Segregation of viral plasmids depends on tethering to chromosomes and is regulated by phosphorylation

Eukaryotic viruses can maintain latency in dividing cells as extrachromosomal nuclear plasmids. Segregation and nuclear retention of DNA is, therefore, a key issue in retaining copy number. The E2 enhancer protein of the papillomaviruses is required for viral DNA replication and transcription. Viral mutants that prevent phosphorylation of the bovine papillomavirus type 1 (BPV) E2 protein are transformation-defective, despite normal viral gene expression and replication function. Cell colonies harboring such mutants show sectoring of viral DNA and are unable to maintain the episome. We find that transforming viral DNA attaches to mitotic chromosomes, in contrast to the mutant genome encoding the E2 phosphorylation mutant. Second-site suppressor mutations were uncovered in both E1 and E2 genes that allow for transformation, maintenance, and chromosomal attachment. E2 protein was also found to colocalize to mitotic chromosomes, whereas the mutant did not, suggesting a direct role for E2 in viral attachment to chromosomes. Such viral hitch-hiking onto cellular chromosomes is likely to provide a general mechanism for maintaining nuclear plasmids.

Identification of novel susceptibility loci for inflammatory bowel disease on chromosomes 1p, 3q, and 4q: Evidence for epistasis between 1p and IBD1

The idiopathic inflammatory bowel diseases, Crohn’s disease (CD) and ulcerative colitis (UC), are chronic, frequently disabling diseases of the intestines. Segregation analyses, twin concordance, and ethnic differences in familial risks have established that CD and UC are complex, non-Mendelian, related genetic disorders. We performed a genome-wide screen using 377 autosomal markers, on 297 CD, UC, or mixed relative pairs from 174 families, 37% Ashkenazim. We observed evidence for linkage at 3q for all families (multipoint logarithm of the odds score (MLod) = 2.29, P = 5.7 × 10−4), with greatest significance for non-Ashkenazim Caucasians (MLod = 3.39, P = 3.92 × 10−5), and at chromosome 1p (MLod = 2.65, P = 2.4 × 10−4) for all families. In a limited subset of mixed families (containing one member with CD and another with UC), evidence for linkage was observed on chromosome 4q (MLod = 2.76, P = 1.9 × 10−4), especially among Ashkenazim. There was confirmatory evidence for a CD locus, overlapping IBD1, in the pericentromeric region of chromosome 16 (MLod = 1.69, P = 2.6 × 10−3), particularly among Ashkenazim (MLod = 1.51, P = 7.8 × 10−3); however, positive MLod scores were observed over a very broad region of chromosome 16. Furthermore, evidence for epistasis between IBD1 and chromosome 1p was observed. Thirteen additional loci demonstrated nominal (MLod > 1.0, P < 0.016) evidence for linkage. This screen provides strong evidence that there are several major susceptibility loci contributing to the genetic risk for CD and UC.

An opsonic function of the neutrophil bactericidal/permeability-increasing protein depends on both its N- and C-terminal domains

The host response to Gram-negative bacterial infection is influenced by two homologous lipopolysaccharide (LPS)-interactive proteins, LPS-binding protein (LBP) and the bacteridical/permeability-increasing protein (BPI). Both proteins bind LPS via their N-terminal domains but produce profoundly different effects: BPI and a bioactive N-terminal fragment BPI-21 exert a selective and potent antibacterial effect upon Gram-negative bacteria and suppress LPS bioactivity whereas LBP is not toxic toward Gram-negative bacteria and potentiates LPS bioactivity. The latter effect of LBP requires the C-terminal domain for delivery of LPS to CD14, so we postulated that the C-terminal region of BPI may serve a similar delivery function but to distinct targets. LBP, holoBPI, BPI-21, and LBP/BPI chimeras were compared for their ability to promote uptake by human phagocytes of an encapsulated, phagocytosis-resistant strain of Escherichia coli. We show that only bacteria preincubated with holoBPI are ingested by neutrophils and monocytes. These findings suggest that, when extracellular holoBPI is bound via its N-terminal domain to Gram-negative bacteria, the C-terminal domain promotes bacterial attachment to neutrophils and monocytes, leading to phagocytosis. Therefore, analogous to the role of the C-terminal domain of LBP in delivery of LPS to CD14, the C-terminal domain of BPI may fulfill a similar function in BPI-specific disposal pathways for Gram-negative bacteria.

Effects of extracellular Ca2+ concentration on hair-bundle stiffness and gating-spring integrity in hair cells

When a hair cell is stimulated by positive deflection of its hair bundle, increased tension in gating springs opens transduction channels, permitting cations to enter stereocilia and depolarize the cell. Ca2+ is thought to be required in mechanoelectrical transduction, for exposure of hair bundles to Ca2+ chelators eliminates responsiveness by disrupting tip links, filamentous interstereociliary connections that probably are the gating springs. Ca2+ also participates in adaptation to stimuli by controlling the activity of a molecular motor that sets gating-spring tension. Using a flexible glass fiber to measure hair-bundle stiffness, we investigated the effect of Ca2+ concentration on stiffness before and after the disruption of gating springs. The stiffness of intact hair bundles depended nonmonotonically on the extracellular Ca2+ concentration; the maximal stiffness of ≈1200 μN⋅m−1 occurred when bundles were bathed in solutions containing 250 μM Ca2+, approximately the concentration found in frog endolymph. For cells exposed to solutions with sufficient chelator capacity to reduce the Ca2+ concentration below ≈100 nM, hair-bundle stiffness fell to ≈200 μN⋅m−1 and no longer exhibited Ca2+-dependent changes. Because cells so treated lost mechanoelectrical transduction, we attribute the reduction in bundle stiffness to tip-link disruption. The results indicate that gating springs are not linearly elastic; instead, they stiffen with increased strain, which rises with adaptation-motor activity at the physiological extracellular Ca2+ concentration.

Effect of reduced myristoylated alanine-rich C kinase substrate expression on hippocampal mossy fiber development and spatial learning in mutant mice: Transgenic rescue and interactions with gene background

The myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent protein kinase C (PKC) substrate in brain that is expressed highly in hippocampal granule cells and their axons, the mossy fibers. Here, we examined hippocampal infrapyramidal mossy fiber (IP-MF) limb length and spatial learning in heterozygous Macs mutant mice that exhibit an ≈50% reduction in MARCKS expression relative to wild-type controls. On a 129B6(N3) background, the Macs mutation produced IP-MF hyperplasia, a significant increase in hippocampal PKCɛ expression, and proficient spatial learning relative to wild-type controls. However, wild-type 129B6(N3) mice exhibited phenotypic characteristics resembling inbred 129Sv mice, including IP-MF hypoplasia relative to inbred C57BL/6J mice and impaired spatial-reversal learning, suggesting a significant contribution of 129Sv background genes to wild-type and possibly mutant phenotypes. Indeed, when these mice were backcrossed with inbred C57BL/6J mice for nine generations to reduce 129Sv background genes, the Macs mutation did not effect IP-MF length or hippocampal PKCɛ expression and impaired spatial learning relative to wild-type controls, which now showed proficient spatial learning. Moreover, in a different strain (B6SJL(N1), the Macs mutation also produced a significant impairment in spatial learning that was reversed by transgenic expression of MARCKS. Collectively, these data indicate that the heterozygous Macs mutation modifies the expression of linked 129Sv gene(s), affecting hippocampal mossy fiber development and spatial learning performance, and that MARCKS plays a significant role in spatial learning processes.

Oligomerization of activated d- and l-guanosine mononucleotides on templates containing d- and l-deoxycytidylate residues

The oligomerization of activated d- and l- and racemic guanosine-5′-phosphoro-2-methylimidazole on short templates containing d- and l-deoxycytidylate has been studied. Results obtained with d-oligo(dC)s as templates are similar to those previously reported for experiments with a poly(C) template. When one l-dC or two consecutive l-dCs are introduced into a d-template, regiospecific synthesis of 3′-5′ oligo(G)s proceeds to the end of the template, but three consecutive l-dCs block synthesis. Alternating d-,l-oligomers do not facilitate oligomerization of the d-, l-, and racemic 2-guanosine-5′-phosphoro-2-methylimidazole. We suggest that once a “predominately d-metabolism” existed, occasional l-residues in a template would not have led to the termination of self-replication.

On the molecular basis and regulation of cellular capacitative calcium entry: Roles for Trp proteins

During the last 2 years, our laboratory has worked on the elucidation of the molecular basis of capacitative calcium entry (CCE) into cells. Specifically, we tested the hypothesis that CCE channels are formed of subunits encoded in genes related to the Drosophila trp gene. The first step in this pursuit was to search for mammalian trp genes. We found not one but six mammalian genes and cloned several of their cDNAs, some in their full length. As assayed in mammalian cells, overexpression of some mammalian Trps increases CCE, while expression of partial trp cDNAs in antisense orientation can interfere with endogenous CCE. These findings provided a firm connection between CCE and mammalian Trps. This article reviews the known forms of CCE and highlights unanswered questions in our understanding of intracellular Ca2+ homeostasis and the physiological roles of CCE.

Solvent effects on the energy landscapes and folding kinetics of polyalanine

The effect of a solvation on the thermodynamics and kinetics of polyalanine (Ala12) is explored on the basis of its energy landscapes in vacuum and in an aqueous solution. Both energy landscapes are characterized by two basins, one associated with α-helical structures and the other with coil and β-structures of the peptide. In both environments, the basin that corresponds to the α-helical structure is considerably narrower than the basin corresponding to the β-state, reflecting their different contributions to the entropy of the peptide. In vacuum, the α-helical state of Ala12 constitutes the native state, in agreement with common helical propensity scales, whereas in the aqueous medium, the α-helical state is destabilized, and the β-state becomes the native state. Thus solvation has a dramatic effect on the energy landscape of this peptide, resulting in an inverted stability of the two states. Different folding and unfolding time scales for Ala12 in hydrophilic and hydrophobic chemical environments are caused by the higher entropy of the native state in water relative to vacuum. The concept of a helical propensity has to be extended to incorporate environmental solvent effects.

Effects of estrogen on growth plate senescence and epiphyseal fusion

Estrogen is critical for epiphyseal fusion in both young men and women. In this study, we explored the cellular mechanisms by which estrogen causes this phenomenon. Juvenile ovariectomized female rabbits received either 70 μg/kg estradiol cypionate or vehicle i.m. once a week. Growth plates from the proximal tibia, distal tibia, and distal femur were analyzed after 2, 4, 6, or 8 weeks of treatment. In vehicle-treated animals, there was a gradual senescent decline in tibial growth rate, rate of chondrocyte proliferation, growth plate height, number of proliferative chondrocytes, number of hypertrophic chondrocytes, size of terminal hypertrophic chondrocytes, and column density. Estrogen treatment accelerated the senescent decline in all of these parameters. In senescent growth plates, epiphyseal fusion was observed to be an abrupt event in which all remaining chondrocytes were rapidly replaced by bone elements. Fusion occurred when the rate of chondrocyte proliferation approached zero. Estrogen caused this proliferative exhaustion and fusion to occur earlier. Our data suggest that (i) epiphyseal fusion is triggered when the proliferative potential of growth plate chondrocytes is exhausted; and (ii) estrogen does not induce growth plate ossification directly; instead, estrogen accelerates the programmed senescence of the growth plate, thus causing earlier proliferative exhaustion and consequently earlier fusion.

Effect of COMT Val108/158 Met genotype on frontal lobe function and risk for schizophrenia

Abnormalities of prefrontal cortical function are prominent features of schizophrenia and have been associated with genetic risk, suggesting that susceptibility genes for schizophrenia may impact on the molecular mechanisms of prefrontal function. A potential susceptibility mechanism involves regulation of prefrontal dopamine, which modulates the response of prefrontal neurons during working memory. We examined the relationship of a common functional polymorphism (Val108/158 Met) in the catechol-O-methyltransferase (COMT) gene, which accounts for a 4-fold variation in enzyme activity and dopamine catabolism, with both prefrontally mediated cognition and prefrontal cortical physiology. In 175 patients with schizophrenia, 219 unaffected siblings, and 55 controls, COMT genotype was related in allele dosage fashion to performance on the Wisconsin Card Sorting Test of executive cognition and explained 4% of variance (P = 0.001) in frequency of perseverative errors. Consistent with other evidence that dopamine enhances prefrontal neuronal function, the load of the low-activity Met allele predicted enhanced cognitive performance. We then examined the effect of COMT genotype on prefrontal physiology during a working memory task in three separate subgroups (n = 11–16) assayed with functional MRI. Met allele load consistently predicted a more efficient physiological response in prefrontal cortex. Finally, in a family-based association analysis of 104 trios, we found a significant increase in transmission of the Val allele to the schizophrenic offspring. These data suggest that the COMT Val allele, because it increases prefrontal dopamine catabolism, impairs prefrontal cognition and physiology, and by this mechanism slightly increases risk for schizophrenia.

New perspectives on forbidden symmetries, quasicrystals, and Penrose tilings

Quasicrystals are solids with quasiperiodic atomic structures and symmetries forbidden to ordinary periodic crystals—e.g., 5-fold symmetry axes. A powerful model for understanding their structure and properties has been the two-dimensional Penrose tiling. Recently discovered properties of Penrose tilings suggest a simple picture of the structure of quasicrystals and shed new light on why they form. The results show that quasicrystals can be constructed from a single repeating cluster of atoms and that the rigid matching rules of Penrose tilings can be replaced by more physically plausible cluster energetics. The new concepts make the conditions for forming quasicrystals appear to be closely related to the conditions for forming periodic crystals.

The effect of cultivation on the size, shape, and persistence of disease patches in fields

Epidemics of soil-borne plant disease are characterized by patchiness because of restricted dispersal of inoculum. The density of inoculum within disease patches depends on a sequence comprising local amplification during the parasitic phase followed by dispersal of inoculum by cultivation during the intercrop period. The mechanisms that control size, shape, and persistence have received very little rigorous attention in epidemiological theory. Here we derive a model for dispersal of inoculum in soil by cultivation that takes account into the discrete stochastic nature of the system in time and space. Two parameters, probability of movement and mean dispersal distance, characterize lateral dispersal of inoculum by cultivation. The dispersal parameters are used in combination with the characteristic area and dimensions of host plants to identify criteria that control the shape and size of disease patches. We derive a critical value for the probability of movement for the formation of cross-shaped patches and show that this is independent of the amount of inoculum. We examine the interaction between local amplification of inoculum by parasitic activity and subsequent dilution by dispersal and identify criteria whereby asymptomatic patches may persist as inoculum falls below a threshold necessary for symptoms to appear in the subsequent crop. The model is motivated by the spread of rhizomania, an economically important soil-borne disease of sugar beet. However, the results have broad applicability to a very wide range of diseases that survive as discrete units of inoculum. The application of the model to patch dynamics of weed seeds and local introductions of genetically modified seeds is also discussed.

On the origin of and phylogenetic relationships among living amphibians

The phylogenetic relationships among the three orders of modern amphibians (Caudata, Gymnophiona, and Anura) have been estimated based on both morphological and molecular evidence. Most morphological and paleontological studies of living and fossil amphibians support the hypothesis that salamanders and frogs are sister lineages (the Batrachia hypothesis) and that caecilians are more distantly related. Previous interpretations of molecular data based on nuclear and mitochondrial rRNA sequences suggested that salamanders and caecilians are sister groups to the exclusion of frogs. In an attempt to resolve this apparent conflict, the complete mitochondrial genomes of a salamander (Mertensiella luschani) and a caecilian (Typhlonectes natans) were determined (16,656 and 17,005 bp, respectively) and compared with previously published sequences from a frog (Xenopus laevis) and several other groups of vertebrates. Phylogenetic analyses of the mitochondrial data supported with high bootstrap values the monophyly of living amphibians with respect to other living groups of tetrapods, and a sister group relationship of salamanders and frogs. The lack of phylogenetically informative sites in the previous rRNA data sets (because of its shorter size and higher among-site rate variation) likely explains the discrepancy between our results and those based on previous molecular data. Strong support of the Batrachia hypothesis from both molecule- and morphology-based studies provides a robust phylogenetic framework that will be helpful to comparative studies among the three living orders of amphibians and will permit better understanding of the considerably divergent vertebral, brain, and digit developmental patterns found in frogs and salamanders.

Perspectives on archaeal diversity, thermophily and monophyly from environmental rRNA sequences.

Phylogenetic analysis of ribosomal RNA sequences obtained from uncultivated organisms of a hot spring in Yellowstone National Park reveals several novel groups of Archaea, many of which diverged from the crenarchaeal line of descent prior to previously characterized members of that kingdom. Universal phylogenetic trees constructed with the addition of these sequences indicate monophyly of Archaea, with modest bootstrap support. The data also show a specific relationship between low-temperature marine Archaea and some hot spring Archaea. Two of the environmental sequences are enigmatic: depending upon the data set and analytical method used, these sequences branch deeply within the Crenarchaeota, below the bifurcation between Crenarchaeota and Euryarchaeota, or even as the sister group to Eukaryotes. If additional data confirm either of the latter two placements, then the organisms represented by these ribosomal RNA sequences would merit recognition as a new kingdom, provisionally named "Korarchaeota."