virus (FIV) proviral DNA inoculation has been shown to be infectious in cats, long-term studies to assess the pathogenic nature of DNA inoculation are lacking. We have recently reported that direct feline leukemia virus (FeLV) DNA inoculation resulted in infection and the development of FeLV-related disease end points with similar temporal expression and virulence to that of cats infected with whole virus. We show in this study that pFIV-PPR DNA inoculation resulted in infection of cats and the development of FIV-related immunologic and neurologic abnormalities. Infected cats demonstrated progressive loss of CD4+ lymphocytes resulting in decreased CD4:CD8 ratios. Neurologic dysfunction was demonstrated by increased bilateral frontal lobe slow-wave activity. Prolongation of the visual evoked potential peak latency onset response pattern also supported a similar progression of abnormal cortical response. Furthermore, histopathologic examination revealed lesions attributed to FIV infection in lymph node, thymus, brain, and lung. Finally, nested polymerase chain reaction detected FIV provirus in brain, bone marrow, mesenteric lymph node, thymus, spleen, tonsil, and liver. These results confirm that FIV DNA inoculation is an efficient model for study of the pathogenic nature of molecular clones in vivo and offers the opportunity to measure temporal genomic stability of a homogeneous challenge material.

Pulmonary veins-left atrial junction: anatomic and histological study.

Pulmonary veins (PVs) are the target of ablation procedures to cure paroxysmal atrial fibrillation (PAF). There are few anatomic and histological studies of PVs. Sixteen human hearts were obtained from autopsies performed at our hospital and cadavers from a local medical school. The anatomic relationship between the PVs and the left atrium (LA) was categorized according to the spatial orientation of the veins within horizontal and vertical planes viewed from the dorsal aspect of the LA. The PVs were sectioned longitudinally, and the sections were stained with hematoxylin and eosin. In addition, selected sections were stained with antismooth muscle antibodies (vessel wall), antipankeratin, and antimyoglobin antibodies (myocardium). The PV-LA junction has variable orientations. Confluent superior and inferior veins, observed in 25% of the hearts, were more common on the left side. A myocardial sleeve extended from the LA onto the adventitial surface of the PV. The sleeve was distinct from the smooth muscle in the PV media, from which it was separated by loose fibrous strands. There was no microscopical boundary between the PV and the LA endocardium. The PV join the LA at variable angles. Each PV is surrounded by a myocardial sleeve extending from the LA.

Frontal lobe neuronal injury correlates to altered function in FIV-infected cats.

Six cats infected intravenously at 8 weeks of age with feline immunodeficiency virus Maryland isolate (FIV-MD), were evaluated at 8 and 14 months of age (6 months and 12 months postinfection, respectively) with high spatial resolution proton magnetic resonance spectroscopy (MRS) of the frontal cortex. Two separate control cat groups were evaluated at 8 months and 16 months of age. Single voxel two-dimensional high-resolution proton magnetic resonance imaging was performed using the PRESS sequence by selecting a 0.125 ml volume of interest in the medial frontal cortex. A significant reduction in both N-acetylaspartate (NAA) and NAA: choline ratio was found in the FIV 14-month-old group compared with FIV 8-month-old cats, and to the respective age-matched control 16-month-old cats. A negative correlation between NAA and CD4 lymphocyte count was seen in the FIV-14 group only. This group of FIV cats also exhibited a higher proportion of quantitative electroencephalographic relative slow wave activity (RSWA) that correlated to lower NAA content in the frontal cortical voxel. Although peripheral blood proviral load increased over time of infection, no correlation was found between proviral blood or lymph node load and NAA values, CD4 lymphocyte counts, or frontal cortical RSWA. Thus, this study demonstrated that neurologic functional disruption of the frontal cortex correlated strongly with neuronal injury and/or loss in FIV-MD-infected cats independent of peripheral proviral load. The ability to define in vivo neurodegeneration further in this animal model helps in understanding the neuropathogenesis of lentivirus infection, and possibly, a means to follow progression and reversibility during the initial stages of brain infection as therapeutic agents are identified.

Midvastus approach in total knee arthroplasty: a description and a cadaveric study determining the distance of the popliteal artery from the patellar margin of the incision.

Total knee arthroplasty (TKA), one of the most efficacious procedures in orthopedics, requires complete exposure of the knee joint for precise instrumentation. Although most orthopedic surgeons agree that TKA is easily performed using a medial parapatellar approach, a large segment of the patellar blood flow is disrupted by this exposure. The southern or subvastus approach addresses these concerns; however, the procedure has the disadvantage of inadequate exposure in certain patients. A compromise between these two approaches, a midvastus approach, has been described. To decrease potential neurovascular injury, this cadaveric study of the midvastus approach determines the proximity of the incision to the popliteal vascular bundle and addresses the innervation pattern of the vastus medialis oblique. A midvastus arthrotomy was performed on 19 female and 15 male adult cadaveric knees. The midpoint of the superior pole of the patella and the superomedial patellar prominence were marked. After determining the midpoint between the 2 previously mentioned landmarks, an incision was made from that point paralleling the fibers of the vastus medialis oblique medially to the popliteal vascular bundle. The length of the incision was measured three times using calipers; measurements were averaged for each individual specimen, then by gender, and, finally, overall. Ninety-five percent confidence intervals were determined. Differences were assessed by an independent t-test with an alpha level of significance at .05. In addition, the terminal branches of the femoral nerve innervating the vastus medialis oblique were dissected in 5 cadavers. The femoral nerve branched extensively to innervate the vastus medialis oblique. The average distance between the patella and the popliteal vessels was 8.8 +/- 1.4 cm. The average distance in males, 9.5 +/- 1.4 cm, was significantly greater than the distance in females, 8.3 +/- 1.2 cm (P < .02). The distance appeared proportionate to the size of the extremity. The midvastus approach is a viable alternative for primary TKA in selected patients who are not obese and who have not had previous arthrotomy or osteotomy. The average distance (8.8 cm) and corresponding range (6.5 cm minimum to 12.3 cm maximum) are sufficient to suggest a maximal safe distance for sharp dissection of 4.5 cm from the patellar margin in an adult. For additional exposure, the muscle can be safely split further with blunt dissection.

Neuronal loss in FIV-MD infected cats.

Previously, this laboratory has shown that the Maryland strain of feline immunodeficiency virus (FIV-MD) causes neurological disease in cats similar to human immunodeficiency virus type 1 (HIV-1) in people. Using morphometrical methods on neocortical histologic sections we now show a significant loss of neurons in FIV-MD infected cats compared to age-matched uninfected controls. The neuronal populations affected resembles those lost in HIV-1 infection of the brain in published reports, providing further evidence for the utility of FIV-MD infection as a model for HIV-1 infections of the brain.

Thiophosphorylated RCM-lysozyme, an active site-directed protein tyrosine phosphatase inhibitor, inhibits G2/M transition during mitotic cell cycle and uncouples MPF activation from G2/M transition.

Microinjection of thiophosphotyrosylated RCM-lysozyme (TRCML), a potent and specific inhibitor of protein tyrosine phosphatases (PTPs) into sea urchin (Lytechinus pictus) eggs prior to fertilization inhibited the first zygotic cell division in a concentration-dependent fashion. Microinjection of TRCML at varying times after fertilization indicated that at least one site of action is late in the first cell cycle near the G2/M boundary. In order to further study the mechanism for the TRCML effect, a cell-free cell cycling system prepared from electrically activated Xenopus eggs was used. The addition of TRCML to cycling extracts delayed the entrance and progression of extracts through mitosis, as indicated by the inhibition of chromatin condensation, nuclear envelope breakdown, M-phase-promoting factor (MPF) inactivation, and cyclin degradation. Surprisingly, TRCML did not inhibit MPF activation. These results suggest that (1) the target(s) of TRCML lies in late G2- or early M-phase before the onset of metaphase, (2) TRCML uncouples MPF activation from progression through M-phase, and (3) there is a potential involvement of a novel PTP(s) in the control of the cell cycle which may act either downstream of the MPF activation or alternatively in an additional but essential mitotic pathway that is parallel to the MPF activation pathway.

Synergistic calcium release in the sea urchin egg by ryanodine and cyclic ADP ribose.

A transient rise in cytoplasmic Ca2+ activity in the sea urchin egg occurs during fertilization due to release from an intracellular store. Two intracellular receptor Ca2+ channels for inositol 1,4,5-trisphosphate (IP3) and ryanodine have been identified by physiological and immunological techniques. While IP3 is the endogenous messenger for the IP3 receptor, a corresponding physiological messenger for the ryanodine receptor is unknown. A variety of recent experimental evidences suggest that cyclic ADP ribose (cADPR) may be a possible candidate. In this study using both egg homogenates and intact eggs, we show that subthreshold concentrations of cADPR and ryanodine can act synergistically to potentiate Ca2+ release. Addition of 10-20 nM cADPR, which causes little net increase in Ca2+, generally enhances the action of subthreshold concentrations of ryanodine. Similarly the addition of 60-80 microM ryanodine causes a slight transient increase but potentiates maximal Ca2+ increase by a subsequent subthreshold addition of cADPR. While the target of Ca2+ release by ryanodine and cADPR may be the ryanodine receptor, their actions appear to be different and more complex than simply opening the release mechanism. There are significant differences in the kinetics of release by the two agonists. In addition we used a poorly metabolized analog of IP3 and an inhibitor of endoplasmic reticulum Ca2+ ATPase activity, to show that the unfertilized egg contains a rapidly filled Ca2+ store, which is commonly released by both IP3-mediated and ryanodine-mediated release mechanisms.

Sources of calcium in sea urchin eggs during the fertilization response.

Shortly after sperm-egg interaction the sea urchin egg is traversed by a Ca2+ wave, which is necessary for metabolic activation of the quiescent cell. Several sources including influx across the egg plasma membrane and release from intracellular stores may contribute to the rise in cytoplasmic Ca2+ activity. Inositol 1,4,5-trisphosphate (InsP3), cyclic ADP ribose (cADPR), and ryanodine have been reported to induce intracellular Ca2+ release. We used confocal laser scanning microscopy to image the Ca2+ transient during fertilization and parthenogenetic activation by microinjection of Ca2+ release agonists. A near instantaneous rise in Ca2+ localized to the egg cortex occurred near the time of sperm-egg binding, followed by a distinctive delay before the onset of the Ca2+ wave. Since the rise in cortical Ca2+ activity was absent when Ca2+ influx was prevented, it appeared that this change in Ca2+ activity was due to the opening of membrane Ca2+ channels. Blocking the influx did not alter the onset of the Ca2+ wave. The Ca2+ wave during the fertilization response seemed to require Ca2+ release mediated by InsP3-, cADPR-, and ryanodine-sensitive mechanisms. Parthenogenetic activation by microinjection of these three agents had different spatiotemporal patterns of Ca2+ release. Most significantly the injection of either InsP3 or cADPR, but not ryanodine, induced an enhanced pronucleus-associated Ca2+ release, which was similar to the Ca2+ response during fertilization.

Synergistic release of calcium in sea urchin eggs by caffeine and ryanodine.

A transient rise in intracellular Ca2+ during fertilization is necessary for activation of the quiescent sea urchin egg. Several mechanisms contribute to the rise in Ca2+ including influx across the egg plasma membrane and release from intracellular stores. The egg contains both IP3-sensitive and -insensitive Ca2+ release mechanisms and in this study we have used single-cell spectrofluorimetry to examine the effects of caffeine and ryanodine on Ca2+ release in eggs preloaded with fura 2. Caffeine induced a small Ca2+ release that was insensitive to heparin or ruthenium red. Ca2+ liberation by caffeine could be augmented by prior treatment with thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ ATPase. Variable Ca2+ releases were observed in response to microinjection of ryanodine. The action of ryanodine appeared to be enhanced by prior injection of heparin and partially inhibited by ruthenium red. The release of Ca2+ by caffeine or ryanodine was generally insufficient to trigger cortical granule exocytosis, thus these eggs could be fertilized and a second Ca2+ release during fertilization was measured. Unlike the caffeine- and ryanodine-sensitive Ca(2+)-induced Ca2+ release mechanism in somatic cells, the graded responses in eggs suggested this caffeine- and ryanodine-sensitive release mechanism is not sensitive to sudden changes in Ca2+. Thus we could examine the combined actions of caffeine and ryanodine on Ca2+ release, which were synergistic. Caffeine treatment of ryanodine-injected eggs or ryanodine injection of caffeine-treated eggs stimulated a Ca2+ release significantly larger than the release by either drug independently. The experiments presented here suggest that sea urchin eggs liberate Ca2+ in response to caffeine and ryanodine; however, the regulation of this release differs from that described for caffeine- and ryanodine-sensitive Ca(2+)-induced Ca2+ release of somatic cells.

A synthetic peptide of the pseudosubstrate domain of protein kinase C blocks cytoplasmic alkalinization during activation of the sea urchin egg.

Multiple second messenger pathways have been proposed for transduction of the sperm-egg fusion event during fertilization of sea urchin eggs. Cytoplasmic alkalinization due to increased Na(+)-H+ antiport has been causally linked to many of the metabolic events during fertilization. Two possible second messenger pathways coupling sperm-egg fusion and antiporter activity are activation of protein kinase C (PKC) and Ca2(+)-calmodulin kinase. A selective inhibitor of PKC is PKC(19-36), a synthetic peptide of the pseudosubstrate domain of the kinase. Injection of PKC(19-36) into unfertilized sea urchin eggs blocked cytoplasmic alkalinization during activation by phorbol 12-myristate 13-acetate, a PKC agonist. The rise in pH during fertilization was partially blocked by PKC(19-36), which suggested that multiple pathways regulate the antiporter during fertilization. The use of fluorescein chromophores to measure intracellular pH in sea urchin eggs is also discussed.