Discovery of the youngest sex chromosomes reveals first case of convergent co-option of ancestral autosomes in turtles.

Most turtle species possess temperature-dependent sex determination (TSD), but genotypic sex determination (GSD) has evolved multiple times independently from the TSD ancestral condition. GSD in animals typically involves sex chromosomes, yet the sex chromosome system of only 9 out of 18 known GSD turtles has been characterized. Here, we combine comparative genome hybridization (CGH) and BAC clone fluorescent in situ hybridization (BAC FISH) to identify a macro-chromosome XX/XY system in the GSD wood turtle Glyptemys insculpta (GIN), the youngest known sex chromosomes in chelonians (8-20 My old). Comparative analyses show that GIN-X/Y is homologous to chromosome 4 of Chrysemys picta (CPI) painted turtles, chromosome 5 of Gallus gallus chicken, and thus to the X/Y sex chromosomes of Siebenrockiella crassicollis black marsh turtles. We tentatively assign the gene content of the mapped BACs from CPI chromosome 4 (CPI-4) to GIN-X/Y. Chromosomal rearrangements were detected in G. insculpta sex chromosome pair that co-localize with the male-specific region of GIN-Y and encompass a gene involved in sexual development (Wt1-a putative master gene in TSD turtles). Such inversions may have mediated the divergence of G. insculpta sex chromosome pair and facilitated GSD evolution in this turtle. Our results illuminate the structure, origin, and evolution of sex chromosomes in G. insculpta and reveal the first case of convergent co-option of an autosomal pair as sex chromosomes within chelonians.

Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease.

One hallmark of Alzheimer disease is the accumulation of amyloid beta-peptide in the brain and its deposition as plaques. Mice transgenic for an amyloid beta precursor protein (APP) mini-gene driven by a platelet-derived (PD) growth factor promoter (PDAPP mice), which overexpress one of the disease-linked mutant forms of the human amyloid precursor protein, show many of the pathological features of Alzheimer disease, including extensive deposition of extracellular amyloid plaques, astrocytosis and neuritic dystrophy. Active immunization of PDAPP mice with human amyloid beta-peptide reduces plaque burden and its associated pathologies. Several hypotheses have been proposed regarding the mechanism of this response. Here we report that peripheral administration of antibodies against amyloid beta-peptide, was sufficient to reduce amyloid burden. Despite their relatively modest serum levels, the passively administered antibodies were able to enter the central nervous system, decorate plaques and induce clearance of preexisting amyloid. When examined in an ex vivo assay with sections of PDAPP or Alzheimer disease brain tissue, antibodies against amyloid beta-peptide triggered microglial cells to clear plaques through Fc receptor-mediated phagocytosis and subsequent peptide degradation. These results indicate that antibodies can cross the blood-brain barrier to act directly in the central nervous system and should be considered as a therapeutic approach for the treatment of Alzheimer disease and other neurological disorders.

Examination of a miniaturized funnel trap for Aedes aegypti (Diptera: Culicidae) larval sampling.

Funnel traps are often used to sample for the presence of Aedes aegypti (L.) (Diptera: Culicidae) larvae in subterranean aquatic habitats. These traps are generally > or = 15 cm in diameter, making them impractical for use in subterranean sites that have narrow (10-cm) access ports, such as those in standard-sized septic tanks. Recent research indicates septic tanks may be important habitats for Ae. aegypti in Puerto Rico and the Caribbean. To sample mosquito larval populations in these sites, a miniaturized funnel trap was necessary. This project describes the use of a smaller funnel trap for sampling larval populations. The effects of larval instar (third and fourth) and population density on trap efficacy also are examined. The trap detected larval presence 83% of the time at a larval density of 0.011 larvae per cm(2) and 100% of the time at densities > or = 0.022 larvae per cm(2). There was a significant trend of increasing percentage of recaptured larvae with higher larval population densities. Although the miniaturized funnel trap is less sensitive at detecting larval presence in low population densities, it may be useful for sampling aquatic environments with restricted access or shallow water, particularly in domestic septic tanks.

Herpes simplex virus infection of human fibroblasts and keratinocytes inhibits recognition by cloned CD8+ cytotoxic T lymphocytes.

CD8+ cytotoxic T lymphocytes (CTL) clones with specificity for herpes simplex virus (HSV) were derived from two donors with genital HSV-2 infection. These CTL clones specifically lysed HSV-infected autologous B lymphoblastoid cells, but not HSV-infected fibroblasts. Exogenous peptide loading sensitized both cell types to lysis by an HSV-specific CTL clone of known specificity. HSV infection rendered fibroblasts refractory to peptide sensitization. HSV infection also rendered fibroblasts and keratinocytes insensitive to lysis by allospecific CD8+ CTL clones. Lysis of B lymphoblastoid cells in this system was only slightly reduced by HSV infection. Reduction of fibroblast allospecific lysis was dose and time dependent and was blocked by acyclovir, indicating the involvement of a late HSV gene product. HSV caused a reduction of fibroblast cell surface HLA class I antigen, at least in part due to reduction of synthesis of heavy chain-beta 2 microglobulin heterodimers. These results suggest that HSV-induced blockade of antigen presentation by cutaneous cells to CD8+ CTL may be a mechanism by which HSV limits or evades the immune response of the host.

The effect of intravenous, subcutaneous, and intranasal GH-RH analog, [Nle27]GHRH(1-29)-NH2, on growth hormone secretion in normal men: dose-response relationships.

A 29 amino acid analog of growth hormone releasing hormone (GH-RH)-40 was given intravenously, subcutaneously, and intranasally to normal men to determine its effectiveness in stimulating growth hormone (GH) release. The GH-RH analog, [Nle27]GH-RH(1-29)-NH2, is an amidated 29 amino acid peptide that has one amino acid substitution at position 27. This peptide stimulates GH secretion when given by the intravenous, subcutaneous, and intranasal routes without adverse effect. The degree of GH stimulation was variable among subjects and the greatest amount of stimulation occurred with the highest doses. GH stimulation occurred in a dose-responsive manner after all three routes of administration. A tenfold higher subcutaneous dose was required to stimulate a comparable amount of GH secretion as compared with intravenous administration, and a thirtyfold higher intranasal than intravenous dose was required to stimulate approximately one fifth the amount of GH release. For comparison, one dose of GH-RH-40, 1 microgram/kg, was administered intravenously. GH secretion after 1 microgram/kg GH-RH-40 and 1 microgram/kg Nle27 GH-RH was comparable between the two groups of subjects. Stimulation of GH secretion by Nle27 GH-RH occurred within 5 minutes of intravenous and within 10 minutes of subcutaneous and intranasal administration; peak GH levels were observed within 30 minutes. GH levels declined and returned to near baseline levels 2 hours after administration of the analog.(ABSTRACT TRUNCATED AT 250 WORDS)

The isolation, characterization and nucleotide sequence of the phosphoglucoisomerase gene of Saccharomyces cerevisiae.

We have isolated the gene which encodes the glycolytic enzyme phosphoglucoisomerase (PGI) from the yeast Saccharomyces cerevisiae by functional complementation of a yeast mutant deficient in PGI activity with DNA from a wild-type yeast genomic library. The cloned gene has been localized by hybridization of specific DNA fragments to total yeast poly(A)+ RNA and by complementation of the mutant phenotype with subclones. The gene is expressed as an abundant mRNA of 1.9-kb and encodes a protein of 554 amino acids with an Mr of 61310. The nucleotide sequence of the gene as well as the 5' and 3' flanking regions are presented. The predicted PGI amino acid sequence shows a high degree of homology with the sequence predicted for human and mouse neuroleukin, a putative neurotropic factor. The codon usage within the coding region is very restricted, characteristic of a highly expressed yeast gene.

A therapeutic vaccine that reduces recurrent herpes simplex virus type 1 corneal disease.

PURPOSE: To investigate the therapeutic efficacy of periocular vaccination with herpes simplex virus (HSV) recombinant glycoprotein D from HSV-1 (gD1) or HSV-2 (gD2) in decreasing HSV-induced recurrent dendritic keratitis and HSV-induced recurrent ocular shedding in rabbits latently infected with HSV-1. METHODS: Rabbits latently infected with HSV-1 were vaccinated periocularly (by subconjunctival injection) with gD1 and adjuvant, gD2 and adjuvant, or adjuvant alone. Eyes were examined daily for 49 days for recurrent herpetic keratitis and for recurrent infectious HSV-1 shedding. RESULTS: In both vaccinated groups, a significantly decreased number of eyes exhibited recurrences of herpetic keratitis compared with recurrences in adjuvant-treated control eyes (gD1 group, 27/1372, [2%]; gD2 group, 24/1274, [2%]; and control, 54/1274 [4%]; P < 0.005). Eyes in the gD1-vaccinated group (44/1308 [3.4%]; P = 0.01), but not those in the gD2-vaccinated group (71/1274 [5.6%]; P = 0.93), had significantly decreased viral shedding (positive cultures compared with total cultures) compared with eyes in the adjuvant-treated control group (69 of 1275 [5.4%]). CONCLUSIONS: Recurrent HSV-1 corneal disease was significantly reduced by therapeutic local periocular vaccination. The vaccine may be more efficacious against HSV-1-induced recurrent corneal disease than against recurrent HSV-1 ocular shedding. Its efficacy against corneal disease appeared to be longer lasting than its efficacy against recurrent spontaneous shedding. The heterotypic gD2 vaccine was as efficacious as the homotypic gD1 vaccine against recurrent corneal disease, whereas the homotypic vaccine was much more efficacious than the heterotypic vaccine against recurrent HSV-1 shedding. This is the first report in any animal model of a successful therapeutic vaccine against recurrent HSV-1-induced corneal disease. These results support the concept that development of a therapeutic vaccine for ocular HSV-1 recurrence in humans may be possible.

Antibodies to the major linear neutralizing domains of cytomegalovirus glycoprotein B among natural seropositives and CMV subunit vaccine recipients.

The gB protein (gpUL55) of human cytomegalovirus (CMV) contains C-terminal (AD-1) and N-terminal (AD-2) linear immunodominant neutralizing domains. To measure antibodies to these epitopes, a modified protein (delta-gB) lacking heavily glycosylated intervening regions, the transmembrane domain, and the cytoplasmic domain, was expressed in recombinant baculovirus-infected cells. Eighty-six percent of 600 naturally CMV-seropositive individuals and 93% of 121 gB vaccine recipients had antibodies to delta-gB as detected by enzyme-linked immunosorbent assay (ELISA). The antibody level in vaccinees (median optical density [OD] = 1.73) exceeded that in natural seropositives (median OD = 0.94; p < .0001). Eleven percent of 95 natural seropositives and 7% of 120 gB vaccinees lacked A-gB antibodies but had neutralizing activity. Among subjects with delta-gB antibody, there were weak correlations between antibody level and neutralizing titer. These data suggest that antibodies to linear neutralizing gB domains are highly prevalent in naturally-infected individuals and regularly develop in gB vaccinees. However, for some individuals, discontinuous and/or linear epitopes not represented on delta-gB may be more important in the generation of neutralizing responses.

Physical and biological characterization of antigen presenting liposome formulations: relative efficacy for the treatment of recurrent genital HSV-2 in guinea pigs.

Antigen presenting liposomes (APLs) containing both liposome encapsulated (44%) and free (56%) recombinant glycoprotein D of herpes simplex virus type-2 (rgD-2) were characterized with respect to the interaction of the antigen with the lipid bilayer and the biological activities provided by each form of rgD-2. We found that free rgD-2 added externally to empty liposomes exhibited some biological activities both in vitro and in vivo, although we could not detect any significant adsorption and/or insertion of this form of rgD-2 into the lipid bilayer. Compared to APLs containing both forms of rgD-2, purified liposomes containing only encapsulated rgD-2 gave only 50% of the relative activity in vitro as measured by their ability to stimulate rgD-2 specific lymphocyte proliferation, and 67% of the relative activity in vivo as measured by their immunotherapeutic effect on recurrent genital HSV-2 disease in guinea pigs (P less than 0.05). These data indicate that while liposome encapsulated rgD-2 is essential for the elicitation of immunogenic responses, the free soluble rgD-2 in the APL formulation also acts in concert to generate an optimum immunotherapeutic efficacy.

Herpes simplex virus glycoprotein immunotherapy of recurrent genital herpes: factors influencing efficacy.

Recombinant herpes simplex virus type 1 (HSV-1) glycoproteins B (gB) and D (gD) were used as immunotherapeutic agents for the treatment of recurrent genital herpes in guinea pigs. Administration of a gBgD vaccine eight to 21 days after intravaginal HSV-2 inoculation significantly increased the titer of anti-HSV antibodies (P less than 0.005) while significantly reducing the frequency of subsequent herpetic recurrences (P less than 0.05). The effectiveness of gBgD immunotherapy was influenced by both the co-administration of adjuvant, the type of adjuvant, and by the timing and route of administration. These data demonstrate that recurrent HSV disease in animals with established latent infection may be favorably altered by the administration of immunogenic viral proteins.

A review of zoonotic disease surveillance supported by the Armed Forces Health Surveillance Center.

The Armed Forces Health Surveillance Center (AFHSC), Division of Global Emerging Infections Surveillance and Response System conducts disease surveillance through a global network of US Department of Defense research laboratories and partnerships with foreign ministries of agriculture, health and livestock development in over 90 countries worldwide. In 2010, AFHSC supported zoonosis survey efforts were organized into four main categories: (i) development of field assays for animal disease surveillance during deployments and in resource limited environments, (ii) determining zoonotic disease prevalence in high-contact species which may serve as important reservoirs of diseases and sources of transmission, (iii) surveillance in high-risk human populations which are more likely to become exposed and subsequently infected with zoonotic pathogens and (iv) surveillance at the human-animal interface examining zoonotic disease prevalence and transmission within and between human and animal populations. These efforts have aided in the detection, identification and quantification of the burden of zoonotic diseases such as anthrax, brucellosis, Crimean Congo haemorrhagic fever, dengue fever, Hantaan virus, influenza, Lassa fever, leptospirosis, melioidosis, Q fever, Rift Valley fever, sandfly fever Sicilian virus, sandfly fever Naples virus, tuberculosis and West Nile virus, which are of military and public health importance. Future zoonotic surveillance efforts will seek to develop local capacity for zoonotic surveillance focusing on high risk populations at the human-animal interface.

Development of a herpes simplex virus subunit glycoprotein vaccine for prophylactic and therapeutic use.

A herpes simplex virus subunit vaccine has been tested for immunogenicity and protective efficacy in animal models. The vaccine is a mixture of the viral glycoproteins gB and gD expressed in mammalian cells as secreted carboxyl terminal truncated derivatives. The antigens have been combined with several adjuvants, including a lipophilic derivative of a muramyl tripeptide, MTP-PE, and administered in a stable, low-oil emulsion. Titers of antibody that are threefold to 15-fold greater than those obtained with alum and within twofold of those generated with complete Freund's adjuvant are elicited in animals, including primates. Prophylactic immunization of guinea pigs provides nearly complete protection against intravaginal challenge with herpes simplex virus type 2. Treatment of previously infected guinea pigs reduces the frequency and severity of recurrent disease, although variations in efficacy dependent on the antigen, adjuvant, and dosing regimen employed are observed. The immunogenicity of this vaccine in animals provides the rationale for further testing in human clinical trials.

Managing risk under CLIA (Clinical Laboratory Improvement Amendments).

By now, physician groups that perform any laboratory testing should have either registered under the Clinical Laboratory Improvement Amendments (CLIA) or applied for a certificate of waiver. Rebecca Burke, in her article, discusses some of the risks associated with CLIA that group practices need to be aware of and how, in some cases, those risks can be reduced.

What physicians need to know about the ADA (Americans with Disabilities Act).

The Americans with Disabilities Act (ADA) has raised a lot of questions and concerns from physicians and medical groups regarding their responsibilities in meeting the public accommodation portions of the act. Rebecca Burke and Peter Thomas answer the most commonly asked questions concerning the ADA.

The helix-coil transition of closed and nicked DNAs in aqueous neutral trichloroacetate solutions.

The melting transition for closed, underwound DNAs and for nicked or linear DNAs was monitored by velocity sedimentation and by absorbance spectroscopy in aqueous NaCCl3CO2 (NaTCA) and RbTCA. The addition of neutral trichloroacetate lowers the midpoint of the helix-coil transition by 26% C/M for RbTCA and by 32% C/M for NaTCA, depressing the denaturation region to near room temperature at neutral pH. The melting of nicked DNA is cooperative, occurring over a temperature range of about 5.6 degrees C. The melting profile for closed DNA is broad and noncooperative with a transition breadth greater than 45 degrees. Closed DNAs undergo a structural alteration, as revealed by velocity sedimentation, resulting in a reduction in the number of superhelical turns at temperatures and salt concentrations substantially below the melting temperatures and salt concentrations substantially below the melting temperature of the nicked DNA. The reduction in the extent of supercoiling continues upon isothermal addition of salt up to the salt concentration at which all superhelical turns are removed. The salt concentration at the principal minimum in the sedimentation velocity profile (3.16 M NaTCA for PM-2 DNA) is approximately the same as that at the midpoint of the helix-coil transition for the nicked DNA.

The early melting of closed duplex DNA: analysis by banding in buoyant neutral rubidium trichloroacetate.

Aqueous RbTCA permits the buoyant banding of both native and denatured DNA at room temperature and neutral pH. A unique property of this solvent is the bouyant resolution of closed circular, underwound DNA (I) from the corresponding nicked (II) species. Conditions are reported here in which PM-2 DNA I is physically resolved from native PM-2 DNA II, the buoyant separation being 1.27 mq/ml in 3.3 M RbTCA at 25 degrees C. The separation between nicked and closed DNAs increases with temperature up to 35.5 degrees C, at which PM-2 DNA II cooperatively melts and subsequently pellets. The isothermal buoyant density of a cloed DNA increases linearly as the linking number (Lk) of the closed DNA decreases. The early melting of closed DNA may be monitored with high precision by buoyant banding in RbTCA, it being possible to detect the disruption of as few as 40 base pairs in PM-2 DNA (10,000 base pairs). The constraint that the linking number be conserved in closed DNA requires that a change in duplex winding be accompanied by a compensating change in supercoiling. We estimate the linking number deficiency of PM-2 DNA I to be 0.094 turns per decibase pair. This result permits the estimation of the EtdBr unwinding angle, phi, by comparison with alternative determinations of the linking number deficiency which depend upom the value of phi. The result obtained here is that phi = 27.7 degrees +/- 0.5 degrees and is approximately independent of temperature over the range 15 degrees-35 degrees.

Measurement of superhelix densities in buoyant dye/CsCl. The use of a standard other than native SV40 DNA.

The dye-induced separation between closed and open duplex DNAs in buoyant CsCl is determined primarily by the superhelix density of the closed DNA, provided that all other experimental variables (such as the solution density and dye concentration) are held constant. The extent of the buoyant separation may be used to estimate the superhelix density of an uncharacterized closed DNA, by comparison with the corresponding separation with native SV40 DNAs under identical conditions. We present here an extension of these quantitative relationships to permit the use of an arbitrarily selected closed duplex DNA of known superhelix density, with the accompanying open form, as a reference. The general result is that the ratio of buoyant separations for any two closed/open DNA pairs remains a linear function of the difference in superhelix densities between the closed DNAs. The value of the proportionality constant depends, however, upon the magnitude of the superhelix density of the closed DNA selected as reference.

The properties of native and denatured DNA in buoyant rubidium trichloroacetate at neutral pH.

Aqueous RbTCA is generally suitable as a buoyant solvent for both native and denatured DNA at neutral pH and room temperature. Native PM-2 DNA II, for example, is buoyant at 3.29 M salt, 25 degrees C; whereas the denatured strands band together at 4.52 M. Two properties of the solvent make this system uniquely useful for separations based upon the extent of secondary structure. First, the melting transition temperature for chemically unaltered DNA is depressed to room temperature or below. Second, the buoyant density increase accompanying denaturation is extraordinarily large, 174 mg/ml for PM-2 DNA II. This value is three times that found in aqueous NaI and ten times that for CsCl. The properties of the RbTCA buoyant solvent presented here include the compositional and buoyant density gradients and the buoyant density dependence upon base composition. The DNA remains chemically unaltered after exposure to RbTCA as shown by the absence of strand scissions for closed circular DNA and by the unimpaired biological activity in transformation assays. Intact virion DNA may be isolated by direct banding of whole virions in RbTCA gradients without prior phenol extraction. Strongly complexed or covalently bound proteins may be detected by their association with the buoyant polymer in the denaturing density gradient.

Disposition of antigen-presenting liposomes in vivo: effect on presentation of herpes simplex virus antigen rgD.

Antigen-presenting liposomes (APLs) containing a lipophilic derivative of muramyl tripeptide (MTP-PE) have previously been shown to enhance the immunotherapeutic effects mediated by HSV recombinant protein gD (rgD) after HSV type 2 infection is established. In this study, both the in vivo disposition of rgD and the immunological activity of in vivo-delivered rgD were determined. Following intravenous administration, most of the liposome-encapsulated rgD accumulated rapidly, mainly in the spleen, while most of the soluble rgD was quickly eliminated through the kidney. We have compared the T-cell stimulatory effects of macrophages, B cells and dendritic cells from the spleens of animals treated with rgD in vivo. Of these antigen-presenting cells, only adherent macrophages, isolated from the spleens of animals treated with rgD encapsulated in APLs for 90 minutes, were capable of stimulating HSV-sensitized autologous T and B cells. Additional in vitro exposure of macrophages to rgD was not required. In contrast, spleen macrophages from HSV-sensitized animals exposed to either empty liposomes or free rgD did not exhibit such immune responses, indicating that the immunobiological effect of the rgD delivered in APLs is antigen- and carrier-specific. The enhanced delivery of antigen to spleen cells, coupled with MTP-PE immunostimulatory activity, may be the key factors for the enhanced therapeutic effects observed in treating HSV-2 disease in guinea pigs. This approach will be useful to enhance the induction of secondary immune responses in postinfection vaccination schemes.