Macaques have served as models for more than 70 human infectious diseases of diverse etiologies, including a multitude of agents-bacteria, viruses, fungi, parasites, prions. The remarkable diversity of human infectious diseases that have been modeled in the macaque includes global, childhood, and tropical diseases as well as newly emergent, sexually transmitted, oncogenic, degenerative neurologic, potential bioterrorism, and miscellaneous other diseases. Historically, macaques played a major role in establishing the etiology of yellow fever, polio, and prion diseases. With rare exceptions (Chagas disease, bartonellosis), all of the infectious diseases in this review are of Old World origin. Perhaps most surprising is the large number of tropical (16), newly emergent (7), and bioterrorism diseases (9) that have been modeled in macaques. Many of these human diseases (e.g., AIDS, hepatitis E, bartonellosis) are a consequence of zoonotic infection. However, infectious agents of certain diseases, including measles and tuberculosis, can sometimes go both ways, and thus several human pathogens are threats to nonhuman primates including macaques. Through experimental studies in macaques, researchers have gained insight into pathogenic mechanisms and novel treatment and vaccine approaches for many human infectious diseases, most notably acquired immunodeficiency syndrome (AIDS), which is caused by infection with human immunodeficiency virus (HIV). Other infectious agents for which macaques have been a uniquely valuable resource for biomedical research, and particularly vaccinology, include influenza virus, paramyxoviruses, flaviviruses, arenaviruses, hepatitis E virus, papillomavirus, smallpox virus, Mycobacteria, Bacillus anthracis, Helicobacter pylori, Yersinia pestis, and Plasmodium species. This review summarizes the extensive past and present research on macaque models of human infectious disease.

Through DNA methylation, histone modifications, and small regulatory RNAs the epigenome systematically controls gene expression during development, both in utero and throughout life. The epigenome is also a very reactive system; its labile nature allows it to sense and respond to environmental perturbations to ensure survival during fetal growth. This pliability can lead to aberrant epigenetic modifications that persist into later life and induce numerous disease states. Endocrine-disrupting compounds (EDCs) are ubiquitous chemicals that interfere with growth and development. Several EDCs also interfere with epigenetic programming. The investigation of the epigenotoxic effects of bisphenol A (BPA), an EDC used in the production of plastics and resins, has further raised concern over the impact of EDCs on the epigenome. Using the Agouti viable yellow (A(vy)) mouse model, dietary BPA exposure was shown to hypomethylate both the A(vy) and the Cabp(IAP) metastable epialleles. This hypomethylating effect was counteracted with dietary supplementation of methyl donors or genistein. These results are consistent with reports of BPA and other EDCs causing epigenetic effects. Epigenotoxicity could lead to numerous developmental, metabolic, and behavioral disorders in exposed populations. The heritable nature of epigenetic changes also increases the risk for transgenerational inheritance of phenotypes. Thus, epigenotoxicity must be considered when assessing these compounds for safety.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is a newly recognized entity. As the incidence of this disease was exceptionally high in Japan, the Japanese Ministry of Public Health and Welfare instituted a special commission for the investigation of this perplexing disease; since 1975 this committee has performed an intensive study of 2100 patients with OPLL in Japan. An epidemiologic study was conducted by this group in Japan and in eastern Asiatic countries. Symptoms and disabilities caused by the disease were described. Roentgenographic findings were classified as continuous, segmental, mixed, or localized. OPLL at the thoracic and lumbar levels combined with ossification of the yellow ligament was described, and the risk of spinal cord damage as well as the importance of tomography and computerized tomographic scanning were stressed. No conclusions were reached concerning etiology, but common findings included a generalized hyperostotic tendency, a tendency for abnormal glucose metabolism, and low enteral calcium absorption. A relatively high hereditary occurrence was noted. Conservative and surgical treatment methods were described, with particular reference to spinal canal-widening operations.

The Yellow Stripe-Like (YSL) family of proteins has been identified based on sequence similarity to maize Yellow Stripe1 (YS1), the transporter responsible for the primary uptake of iron from the soil. YS1 transports iron that is complexed by specific plant-derived Fe(III) chelators called phytosiderophores (PS). Non-grass species of plants neither make nor use PS, yet YSL family members are found in non-grass species (monocot, dicot, gymnosperm, and moss species) including Arabidopsis thaliana. YSLs in non-grasses have been hypothesized to transport metals complexed by nicotianamine (NA), an iron chelator that is structurally similar to PS and which is found in all higher plants. Here we show that Arabidopsis YSL2 (At5g24380) transports iron and copper when these metals are chelated by NA. YSL2 is expressed in many cell types in both roots and shoots, suggesting that diverse cell types obtain metals as metal-NA complexes. YSL2 transcription is regulated by the levels of iron and copper in the growth medium. Based on its expression pattern, a major function of the YSL2 appears to be in the lateral movement of metals in the vasculature.

In mammalian retina, the rod bipolar cells synapse on the AII amacrine cells, which are therefore the third-order neurons in the rod-signal pathway. The AII amacrine cells are connected by gap junctions, both to each other and to fourth-order, On-center cone bipolar cells. They also receive synaptic input from the dopaminergic amacrine cells, and in this study, we investigated whether dopamine modulates the permeability of the gap junctions between AII amacrine cells in the isolated rabbit retina. The small biotinylated tracer Neurobiotin was injected into nuclear yellow-labeled AII cells under direct microscopic control. The extent of tracer coupling to neighboring AII cells, 40-60 min after Neurobiotin injection (0.5 nA for 60 sec), provided a standard measure of the permeability of the homologous gap junctions. Under control conditions, individual AII amacrine cells were coupled to 73 +/- 15 neighboring cells, and this was unaffected by changes in pH from 6.6 to 7.8. Exogenous dopamine significantly reduced the tracer coupling at concentrations as low as 10 nM (26 +/- 16 cells), with the effect increasing with dopamine concentration up to 10 microM (6 +/- 4 cells). The uncoupling effect of dopamine was both blocked by the selective D1 antagonist SCH-23390 (10 microM) and mimicked by the specific D1 agonist SKF-38393 (500 microM). Moreover, the AII amacrine cells were also uncoupled when the retina was incubated in forskolin (60 microM) and isobutylmethylxanthine (200 microM). Taken together, these results indicated that the uncoupling was mediated by a D1-like receptor that stimulates cAMP production. Although the selective D1 antagonist on its own did not increase tracer coupling, suggesting that there was little release of endogenous dopamine in the superfused photo-bleached retina, veratridine-evoked release of endogenous transmitters did uncouple the AII amacrine cells, and this effect was blocked by the specific D1 antagonist.

West Nile (WN) virus is an important cause of febrile exanthem and encephalitis. Since it invaded the U.S. in 1999, >19,000 human cases have been reported. The threat of continued epidemics has spurred efforts to develop vaccines. ChimeriVax-WN02 is a live, attenuated recombinant vaccine constructed from an infectious clone of yellow fever (YF) 17D virus in which the premembrane and envelope genes of 17D have been replaced by the corresponding genes of WN virus. Preclinical tests in monkeys defined sites of vaccine virus replication in vivo. ChimeriVax-WN02 and YF 17D had similar biodistribution but different multiplication kinetics. Prominent sites of replication were skin and lymphoid tissues, generally sparing vital organs. Viruses were cleared from blood by day 7 and from tissues around day 14. In a clinical study, healthy adults were inoculated with 5.0 log(10) plaque-forming units (PFU) (n = 30) or 3.0 log10 PFU (n = 15) of ChimeriVax-WN02, commercial YF vaccine (YF-VAX, n = 5), or placebo (n = 30). The incidence of adverse events in subjects receiving the vaccine was similar to that in the placebo group. Transient viremia was detected in 42 of 45 (93%) of ChimeriVax-WN02 subjects, and four of five (80%) of YF-VAX subjects. All subjects developed neutralizing antibodies to WN or YF, respectively, and the majority developed specific T cell responses. ChimeriVax-WN02 rapidly elicits strong immune responses after a single dose, and is a promising candidate warranting further evaluation for prevention of WN disease.

Epigenetics is the study of the heritable changes in gene expression that occur without a change in the DNA sequence itself. These heritable epigenetic changes include chromatin folding and attachment to the nuclear matrix, packaging of DNA around nucleosomes, histone modifications, and DNA methylation. The epigenome is particularly susceptible to dysregulation during gestation, neonatal development, puberty, and old age. Nevertheless, it is most vulnerable to environmental factors during embryogenesis because the DNA synthetic rate is high, and the elaborate DNA methylation patterning and chromatin structure required for normal tissue development is established during early development. Metastable epialleles are alleles that are variably expressed in genetically identical individuals due to epigenetic modifications established during early development and are thought to be particularly vulnerable to environmental influences. The viable yellow agouti (A(vy)) allele, whose expression is correlated to DNA methylation, is a murine metastable epiallele, which has been used as an epigenetic biosensor for environmental factors affecting the fetal epigenome. In this review, we introduce epigenetic gene regulation, describe important epigenetic phenomenon in mammals, summarize literature linking the early environment to developmental plasticity of the fetal epigenome, and promote the necessity to identify epigenetically labile genes in the mouse and human genomes.

The visualization of live cell behaviors operating in situ combined with the power of mouse genetics represents a major step toward understanding the mechanisms regulating embryonic development, homeostasis, and disease progression in mammals. The availability of genetically encoded fluorescent protein reporters, combined with improved optical imaging modalities, have led to advances in our ability to examine cells in vivo. We developed a series of lipid-modified fluorescent protein fusions that are targeted to and label the secretory pathway and the plasma membrane, and that are amenable for use in mice. Here we report the generation of two strains of mice, each expressing a spectrally distinct lipid-modified GFP-variant fluorescent protein fusion. The CAG::GFP-GPI strain exhibited widespread expression of a glycosylphosphatidylinositol-tagged green fluorescent protein (GFP) fusion, while the CAG::myr-Venus strain exhibited widespread expression of a myristoyl-Venus yellow fluorescent protein fusion. Imaging of live transgenic embryonic stem (ES) cells, either live or fixed embryos and postnatal tissues demonstrated that glycosylphosphatidyl inositol- and myristoyl-tagged GFP-variant fusion proteins are targeted to and serve as markers of the plasma membrane. Moreover, our data suggest that these two lipid-modified protein fusions are dynamically targeted both to overlapping as well as distinct lipid-enriched compartments within cells. These transgenic strains not only represent high-contrast reporters of cell morphology and plasma membrane dynamics, but also may be used as in vivo sensors of lipid localization. Furthermore, combining these reporters with the study of mouse mutants will be a step forward in understanding the inter- and intracellular behaviors underlying morphogenesis in both normal and mutant contexts.

Molecular analysis of a Drosophila minichromosome, Dp(1;f)1187, revealed a relationship between position-effect variegation and the copy number reductions of heterochromatic sequences that occur in polytene cells. Heterochromatin adjacent to a defined junction with euchromatin underpolytenized at least 60-fold. Lesser reductions were observed in euchromatic sequences up to 103 kb from the breakpoint. The copy number changes behaved in all respects like the expression of yellow, a gene located within the affected region. Both copy number and yellow expression displayed a cell-by-cell mosaic pattern of reduction, and adding a Y chromosome, a known suppressor of variegation, increased both substantially. We discuss the possibility that changes in replication alter copy number locally and also propose an alternative model of position-effect variegation based on the somatic elimination of heterochromatic sequences.

OBJECTIVE

Recent advances in understanding the neuroendocrine pathways regulating appetite, metabolism and body weight afford an opportunity to explore further the mechanisms by which obesity influences arterial pressure. ob/ob(Lep(ob)/Lep(ob)) mice have a mutation in the ob gene and are leptin-deficient. Leptin possesses pressor actions and has been shown to increase arterial pressure when infused chronically or over-expressed transgenically. In contrast, agouti yellow obese(Ay) mice have overexpression of an agouti peptide that blocks melanocortin receptors. Stimulation of melanocortin receptors by alpha-melanocyte-stimulating hormone decreases arterial pressure.

METHODS

This study measured arterial pressure in leptin-deficient ob/ob mice, agouti yellow obese mice and their lean controls to test the hypothesis that the effects of obesity on arterial pressure are importantly influenced by the genetic and neuroendocrine mechanisms causing the obesity. We measured arterial pressure directly in conscious ob/ob mice (n = 14), agouti yellow obese mice (n = 6) and the same number of lean littermates.

RESULTS

Body weight was nearly twice as high in ob/ob mice as in their lean controls, but mean arterial pressure was significantly lower in ob/ob mice (92+/-3 mmHg) compared with their lean controls (106+/-2 mmHg; P = 0.00017). In contrast, mean arterial pressure was significantly higher in agouti yellow obese mice (124+/-3 mmHg) than in their lean controls (99+/-1 mmHg; P = 0.000002) despite the fact that the agouti mice had milder obesity.

CONCLUSIONS

This study prompts three conclusions: (1) leptin-deficient ob/ob mice and agouti yellow obese mice have contrasting blood pressure responses to obesity, (2) obesity does not invariably increase arterial pressure in mice, and (3) the arterial pressure response to obesity may depend critically on the underlying genetic and neuroendocrine mechanisms.

Recent biochemical studies indicate that the serotonin transporter can form oligomers. We investigated whether the human serotonin transporter (hSERT) can be visualized as an oligomer in the plasma membrane of intact cells. For this purpose, we generated fusion proteins of hSERT and spectral variants of the green fluorescent protein (cyan and yellow fluorescent proteins, CFP and YFP, respectively). When expressed in human embryonic kidney 293 cells, the resulting fusion proteins (CFP-hSERT and YFP-hSERT) were efficiently inserted into the plasma membrane and were functionally indistinguishable from wild-type hSERT. Oligomers were visualized by fluorescence resonance energy transfer microscopy in living cells using two complementary methods, i.e. ratio imaging and donor photobleaching. Interestingly, oligomerization was not confined to hSERT; fluorescence resonance energy transfer was also observed between CFP- and YFP-labeled rat gamma-aminobutyric acid transporter. The bulk of serotonin transporters was recovered as high molecular weight complexes upon gel filtration in detergent solution. In contrast, the monomers of CFP-hSERT and YFP-hSERT were essentially undetectable. This indicates that the homo-oligomeric form is the favored state of hSERT in living cells, which is not significantly affected by coincubation with transporter substrates or blockers. Based on our observations, we conclude that constitutive oligomer formation might be a general property of Na(+)/Cl(-)-dependent neurotransmitter transporters.

We have recovered terminal chromosome deletions of the X chromosome of Drosophila [Df(1)RT; RT = receding tips] that break in various positions of the yellow gene (y) region and delete all distal DNA sequences. Terminal DNA fragments are heterogeneous in length. Molecular cloning and sequencing of the terminal DNA fragments revealed that the broken ends of the deleted chromosomes do not carry any telomeric DNA sequences, yet the broken chromatids do not fuse to one another. Moreover, we confirmed by sequence analysis of 49 independently cloned terminal DNA fragments from two RT lines collected at different times that they lose DNA sequences from their distal ends at a rate of 70-75 base pairs per fly generation. We calculate that the rate of loss from these ends is consistent with the removal of an octanucleotide RNA primer at each round of DNA replication in the germ line.

Bipolar, amacrine, and ganglion cells of carp retina, stained intracellularly with Procion yellow, can be divided into types a and b, according to the destination of terminals and dendritic trees in the inner plexiform layer (sublamina a and b, respectively). Type a cells showed hyperpolarizing, or off, responses and type b cells depolarizing, or on, responses. Carp thus resembles cat in the basic organization of on and off pathways in the retina.

We have purified biosynthetically labeled alpha-factor secreted from transformed yeast alpha cells. This alpha-factor binds specifically to a cells and is internalized by a time-, temperature-, and energy-dependent process. alpha-factor is internalized in an intact form and then rapidly degraded. Two yeast mutants defective in the accumulation of an endocytotic marker, lucifer yellow CH, in the vacuole have been isolated. end1 accumulates invaginations of the plasma membrane, and end2, an internal membrane-bound organelle. One of these mutants, end1, is defective for internalization of alpha-factor. Both of these mutants are defective in pheromone response.

The mouse connexin 36 (Cx36) gene was mapped on chromosome 2 and an identical transcriptional start site was determined in brain and retina on exon I. Rabbit polyclonal antibodies to the presumptive cytoplasmic loop of the Cx36 protein recognized in immunohistochemical analyses Cx36 expression in the retina, olfactory bulb, hippocampus, inferior olive and cerebellum. In olivary neurons strong punctate labeling at dendritic cell contacts and weaker labeling in the cytoplasm of dendrites were shown by immuno electron microscopy. After expression of mouse Cx36 cDNA in human HeLa cells, neurobiotin transfer was increased 1.8-fold and electrical conductance at least 15-fold compared to untransfected HeLa cells. No Lucifer Yellow transfer was detected in either untransfected or Cx36 transfected HeLa cells. Single Cx36 channels in transfected HeLa cells showed a unitary conductance of 14.3 + or - 0. 8 pS. The sensitivity of Cx36 channels to transjunctional voltage was low in both HeLa-Cx36 cells and Xenopus oocytes expressing mouse Cx36. No increased transfer of neurobiotin was detected in heterotypic gap junctions formed by Cx36 and 9 other connexins expressed in HeLa cells. Our results suggest that Cx36 channels function as electrical synapses for transmission of electrical and metabolic signals between neurons in the central nervous system.

Two seemingly opposite evolutionary patterns of clinal variation in body size and associated life history traits exist in nature. According to Bergmann's rule, body size increases with latitude, a temperature effect. According to the converse Bergmann rule, body size decreases with latitude, a season length effect. A third pattern causally related to the latter is countergradient variation, whereby populations of a given species compensate seasonal limitations at higher latitudes by evolving faster growth and larger body sizes compared to their low latitude conspecifics. We discuss these patterns and argue that they are not mutually exclusive because they are driven by different environmental causes and proximate mechanisms; they therefore can act in conjunction, resulting in any intermediate pattern. Alternatively, Bergmann and converse Bergmann clines can be interpreted as over- and undercompensating countergradient variation, respectively. We illustrate this with data for the wide-spread yellow dung fly, Scathophaga stercoraria (Diptera: Scathophagidae), which in Europe shows a Bergmann cline for size and a converse Bergmann cline (i.e., countergradient variation) for development time. A literature review of the available evidence on arthropod latitudinal clines further shows a patterned continuum of responses. Converse Bergmann clines due to end-of-season time limitations are more common in larger species with longer development times. Our study thus provides a synthesis to the controversy about the importance of Bergmann's rule and the converse Bergmann rule in nature.

Maize (Zea mays) plants make different types of vegetative or reproductive branches during development. Branches develop from axillary meristems produced on the flanks of the vegetative or inflorescence shoot apical meristem. Among these branches are the spikelets, short grass-specific structures, produced by determinate axillary spikelet-pair and spikelet meristems. We investigated the mechanism of branching in maize by making transgenic plants expressing a native expressed endogenous auxin efflux transporter (ZmPIN1a) fused to yellow fluorescent protein and a synthetic auxin-responsive promoter (DR5rev) driving red fluorescent protein. By imaging these plants, we found that all maize branching events during vegetative and reproductive development appear to be regulated by the creation of auxin response maxima through the activity of polar auxin transporters. We also found that the auxin transporter ZmPIN1a is functional, as it can rescue the polar auxin transport defects of the Arabidopsis (Arabidopsis thaliana) pin1-3 mutant. Based on this and on the groundbreaking analysis in Arabidopsis and other species, we conclude that branching mechanisms are conserved and can, in addition, explain the formation of axillary meristems (spikelet-pair and spikelet meristems) that are unique to grasses. We also found that BARREN STALK1 is required for the creation of auxin response maxima at the flanks of the inflorescence meristem, suggesting a role in the initiation of polar auxin transport for axillary meristem formation. Based on our results, we propose a general model for branching during maize inflorescence development.

The bursa aurealis transposon has been used to create transposon insertion libraries of Bacillus anthracis and Staphylococcus aureus. To provide a set of genetic tools to enhance the utility of these libraries, we generated an allelic-exchange system that allows for the replacement of the transposon with useful genetic markers and fluorescent reporter genes. These tools were tested in the Nebraska Transposon Mutant Library (NTML), containing defined transposon insertions in 1,952 nonessential S. aureus genes. First, we generated a plasmid that allows researchers to replace the genes encoding green fluorescent protein (GFP) and erythromycin resistance in the transposon with a noncoding DNA fragment, leaving a markerless mutation within the chromosome. Second, we produced allelic-exchange plasmids to replace the transposon with alternate antibiotic resistance cassettes encoding tetracycline, kanamycin, and spectinomycin resistance, allowing for the simultaneous selection of multiple chromosomal mutations. Third, we generated a series of fluorescent reporter constructs that, after allelic exchange, generate transcriptional reporters encoding codon-optimized enhanced cyan fluorescent protein (ECFP), enhanced yellow fluorescent protein (EYFP), DsRed.T3(DNT), and eqFP650, as well as superfolder green fluorescent protein (sGFP). Overall, combining the NTML with this allelic-exchange system provides an unparalleled resource for the study of S. aureus.

Recording of electrical responses from isolated small intestine of mice using conventional microelectrodes revealed two types of potential, a pacemaker potential and a slow wave, both with rapid rising primary components and following plateau components. The rate of rise and peak amplitude were greater for pacemaker potentials than for slow waves, and the plateau component was smaller in slow waves than in pacemaker potentials. Both potentials oscillated at a similar frequency (20-30 min-1). Unitary potentials often discharged during the interval between pacemaker potentials. Infusion of Lucifer Yellow allowed visualization of the recorded cells; pacemaker potentials were recorded from myenteric interstitial cells of Cajal (ICC-MY) while slow waves were recorded from circular smooth muscle cells. Pacemaker potentials were characterized as follows: the primary component was inhibited by Ni2+, Ca2+-free solution or depolarization with high-K+ solution, the plateau component was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), an inhibitor of Ca2+-activated Cl- channels, low [Cl-]o solution or Ca2+-free solution, and the generation of potentials was abolished by co-application of Ni2+and DIDS or by chelating intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM). These results indicate that in the mouse small intestine ICC-MY generate pacemaker potentials with two components in situ; the primary and plateau components may be generated by activation of voltage-dependent Ca2+-permeable channels and Ca2+-activated Cl- channels, respectively. Slow waves are generated in circular smooth muscles via electrotonic spread of pacemaker potentials. These properties of intestinal pacemaker potentials are considered essentially similar to those of gastric pacemaker potentials.

Pigment patterns of fishes are a tractable system for studying the genetic and cellular bases for postembryonic phenotypes. In the zebrafish Danio rerio, neural crest-derived pigment cells generate different pigment patterns during different phases of the life cycle. Whereas early larvae exhibit simple stripes of melanocytes and silver iridophores in a background of yellow xanthophores, this pigment pattern is transformed at metamorphosis into that of the adult, comprising a series of dark melanocyte and iridophore stripes, alternating with light stripes of iridophores and xanthophores. Although several genes have been identified in D. rerio that contribute to the development of both early larval and adult pigment patterns, comparatively little is known about genes that are essential for pattern formation during just one or the other life cycle phase. In this study, we identify the gene responsible for the rose mutant phenotype in D. rerio. rose mutants have wild-type early larval pigment patterns, but fail to develop normal numbers of melanocytes and iridophores during pigment pattern metamorphosis and exhibit a disrupted pattern of these cells. We show that rose corresponds to endothelin receptor b1 (ednrb1), an orthologue of amniote Ednrb genes that have long been studied for their roles in neural crest and pigment cell development. Furthermore, we demonstrate that D. rerio ednrb1 is expressed both during pigment pattern metamorphosis and during embryogenesis, and cells of melanocyte, iridophore, and xanthophore lineages all express this gene. These analyses suggest a phylogenetic conservation of roles for Ednrb signaling in the development of amniote and teleost pigment cell precursors. As murine Ednrb is essential for the development of all neural crest derived melanocytes, and D. rerio ednrb1 is required only by a subset of adult melanocytes and iridophores, these analyses also reveal variation among vertebrates in the cellular requirements for Ednrb signaling, and suggest alternative models for the cellular and genetic bases of pigment pattern metamorphosis in D. rerio.

The authors have reviewed 158 eyes with evolving or completed idiopathic macular holes. Observations of these patients suggest that prefoveal vitreous cortex contraction is probably the cause of idiopathic macular holes. The earliest sign of an impending macular hole (stage 1) appears to be the development of a yellow spot or halo associated with loss of the normal anatomic foveal depression. No vitreous separation is present. This may resolve or progress to a small, early macular hole (stage 2). This hole gradually enlarged to a diameter of approximately 485 micron. The vitreous usually remained attached or a vitreofoveal separation developed (stage 3). Some eyes had complete posterior vitreous separation (stage 4). The implications for surgical intervention are discussed. A prospective study should be undertaken to confirm these findings and to investigate the feasibility of vitrectomy intervention to peel the prefoveal vitreous cortex in eyes with a stage 1 lesion.

Aprataxin and polynucleotide kinase (PNK) are DNA end processing factors that are recruited into the DNA single- and double-strand break repair machinery through phosphorylation-specific interactions with XRCC1 and XRCC4, respectively. These interactions are mediated through a divergent class of forkhead-associated (FHA) domain that binds to peptide sequences in XRCC1 and XRCC4 that are phosphorylated by casein kinase 2 (CK2). Here, we identify the product of the uncharacterized open reading frame C2orf13 as a novel member of this FHA domain family of proteins and we denote this protein APLF (aprataxin- and PNK-like factor). We show that APLF interacts with XRCC1 in vivo and in vitro in a manner that is stimulated by CK2. Yeast two-hybrid analyses suggest that APLF also interacts with the double-strand break repair proteins XRCC4 and XRCC5 (Ku86). We also show that endogenous and yellow fluorescent protein-tagged APLF accumulates at sites of H(2)O(2) or UVA laser-induced chromosomal DNA damage and that this is achieved through at least two mechanisms: one that requires the FHA domain-mediated interaction with XRCC1 and a second that is independent of XRCC1 but requires a novel type of zinc finger motif located at the C terminus of APLF. Finally, we demonstrate that APLF is phosphorylated in a DNA damage- and ATM-dependent manner and that the depletion of APLF from noncycling human SH-SY5Y neuroblastoma cells reduces rates of chromosomal DNA strand break repair following ionizing radiation. These data identify APLF as a novel component of the cellular response to DNA strand breaks in human cells.

A newly discovered infectious disease of amphibians, chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis, is implicated in population declines and possible extinctions throughout the world. The purpose of our study was to examine the effects of B. dendrobatidis on the mountain yellow-legged frog (Rana muscosa) in the Sierra Nevada of California (USA). We (1) quantified the prevalence and incidence of B. dendrobatidis through repeat surveys of several hundred R. muscosa populations in the southern Sierra Nevada; (2) described the population-level effects of B. dendrobatidis on R. muscosa population abundance; and (3) compared the mortality rates of infected and uninfected R. muscosa individuals from pre- through post-metamorphosis using both laboratory and field experiments. Mouthpart inspections conducted in 144 and 132 R. muscosa populations in 2003 and 2004, respectively, indicated that 19% of R. muscosa populations in both years showed indications of chytridiomycosis. Sixteen percent of populations that were uninfected in 2003 became infected by 2004. Rana muscosa population sizes were reduced by an average of 88% following B. dendrobatidis outbreaks at six sites, but at seven B. dendrobatidis-negative sites, R. muscosa population sizes increased by an average of 45% over the same time period. In the laboratory, all infected R. muscosa developed fatal chytridiomycosis after metamorphosis, while all uninfected individuals remained healthy. In the field experiment in which R. muscosa tadpoles were caged at infected and uninfected sites, 96% of the individuals that metamorphosed at infected sites died vs. 5% at the uninfected sites. These studies indicate that chytridiomycosis causes high mortality in post-metamorphic R. muscosa, that this emerging disease is the proximate cause of numerous observed R. muscosa population declines, and that the disease threatens this species with extirpation at numerous sites in California's Sierra Nevada.

We present fine mapping of a cis-acting nucleotide sequence found in the 5' region of yellow fever virus genomic RNA that is required for RNA replication. There is evidence that this sequence interacts with a complementary sequence in the 3' region of the genome to cyclize the RNA. Replicons were constructed that had various deletions in the 5' region encoding the capsid protein and were tested for their ability to replicate. We found that a sequence of 18 nucleotides (residues 146 to 163 of the yellow fever virus genome, which encode amino acids 9 to 14 of the capsid protein) is essential for replication of the yellow fever virus replicon and that a slightly longer sequence of 21 nucleotides (residues 146 to 166, encoding amino acids 9 to 15) is required for full replication. This region is larger than the core sequence of 8 nucleotides conserved among all mosquito-borne flaviviruses and contains instead the entire sequence previously proposed to be involved in cyclization of yellow fever virus RNA.