Blastomycosis in indoor cats: Suburban Chicago, Illinois, USA

Blastomyces dermatitidis, the etiologic agent of blastomycosis, a potentially life-threatening systemic mycosis of humans and animals, is acquired from a yet incompletely defined environmental niche. There is controversy regarding the potential for contact with the fungus in or near one’s home, particularly in urban areas. We investigated an outbreak of blastomycosis among five urban, indoor cats diagnosed at three veterinary clinics March 3–July 13, 2005, in suburban Chicago, Illinois, by owner interviews, site visits, environmental cultures for B. dermatitidis, GIS analysis, and analysis of local weather data. There were no environmental exposures common to the five cats that lived a median of 300 m from nearest body of water, in homes on a loam soil. Closest and farthest case home sites were 3.4 and 26.1 km, respectively. All cats were confined indoors except one cat that averaged 15 min/week in his backyard and was exposed to excavation. B. dermatitidis was not isolated from any of 60 environmental samples. The annualized incidence rate March through July 2005 among 6,761 cats in these practices was 178/100,000, compared to none in the previous 4 years, and 0.14/100,000 cat visits from a nationwide animal hospital registry. Precipitation January through June 2005 was 9.30 versus period mean of 14.05 ± 1.69 inches the previous 4 years (P = 0.01). Circumstantial evidence suggests acquisition of B. dermatitidis from the home site environment in five cats. Relative drought may have contributed to an apparent outbreak of blastomycosis in this urban locale.     

A microaliquoting technique for precise histological annotation and optimization of cell content in frozen tissue specimens

Knowledge of the exact cell content of frozen tissue samples is of growing importance in genomic research. We developed a microaliquoting technique to measure and optimize the cell composition of frozen tumor specimens for molecular studies. Frozen samples of 31 mesothelioma cases were cut in alternating thin and thick sections. Thin sections were stained and evaluated visually. Thick sections, i.e., microaliquots, were annotated using bordering stained sections. A range of cellular heterogeneity was observed among and within samples. Precise annotation of samples was obtained by integration and compared to conventional single face and “front and back” section estimates of cell content. Front and back estimates were more highly correlated with block annotation by microaliquoting than were single face estimates. Both methods yielded discrepant estimates, however, and for some studies may not adequately account for the heterogeneity of mesothelioma or other malignancies with variable cellular composition. High yield and quality RNA was extracted from precision annotated, tumor-enriched subsamples prepared by combining individual microaliquots with the highest tumor cellularity estimates. Microaliquoting provides accurate cell content annotation and permits genomic analysis of enriched subpopulations of cells without fixation or amplification.     

Base-CP proteasome can serve as a platform for stepwise lid formation

26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid sub-complexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base sub-complexes and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP. A key role for Regulatory particle non-ATPase 11 (Rpn11) in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11–m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of five module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Re-introducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11–Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes.     

Nuclear import of an intact preassembled proteasome particle

The 26S proteasome is a conserved 2.5 MDa protein degradation machine that localizes to different cellular compartments, including the nucleus. Little is known about the specific targeting mechanisms of proteasomes in eukaryotic cells. We used a cell-free nuclear reconstitution system to test for nuclear targeting and import of distinct proteasome species. Three types of stable, proteolytically active proteasomes particles were purified from Xenopus egg cytosol. Two of these, the 26S holoenzyme and the 20S core particle, were targeted to the nuclear periphery but did not reach the nucleoplasm. This targeting depends on the presence of mature nuclear pore complexes (NPCs) in the nuclear envelope. A third, novel form, designated here as 20S+, was actively imported through NPCs. The 20S+ proteasome particle resembles recently described structural intermediates from other systems. Nuclear import of this particle requires functional NPCs, but it is not directly regulated by the Ran GTPase cycle. The mere presence of the associated "+" factors is sufficient to reconstitute nuclear targeting and confer onto isolated 20S core particles the ability to be imported. Stable 20S+ particles found in unfertilized eggs may provide a means for quick mobilization of existing proteasome particles into newly formed nuclear compartments during early development.     

Dual function of Rpn5 in two PCI complexes, the 26S proteasome and COP9 signalosome

Subunit composition and architectural structure of the 26S proteasome lid is strictly conserved between all eukaryotes. This eight-subunit complex bears high similarity to the eukaryotic translation initiation factor 3 and to the COP9 signalosome (CSN), which together define the proteasome CSN/COP9/initiation factor (PCI) troika. In some unicellular eukaryotes, the latter two complexes lack key subunits, encouraging questions about the conservation of their structural design. Here we demonstrate that, in Saccharomyces cerevisiae, Rpn5 plays dual roles by stabilizing proteasome and CSN structures independently. Proteasome and CSN complexes are easily dissected, with Rpn5 the only subunit in common. Together with Rpn5, we identified a total of six bona fide subunits at roughly stoichiometric ratios in isolated, affinity-purified CSN. Moreover, the copy of Rpn5 associated with the CSN is required for enzymatic hydrolysis of Rub1/Nedd8 conjugated to cullins. We propose that multitasking by a single subunit, Rpn5 in this case, allows it to function in different complexes simultaneously. These observations demonstrate that functional substitution of subunits by paralogues is feasible, implying that the canonical composition of the three PCI complexes in S. cerevisiae is more robust than hitherto appreciated.     

Apo-E genotypes and cardiovascular diseases: a sensitivity study using cross-validatory criteria

The Apolipoprotein-E (Apo-E) gene, a gene that produces proteins which help to regulate lipid levels in the bloodstream, is of interest in the study of cardiovascular diseases. An approach to making inferences about the genetic effects of the Apo-E gene has been developed by Glickman and Gagnon (2002). The framework describes the role of genetic and risk factors on the onset ages of multiple diseases, and accounts for the possibility that an individual was censored for reasons related to the diseases of interest. The framework also allows for missing genetic information, so that subjects censored prior to genetic sampling, and therefore missing such information, may still be included in the analysis. We apply an extension to this framework to the original cohort of the Framingham Heart Study for measuring the effects of different Apo-E genotypes on the onset age of various cardiovascular disease events. In particular, we compare the fit of univariate versus multivariate onset age components to the model, whether to incorporate health covariates measured at baseline or at a point later in the study, and whether to assume a heritability model for Apo-E genotype frequencies. The results of the best fitting model are presented.     

The ontogeny of the urogenital system of the spotted hyena (Crocuta crocuta Erxleben)

Studies were conducted to elucidate the importance of androgen-mediated induction of the extreme masculinization of the external genitalia in female spotted hyenas. Phallic size and shape; androgen receptor (AR) and alpha-actin expression; and sex-specific differences in phallic retractor musculature, erectile tissue, tunica albuginea, and urethra/urogenital sinus were examined in male and female fetuses from Day 30 of gestation to term. Similar outcomes were assessed in fetuses from dams treated with an AR blocker and a 5alpha-reductase inhibitor (antiandrogen treatment). Clitoral and penile development were already advanced at Day 30 of gestation and grossly indistinguishable between male and female fetuses throughout pregnancy. Sex-specific differences in internal phallic organization were evident at Gestational Day 45, coincident with AR expression and testicular differentiation. Antiandrogen treatment inhibited prostatic development in males and effectively feminized internal penile anatomy. We conclude that gross masculinization of phallic size and shape of male and female fetuses is androgen-independent, but that sexual dimorphism of internal phallic structure is dependent on fetal testicular androgens acting via AR in the relevant cells/tissues. Androgens secreted by the maternal ovaries and metabolized by the placenta do not appear to be involved in gross masculinization or in most of the sex differences in internal phallic structure.     

Sexual differentiation in three unconventional mammals: spotted hyenas, elephants and tammar wallabies

The present review explores sexual differentiation in three non-conventional species: the spotted hyena, the elephant and the tammar wallaby, selected because of the natural challenges they present for contemporary understanding of sexual differentiation. According to the prevailing view of mammalian sexual differentiation, originally proposed by Alfred Jost, secretion of androgen and anti-Mullerian hormone (AMH) by the fetal testes during critical stages of development accounts for the full range of sexually dimorphic urogenital traits observed at birth. Jost's concept was subsequently expanded to encompass sexual differentiation of the brain and behavior. Although the central focus of this review involves urogenital development, we assume that the novel mechanisms described in this article have potentially significant implications for sexual differentiation of brain and behavior, a transposition with precedent in the history of this field. Contrary to the "specific" requirements of Jost's formulation, female spotted hyenas and elephants initially develop male-type external genitalia prior to gonadal differentiation. In addition, the administration of anti-androgens to pregnant female spotted hyenas does not prevent the formation of a scrotum, pseudoscrotum, penis or penile clitoris in the offspring of treated females, although it is not yet clear whether the creation of masculine genitalia involves other steroids or whether there is a genetic mechanism bypassing a hormonal mediator. Wallabies, where sexual differentiation occurs in the pouch after birth, provide the most conclusive evidence for direct genetic control of sexual dimorphism, with the scrotum developing only in males and the pouch and mammary glands only in females, before differentiation of the gonads. The development of the pouch and mammary gland in females and the scrotum in males is controlled by genes on the X chromosome. In keeping with the "expanded" version of Jost's formulation, secretion of androgens by the fetal testes provides the best current account of a broad array of sex differences in reproductive morphology and endocrinology of the spotted hyena, and androgens are essential for development of the prostate and penis of the wallaby. But the essential circulating androgen in the male wallaby is 5alpha androstanediol, locally converted in target tissues to DHT, while in the pregnant female hyena, androstenedione, secreted by the maternal ovary, is converted by the placenta to testosterone (and estradiol) and transferred to the developing fetus. Testicular testosterone certainly seems to be responsible for the behavioral phenomenon of musth in male elephants. Both spotted hyenas and elephants display matrilineal social organization, and, in both species, female genital morphology requires feminine cooperation for successful copulation. We conclude that not all aspects of sexual differentiation have been delegated to testicular hormones in these mammals. In addition, we suggest that research on urogenital development in these non-traditional species directs attention to processes that may well be operating during the sexual differentiation of morphology and behavior in more common laboratory mammals, albeit in less dramatic fashion.     

Biochemical and genetic analysis of the four DNA ligases of mycobacteria

Mycobacterium tuberculosis encodes an NAD(+)-dependent DNA ligase (LigA) plus three distinct ATP-dependent ligase homologs (LigB, LigC, and LigD). Here we purify and characterize the multiple DNA ligase enzymes of mycobacteria and probe genetically whether the ATP-dependent ligases are required for growth of M. tuberculosis. We find significant differences in the reactivity of mycobacterial ligases with a nicked DNA substrate, whereby LigA and LigB display vigorous nick sealing activity in the presence of NAD(+) and ATP, respectively, whereas LigC and LigD, which have ATP-specific adenylyltransferase activity, display weak nick joining activity and generate high levels of the DNA-adenylate intermediate. All four of the mycobacterial ligases are monomeric enzymes. LigA has a low K(m) for NAD(+) (1 microm) and is sensitive to a recently described pyridochromanone inhibitor of NAD(+)-dependent ligases. LigA is able to sustain growth of Saccharomyces cerevisiae in lieu of the essential yeast ligase Cdc9, but LigB, LigC, and LigD are not. LigB is distinguished by its relatively high K(m) for ATP (0.34 mm) and its dependence on a distinctive N-terminal domain for nick joining. None of the three ATP-dependent ligases are essential for mycobacterial growth. M. tuberculosis ligDDelta cells are defective in nonhomologous DNA end joining.     

Inhibition of Dengue Virus Polymerase by Blocking of the RNA Tunnel

Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Neither vaccine nor antiviral therapy is currently available for DENV. We report here that N-sulfonylanthranilic acid derivatives are allosteric inhibitors of DENV RNA-dependent RNA polymerase (RdRp). The inhibitor was identified through high-throughput screening of one million compounds using a primer extension-based RdRp assay [substrate poly(C)/oligo(G)₂₀]. Chemical modification of the initial “hit” improved the compound potency to an IC₅₀ (that is, a concentration that inhibits 50% RdRp activity) of 0.7 μM. In addition to suppressing the primer extension-based RNA elongation, the compound also inhibited de novo RNA synthesis using a DENV subgenomic RNA, but at a lower potency (IC₅₀ of 5 μM). Remarkably, the observed anti-polymerase activity is specific to DENV RdRp; the compound did not inhibit WNV RdRp and exhibited IC₅₀s of >100 μM against hepatitis C virus RdRp and human DNA polymerase α and β. UV cross-linking and mass spectrometric analysis showed that a photoreactive inhibitor could be cross-linked to Met343 within the RdRp domain of DENV NS5. On the crystal structure of DENV RdRp, Met343 is located at the entrance of RNA template tunnel. Biochemical experiments showed that the order of addition of RNA template and inhibitor during the assembly of RdRp reaction affected compound potency. Collectively, the results indicate that the compound inhibits RdRp through blocking the RNA tunnel. This study has provided direct evidence to support the hypothesis that allosteric pockets from flavivirus RdRp could be targeted for antiviral development.The family Flaviviridae consists of three genera: Flavivirus, Pestivirus, and Hepacivirus. The genus Flavivirus contains about 73 viruses, many of which are arthropod-borne and pose major public health threats worldwide (15). The four serotypes of dengue virus infect 50 to 100 million people each year, with approximately 500,000 cases developing into life-threatening dengue hemorrhage fever (DHF) and dengue shock syndrome (DSS), leading to about 20,000 deaths. In addition to DENV, West Nile virus (WNV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), and tick-borne encephalitis virus (TBEV) also cause significant human diseases. No antiviral therapy is currently available for treatment of flavivirus infections. Human vaccines are only available for YFV, JEV, and TBEV (15). Development of antiviral therapy and new vaccines is urgently needed for flaviviruses.The flavivirus genome is a single-stranded RNA of plus-sense polarity. The genomic RNA contains a 5′ untranslated region (UTR), a single open reading frame, and a 3′ UTR. The single open reading frame encodes a long polyprotein that is processed by viral and host proteases into 10 mature viral proteins. Three structural proteins (Capsid [C], premembrane [prM], and envelope [E]) are components of virus particles. Seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) are responsible for viral replication (40), virion assembly (19, 21, 24, 33), and innate immunity antagonism (4, 16, 23, 29, 30). Two viral proteins encode enzymatic activities that have been targeted for antiviral development. NS3 functions as a protease (with NS2B as a cofactor), helicase, 5′-RNA triphosphatase, and nucleoside triphosphatase (7, 14, 42). The N-terminal part of NS5 is a methyltransferase that methylates the N7 and 2′-O positions of the viral RNA cap structure (13, 18, 37); the C-terminal part of NS5 has an RNA-dependent RNA polymerase (RdRp) activity (1, 39). The RdRp activity is unique to RNA viruses and therefore represents an attractive antiviral target.Two types of inhibitors could be developed to suppress viral polymerases. Type 1 inhibitors are nucleoside/nucleotide analogs that function as RNA or DNA chain terminators; about half of the current antiviral drugs are nucleotide analogs (10). For flaviviruses, a nucleoside analog (7-deaza-2′-C-methyl-adenosine), originally developed for hepatitis C virus (HCV) RdRp, showed anti-DENV activity (32, 38). We recently reported a similar adenosine analog (7-deaza-2′-C-acetylene-adenosine) that potently inhibited DENV both in cell culture and in mice; unfortunately, this compound showed side effects during a 2-week in vivo toxicity study (44). Nevertheless, these studies have proved the concept that nucleoside analogs could potentially be developed for flavivirus therapy. Type 2 inhibitors are non-nucleoside inhibitors (NNI) which bind to allosteric pockets of protein to block enzymatic activities; the mechanism of action of NNI includes structural alteration of polymerase to an inactive conformation, blocking the conformational switch from polymerase initiation to elongation, or impeding the processivity of polymerase elongation (11). A broad range of chemical classes have been identified as NNI, including inhibitors of HIV (9, 35) and HCV (3, 5, 11, 25).In the present study, we performed high-throughput screening (HTS) to search for NNI of DENV RdRp. The HTS and chemistry synthesis led to the identification of N-sulfonylanthranilic acid derivatives as inhibitors of DENV RdRp. The compounds specifically inhibit DENV RdRp. UV cross-linking experiments mapped the compound binding site to the RdRp domain of DENV NS5. Amino acid Met343, located at the entrance of RNA template tunnel of the DENV RdRp, was cross-linked to the compound. These results, together with biochemistry experiments, suggest that the compound blocks the RdRp activity through binding to the RNA template tunnel of the polymerase.