Two Rhabdoviridae: Dillard's Draw virus, a putative new virus, and Merida virus from Culex tarsalis (Diptera: Culicidae) in New Mexico, USA.

The U.S. Air Force conducts vector and arbovirus surveillance worldwide. We report on two Rhabdoviridae detected at Holloman Air Force Base in Otero County, New Mexico including the nearly complete 11-kb genome of Merida virus, which was not previously known from the USA, and a novel virus tentatively named Dillard's Draw virus. Merida virus was previously only known from Mexico. Dillard's Draw virus represents a novel arbovirus most closely related to an avian pathogen from Australia and shares some genetic similarity to Durham virus from the eastern United States.

Transcription efficiency of different chicken mannose-binding lectin promoter alleles.

The serum collectin mannose-binding lectin (MBL) plays a major role in innate immunity by activation of the lectin complement pathway or by acting as an opsonin. The serum levels of human and animal MBL are associated with susceptibility to a wide range of infections, and the variation of MBL in serum is genetically determined. In the chicken, 14 single nucleotide polymorphisms (SNPs) have so far been found in the MBL promoter region. In this study, the transcription activity of a 670-bp promoter region covering all 14 SNPs from the four MBL promoter alleles A1 to A4 was assessed using a dual-luciferase assay. Of the analysed alleles, A1 showed the highest transcription activity although this allele is frequently found in chickens with low MBL mRNA expression.

Kennedy, the early sixties, and visitation by the angel of death.

The inaugural issue of Pathologia Veterinaria in 1964 contained the first detailed account of lesions in aborted fetuses following natural, experimental, and postvaccinal infection with bovine herpesvirus 1 (BoHV-1). The article, written by pathologists Kennedy and Richards, described diagnostic gross and histologic features in 13 bovine fetuses. The authors provided clinical and epidemiologic features of 1 postvaccination outbreak, including the absence of clinical signs in infected dams and the propensity for abortions to occur after 6 months' gestation. Subsequent field and experimental studies corroborated and expanded these observations. As a result of this and later reports, veterinarians became alert to the association between infectious bovine rhinotracheitis and abortion, including the risks of exposing pregnant cattle to live vaccinal BoHV-1. Methods were developed to corroborate a morphologic diagnosis of herpetic abortion in cattle, including immunofluorescence, immunohistochemistry, and polymerase chain reaction methods. Outbreaks of postvaccinal BoHV-1 abortion in the United States began to be reported with apparently increased frequency in the early 2000s. This coincided with licensure in 2003 of modified live BoHV-1 vaccines intended for use in pregnant cattle, which are now sold by 3 manufacturers. Ten recent herd episodes of postvaccinal BoHV-1 abortion are reported. All 10 BoHV-1 isolates had single-nucleotide polymorphism (SNPs) profiles previously identified in a group of BoHV-1 isolates that contains vaccine strains, based on a BoHV-1 SNP classification system. They lacked SNP features typical of those in characterized field-type strains of BoHV-1.

The novel HLA-B*15:180 allele appears to be a recombinant B*08/B*15 allele.

Human leukocyte antigen B-*15:180 is a B*08/B*15 recombinant allele similar to B*15:29 with substitutions positions at 97, 292, 538, 539.

Control of Culicoides sonorensis (Diptera: Ceratopogonidae) blood feeding on sheep with long-lasting repellent pesticides.

Culicoides sonorensis is the primary vector of bluetongue and epizootic hemorrhagic disease viruses in North America. Bluetongue disease is one of the most economically important arthropod-borne diseases of sheep in North America, because it causes significant morbidity and mortality and can lead to local quarantines and international trade restrictions. Long-lasting repellent pesticides could be applied to sheep as they are moved down from mountain pastures to protect them from biting midges until the 1st frost. We tested long-lasting pesticides on sheep as repellents against C. sonorensis. Both Python ear tags with 10% zeta-cypermethrin (9.8 g/tag) synergized with 20% piperonyl butoxide (PBO) and a 12-ml low-volume spray application of ready-to-use sheep insecticide (Y-TEX) with 2.5% permethrin and 2.5% PBO in an oil-based formulation were repellent to C. sonorensis for at least 3-5 wk after a single application.

Vitiligo: Patient stories, self-esteem, and the psychological burden of disease.

Vitiligo is a relatively common disorder that is characterized by depigmented patches of skin. Multiple studies characterize the overwhelming psychological burden that is experienced by many patients around the globe. This review examines personal patient stories and the impacts of age, culture, sex, race, and ethnicity in relationship to altered self-esteem and quality of life in patients who live with vitiligo.

RNA-binding properties of the mitochondrial Y-box protein RBP16.

We have previously identified a mitochondrial Y-box protein in Trypanosoma brucei that we designated RBP16. The predicted RBP16 amino acid sequence revealed the presence of a cold-shock domain at its N-terminus and a glycine- and arginine-rich C-terminus reminiscent of an RGG RNA-binding motif. Since RBP16 is capable of interacting with different guide RNAs (gRNAs) in vitro and in vivo primarily via the oligo(U) tail, as well as with ribosomal RNAs, possible functions of RBP16 may be in kinetoplastid RNA editing and/or translation. Herein, we report experiments that further define the RNA-binding properties of RBP16. RBP16 forms a single stable complex with the gRNA gA6[14] at low protein concentration, while at higher protein concentration two stable complexes that possibly represent two different conformations are observed. Both complexes are stable at relatively high salt and moderate heparin concentrations indicating that the binding of RBP16 to gA6[14] does not rely primarily on ionic interactions. Phenylglyoxal treatment of the protein indicates that arginine residues are important in RNA binding. The minimal length of RNA sequence necessary for the binding of RBP16 was assessed by gel retardation and UV cross-linking competition assays using oligo(U) ribonucleotides of varying lengths (4-40 nt). Although RBP16 can bind to oligonucleotides as small as U(4), its affinity increases with the length of the oligo(U) ribonucleotide, with a dramatic increase in binding efficiency observed when the length is increased to 10 nt. Gel retardation assays employing T.brucei mRNAs demonstrated that, although it acts as a major binding determinant, a 3' U tail is not an absolute requirement for efficient RBP16-RNA binding. Experiments with oligonucleotides containing U stretches embedded at different positions in oligo(dC) indicated that high-affinity binding requires both a uridine stretch, as well as 5' and 3' non-specific sequences. These results suggest a model for the molecular interactions involved in RBP16-RNA binding.

The trypanosome homolog of human p32 interacts with RBP16 and stimulates its gRNA binding activity.

RBP16 is a guide RNA (gRNA)-binding protein that was shown through immunoprecipitation experiments to interact with approximately 30% of total gRNAs in Trypanosoma brucei mitochondria. To gain insight into the biochemical function of RBP16, we used affinity chromatography and immunoprecipitation to identify RBP16 protein binding partners. By these methods, RBP16 does not appear to stably interact with the core editing machinery. However, fractionation of mitochondrial extracts on MBP-RBP16 affinity columns consistently isolated proteins of 12, 16, 18 and 22 kDa that were absent from MBP control columns. We describe here our analysis of one RBP16-associated protein, p22. The predicted p22 protein has significant sequence similarity to a family of multimeric, acidic proteins that includes human p32 and Saccharomyces cerevisiae mam33p. Glutaraldehyde crosslinking of recombinant p22 identified homo-multimeric forms of the protein, further substantiating its homology to p32. We confirmed the p22-RBP16 interaction and demonstrated that the two proteins bind each other directly by ELISA utilizing recombinant p22 and RBP16. p32 family members have been reported to modulate viral and cellular pre-mRNA splicing, in some cases by perturbing interaction of their binding partners with RNA. To determine whether p22 similarly affects the gRNA binding properties of RBP16, we titrated recombinant p22 into UV crosslinking assays. These experiments revealed that p22 significantly stimulates the gRNA binding capacity of RBP16. Thus, p22 has the potential to be a regulatory factor in T.brucei mitochondrial gene expression by modulating the RNA binding properties of RBP16.

Why do we need to conserve what we have? A post-genome sequencing perspective on existing chicken strains.

The recent publication of the chicken genome sequence along with the extensive single nucleotide polymorphism and physical map open exciting avenues for defining gene function and for understanding the genotypic basis of phenotypic variation in the chicken. The number of genes identified on the sequence map is growing rapidly. Genetically uniform lines and crosses derived from them will allow identification of gene function and gene interactions that contribute to traits such as immunity, disease resistance, growth, production, and behavior. Selected, inbred, and congenic lines will continue to be essential in defining the genetics of many traits. Although dwindling under budgetary pressures, a number of well characterized lines and genetic strains remain. If preserved, these can be used to address questions regarding newly mapped candidate genes defining their importance in a variety of problems in basic, biomedical, and applied avian biology. If lost, years of breeding and selection will be required to replace them.

Killer Ig-like receptor (KIR) compatibility plays a role in the prevalence of acute GVHD in unrelated hematopoietic cell transplants for AML.

Killer Ig-like receptor (KIR) is a major cluster of the natural killer cell receptors and may play a role in the outcome of hematopoietic cell transplants. A total of 65 AML cases transplanted with T-replete hematopoietic cells from unrelated donors were retrospectively KIR-genotyped by a multiplex PCR method of our own design. The KIR gene frequency and genotype patterns in these 130 samples were consistent with the data in the literature. Based upon overall inhibitory and activating KIR genes in both donors and patients, we developed an algorithm to calculate a compatibility score for each transplant case as plus, zero or minus. Significantly higher incidence (18/20, 90%) of acute (a) GVHD (grade II-IV) was found in the transplant cases with plus scores than that (25/45, 56%) in the cases with zero or minus scores (P < 0.01). When the scores are sorted in the opposite way, fewer cases (13/26, 50%) of aGVHD were found in the transplants with minus scores than that (30/39, 77%) in the transplants with zero or plus scores (P < 0.05). The difference of aGVHD prevalence between the plus score and minus score groups is highly significant (P < 0.01). KIR genotype compatibility calculated by this algorithm may predict aGVHD incidence and be helpful in choosing donors.

Bovine herpesvirus-1: evaluation of genetic diversity of subtypes derived from field strains of varied clinical syndromes and their relationship to vaccine strains.

Bovine herpesvirus-1 (BoHV-1) causes significant disease in cattle. Control programs in North America incorporate vaccination with modified live viral (MLV) or killed (KV) vaccine. BoHV-1 strains are isolated from diseased animals or fetuses after vaccination. There are markers for differentiating MLV from field strains using whole-genome sequencing and analysis identifying single nucleotide polymorphisms (SNPs). Using multiple primer sets and sequencing of products permits association of BoHV-1 isolates with vaccines. To determine association between vaccine virus and strains isolated from clinical cases following vaccination, we analyzed 12 BoHV-1 isolates from animals with various clinical syndromes; 9 corresponded to BoHV-1.1 respiratory group. The remaining three corresponded to BoHV-1.2b, typically found in genital tracts of cattle. Four BoHV-1 isolates were identical to a vaccine strain; three were from post-vaccination abortion episodes with typical herpetic lesions whose dams had received MLV vaccine during pregnancy, and one from a heifer given a related MLV vaccine; Sequences of two respiratory isolates perfectly matched mutations characterizing RLB106 strain, a temperature sensitive mutant used in intranasal and parenteral vaccines. The last three respiratory strains clearly appeared related to a group of MLV vaccines. Previously the MLV vaccines were grouped into four groups based on SNPs patterns. In contrast with above-mentioned isolates that closely matched SNP patterns of their respective MLV vaccine virus, these 3 strains both lacked some and possessed a number of additional mutations compared to a group of MLV vaccine viral genome. Finding BoHV-1.2b in respiratory cases indicates focus should be given BoHV-1.2b as an emerging virus or a virus not recognized nor fully characterized in BRD.

Age-related alterations in the expression of prohormone convertase messenger ribonucleic acid (mRNA) levels in hypothalamic proopiomelanocortin mRNA neurons in the female C57BL/6J mouse.

POMC processing is mediated by the prohormone convertases (PC1 and PC2). The cleavage of beta-endorphin-(1-31) is mediated by PC2. PC2 can also further cleave beta-endorphin-(1-31) to beta-endorphin-(1-27). We previously reported a significant increase in the proportion of beta-endorphin-(1-27) and -(1-27) forms in the arcuate nucleus (ARC) of the hypothalamus in middle-aged females with irregular estrous cycles (5-7 days) compared to young female C57BL/6J mice with regular cycles (4-5 days). Changes in processing enzymes may be a mechanism underlying this change. We compared ARC messenger RNA (mRNA) levels of PC1, PC2, and furin by Northern blot and in situ hybridization analyses in young, middle-aged, and old mice. Antisense complementary RNA probes to mouse PC1, PC2, and furin were radiolabeled and used in single label studies, alone or in combination with a mouse POMC digoxigenin-labeled complementary RNA probe for double label studies. For Northern blot analysis, young (4- to 5-month-old) normally cycling (4-5 days) mice at diestrus were compared to middle-aged (12- to 13-month-old) irregularly cycling (5-7 days) mice at diestrus. By Northern blot analysis, a significant increase (P < 0.05) in ARC PC2 mRNA levels was detected in middle-aged compared to young mice, but ARC PC1 and furin mRNA levels were unaltered. Single label in situ hybridization analysis confirmed these findings in the general neuron population. We also observed a significant reduction in ARC furin mRNA levels in old mice compared to either young or middle-aged mice. Double labeling in situ hybridization histochemistry demonstrated that PC2 mRNA levels were significantly increased (at least 2-fold) in POMC mRNA-containing neurons of middle-aged compared to young mice. Selective changes in PC2 mRNA levels in ARC POMC neurons are correlated with changes in beta-endorphin-(1-31) processing to beta-endorphin-(1-27)/(1-26) in middle-aged animals. Our data suggest that the natural age-related shift in beta-endorphin peptide processing is mediated by PC2.

Liposome entrapment enhances the hypocalcemic action of parenterally administered calcitonin.

The hypocalcemic effect of liposomal-entrapped calcitonin (CT) was evaluated in rats. Salmon CT and human CT were entrapped in liposomes composed of egg phosphatidylcholine with or without an equimolar amount of cholesterol. The liposomes were separated by Sepharose 4B chromatography into fractions consisting of large multilamellar vesicles and small unilamellar vesicles. The incorporation of CT was monitored by counting [125I]CT and by specific RIA. Liposomal entrapment enhanced the hypocalcemic potency of parenterally administered salmon CT and human CT. After iv administration, the large multilamellar vesicles were more potent than small unilamellar vesicles in their hypocalcemic effect; cholesterol inclusion in the MLV liposome preparation prolonged the hypocalcemia. However, with im administration, the cholesterol-free liposomes were more potent than their cholesterol-containing counterparts regardless of size. These studies demonstrate that liposomal entrapment can be used to enhance the hypocalcemic potency of CT. It appears that both the size and composition of the liposome preparation are important in this effect, as is the route of administration. It may be possible to produce liposome-CT preparations with advantageous pharmacological characteristics.

Evidence for the presence of the estrogen receptor in the ovary of the baboon (Papio anubis).

Although intraovarian estrogen has been firmly established as an important factor in the regulation of ovarian follicular development and function in the rat, an autocrine-paracrine role for estrogen in the primate ovary is not yet established. Immunocytochemical identification of an estrogen receptor in the monkey follicle was negative, but it was positive for the granulosa cells of antral follicles of the human ovary. In the present study baboon ovaries obtained during the follicular phase were examined for the presence of estrogen receptor by immunocytochemical analysis of frozen sections and Northern blot analysis of RNA extracts of the ovaries. Immunocytochemistry identified the estrogen receptor in the granulosa cells of healthy appearing and atretic or cystic-like antral follicles and in occasional, but rare, thecal cells. The ovaries contained a prominent mRNA species for the estrogen receptor, approximately 7 kilobases in size, which was present in relatively low abundance compared to that in the nonpregnant baboon uterus, but in a similar abundance to the estrogen receptor mRNA content of the pregnant endometrium. These studies are the first to report the presence of estrogen receptor mRNA in the ovary of a primate. These results in conjunction with the immunocytochemical studies firmly establish the presence of the estrogen receptor in the primate ovary and suggest an autocrine-paracrine role for intraovarian estrogen in primate ovarian physiology.

CV 205-502 treatment of hyperprolactinemia.

CV 205-502 is a nonergot oral dopamine agonist with specific D2 activity, which has a prolonged suppressive effect on serum PRL and may have fewer side-effects than other dopamine agonists. We treated 26 hyperprolactinemic women with this compound given as a single bedtime (hs) dose for up to 12 weeks. All had gonadal dysfunction, either amenorrhea or oligomenorrhea, and 15 had galactorrhea. The initial and subsequent doses were administered in a randomized fashion; the initial dose ranged from 0.01-0.05 mg, and the dose at 12 weeks ranged from 0.03-0.09 mg. The women were evaluated every 2 weeks, and the dose was increased by 0.02 mg every 4 weeks if the serum PRL level was greater than 20 micrograms/L. Of the 26 women initially enrolled, 24 completed 12 weeks of therapy, and 2 discontinued therapy because of side-effects. Thirteen women (54%) had return of menses, and 12 (80%) had either a decrease in or disappearance of galactorrhea. Serum PRL concentrations decreased to a variable degree in all patients; 13 (54%) achieved a normal serum PRL level (less than or equal to 20 micrograms/L). The mean (+/- SE) pretreatment serum PRL concentration was 129 +/- 34, and it was 29.9 +/- 5.9 micrograms/L after 12 weeks of treatment (P = 0.005). The mean (+/- SE) percent reduction in serum PRL was 66.5 +/- 5.0% (median, 78.0%). A dose response was not demonstrated (r = -0.08; P = 0.70) among the 6 dose groups during the last 4 weeks of therapy. In 5 women, serum PRL levels, measured frequently for 24 h after treatment remained low. Side-effects after the initiation of therapy included nausea, headache, and morning fatigue in 10 women. These symptoms caused 2 women to discontinue therapy; they subsided in the other women. An optimal dose was not determined and will probably need to be determined by titration in each patient. CV 205-502, given once daily, appears to be a safe and effective alternative to other dopamine agonists in the treatment of hyperprolactinemia.

Progesterone regulated expression of flavin-containing monooxygenase 5 by the B-isoform of progesterone receptors: implications for tamoxifen carcinogenicity.

Progesterone is a key developmental, proliferative, and differentiative hormone in the breast and endometrium, and it can accelerate carcinogenesis in the mammary gland epithelium. In the breast and uterus, progesterone acts through two coexpressed isoforms of progesterone receptors, the B- and A-receptors. To study the function of each isoform in isolation, we previously constructed two breast cancer cell lines that stably and independently express either B-receptors (YB cells) or A-receptors (YA cells). In the present study, YA or YB cells were left untreated, or were treated with the synthetic progestin R5020, and the messages present in each cell line under the two conditions were analyzed by differential display. Two message species are described that are regulated only by B-receptors. One of these is regulated in a ligand-independent manner. A third set of messages, encoding flavin-containing monooxygenase 5 (FMO5), was induced by R5020 only in YB cells. A-receptors appear to be inhibitory. FMOs are involved in the metabolic activation of drugs and xenobiotic compounds, including the antiestrogen tamoxifen, to carcinogenic intermediates. It is possible, therefore, that by upregulating the levels of FMO5, progesterone enhances the carcinogenicity of tamoxifen in target tissues that overexpress progesterone B-receptors.

Aging changes in the beta-endorphin neuronal system in the preoptic area of the C57BL/6J mouse: ultrastructural analysis.

In hypothalami of aging rodents, beta-endorphin (beta-EP) neuron number and content are reduced. The objectives of this study were: first, to analyze ultrastructurally the population of neuronal elements in a selected region of the preoptic area (POA) in young and old mice; second, to study the beta-EP neuronal system in the same region to determine whether or not this population remains stable with age. Vibratome sections from the most caudal POA through the diagonal band of Broca were examined by light microscopy and immunocytochemistry in mature, cycling (5-6 months old) and old, acyclic, disease-free (24-26 months old) mice. A subset of beta-EP-like perikarya and associated structures was observed in the periventricular POA. When this subregion was examined at the ultrastructural level, there was a significant decrease in the number of recipient dendrites [3.78 +/- 0.04 SEM/micron 2 young vs. 0.82 +/- 0.03/micron 2 old; p < 0.007, analysis of variance (ANOVA)], but a significant increase in the number of nonmyelinated axons (20.0 +/- 2.6/micron 2 young vs. 26.8 +/- 0.7/micron 2 old; p < 0.05). Immunolabeled terminals that contained a synapse comprised 2.56 +/- 0.08% of all terminals with synapses in young mice but only 0.34 +/- 0.04% in old ones when corrected for surface area examined (p < 0.03). A significant age-related loss was also observed in the nonmyelinated beta-EP-labeled axon population (1.50 +/- 0.10% young vs. 0.40 +/- 0.01% old; p < 0.009, ANOVA). We conclude that there are critical changes in the microenvironment of the POA in old, noncycling female mice that are likely to affect neuron function.

Ovariectomy and age alter gonadotropin hormone releasing hormone-noradrenergic interactions.

The noradrenergic (NA) system influences gonadotropin hormone releasing hormone (GnRH) neurons resulting in the luteinizing hormone surge. Direct neuroanatomical interactions between preoptic area (POA) GnRH neuronal elements and NA [i.e., dopamine-beta-hydroxylase (DBH)] terminals; effects of short-term ovariectomy (S-OVX) on these interactions; and the stability of these interactions with age were studied in young (5-month-old) proestrous, 5-month-old S-OVX (6d), old (24-month-old) constant diestrous, and 24-month-old long-term (L)-OVX mice. Proestrous females demonstrated direct interactions between NA terminals and GnRH neuronal elements. The percentage of GnRH dendritic profiles contacted by DBH terminals at proestrous (6.96 +/- 1.07%) did not differ versus young S-OVX females (4.55 +/- 0.91%). No GnRH-NA interactions were observed in old mice. S-OVX resulted in a decrease in DBH terminals but an increase in dendrite and nonmyelinated axon number versus young proestrous females (p < or = 0.05 ANOVA). Findings show direct GnRH-DBH interactions, confirm S-OVX effects on neural ultrastructure, and demonstrate some S-OVX changes resembling those in older mice. L-OVX failed to prevent aging changes. Diminished capacity to produce normal estrous cycles in aging females could result in part from absence of direct GnRH-DBH interactions.

Loss during aging of beta-endorphinergic neurons in the hypothalamus of female C57BL/6J mice.

Beta-endorphin (B-EP) content is often reduced in hypothalami of aging rodents. The objective of this study was to determine whether reduced B-EP content is associated with a reduced number of B-EP immunoreactive neurons. Serial coronal sections extending from the caudal hypothalamus through the retrochiasmatic area were examined by quantitative light microscopy in mature (5-6 month) and senescent (24-28 month) mice that had been ovariectomized 1 week earlier and injected with colchicine 24-48 h before sacrifice. Old mice were acyclic. As expected, B-EP immunoreactive cell bodies were restricted to the region of the arcuate nucleus. There was a 35% loss of B-EP immunopositive neurons in old, macroscopically disease-free animals. By contrast, some old animals with pituitary tumors had no loss of B-EP neurons. These results suggest that a subpopulation of B-EP neurons either die or stop synthesizing detectable concentrations of B-EP in aged mice. The basis for the absence of reduced B-EP neurons in some mice with pituitary tumors is unclear, but this observation underscores the importance of distinguishing age-related changes associated with diseases of aging from those that are independent of such diseases.