A phylogeny of frugivorous hornbills linked to the evolution of Indian plants within Asian rainforests.

Understanding the origin and radiation of modern Asian hornbills and the influential ecological roles they play as seed dispersal agents within Asian rainforests should help reveal the evolution of these roles. We constructed a dated phylogeny of hornbills using mitochondrial DNA sequences of the cytochrome b gene and discovered that all clades leading to frugivorous hornbills originated in the mid-Eocene ∼48 Ma. This 'explosive' radiation coincided with a remarkable floral invasion of Asian rainforests from the Indian microcontinent. Analysis of phylogenetic data, in conjunction with palaeontological events, suggests that the invasion of distinctive flora comprised two waves, one during the mid-Eocene, when India was offshore of the Sunda Shelf, and the other late Eocene, when India collided with the Asian mainland. We propose that frugivorous vertebrates, such as hornbills, were present during the first wave and assisted rapid colonization of the Asian flora.

Chemotherapy of surra in horses and mules with diminazene aceturate.

During June-July 2000, an outbreak of surra occurred on an equine breeding farm in Khonkaen Province, Thailand. Forty-two percent of pregnant mares aborted or gave stillbirth and 40% (19/47) of horses and 10% (1/10) of mules died from surra. In August 2000 Trypanosoma evansi were detected in the remaining animals (28 horses and nine mules) on the farm by blood smear and/or the haematocrit centrifuge technique. All animals were treated with diminazene aceturate at 3.5 mg/kg body weight by intramuscular injection on days 0 and 41 of the study. Blood samples of eight randomly selected horses and mules were collected on days 0, 1 and once a week until day 56 and examined for T. evansi by various parasitological techniques. The sera were tested for antibodies against T. evansi using an indirect enzyme linked immunosorbent assay (ELISA).The results revealed that diminazene aceturate at 3.5 mg/kg appeared to be effective in the first treatment of horses and mules infected with T. evansi. Parasites were cleared from the peripheral blood of horses on days 1 and 7 and mules on days 1 and 14. Thereafter the number of positive animals increased. After the second treatment, 50% of horses and 25% of mules were still positive to surra 24 h after treatment demonstrating that diminazene had no protective effect. Mild to severe toxicity of diminazene was seen in the horses and mules after injection.

UNCL, the mammalian homologue of UNC-50, is an inner nuclear membrane RNA-binding protein.

We isolated a mammalian homologue of the C. elegans gene unc-50 that we have named UNCL. The 777 kb rat UNCL cDNA encodes a 259 amino acid protein that is expressed in a wide variety of tissues with highest mRNA levels in brain, kidney and testis. Hydropathy plot analysis and in vitro translation experiments with microsomal membranes indicate that UNCL is a transmembrane protein. Hemagglutinin tagged UNCL was stably transfected into SaOS-2 osteosarcoma cells and exhibited a nuclear rim staining pattern which was retained following extraction with 1% Triton X-100, suggesting that UNCL localizes to the inner nuclear membrane. UNCL-HA was extractable in 350 mM NaCl, suggesting that UNCL is not associated with the nuclear matrix. Homopolymer RNA-binding assays performed on in vitro translated UNCL protein and 'structural modeling by homology' suggest that UNCL binds RNA via an amino-terminal RNA Recognition-like Motif. Since unc-50 is required for expression of assembled muscle-type nicotinic receptors in the nematode we investigated whether UNCL had a similar function for mammalian nicotinic receptors. When UNCL was co-expressed with neural nicotinic receptors in Xenopus oocytes or COS cells it increased expression of functional cell surface receptors up to 1. 6-fold. We conclude that UNCL is a novel inner nuclear membrane protein that associates with RNA and is involved in the cell-surface expression of neuronal nicotinic receptors. UNCL plays a broader role because UNCL homologues are present in two yeast and a plant species, none of which express nicotinic receptors and it is also found in tissues that lack nicotinic receptors.

Sequence and expression analysis of a surface antigen gene family of the rickettsia Anaplasma marginale.

The tick-borne rickettsial organism, Anaplasma marginale, causes a disease in cattle of world-wide economic significance. This disease, anaplasmosis, is characterized by severe hemolytic anemia, high levels of rickettsemia and, often, death in animals over 3years of age. Animals that survive acute infection remain carriers, with continuous sub-microscopic cycles of rickettsemia that can persist for the lifetime of the animal. In the search for potential recombinant immunogens, it was discovered that several surface proteins of A. marginale encode polymorphic multigene families. Despite the small size of the genome (approx. 1250kb), these surface antigen gene families comprise greater than 2% of the genome. We present here a mapping, sequencing and expression analysis of five complete or partial genes encoding MSP1b in a Florida strain of A. marginale. Two genes are complete; they encode mRNA that is translated into polypeptide products. Three genes are incomplete and appear to be derived from the complete genes by a series of segmental intragenic recombinations. In two of the incomplete genes, 5' sequence in the incomplete genes is 3' sequence in the complete genes. Recombination within these gene families may generate diversity in surface antigens through combinatorial rearrangements. This could contribute to persistence in the chronic infections caused by A. marginale and related rickettsiae.

Pharmacological similarities between native brain and heterologously expressed alpha4beta2 nicotinic receptors.

1 We studied the pharmacological properties of native rat brain and heterologously expressed rat alpha4beta2 nicotinic receptors immunoprecipitated onto a fixed substrate with the anti-alpha4 antibody mAb 299. 2 Immunodepletion with the anti-beta2 antibody mAb 270 showed that 89% of the mAb-299-precipitated rat brain receptors contained beta2. 3 The association and dissociation rate constants for 30 pM +/-[3H]-epibatidine binding to alpha4beta2 receptors expressed in oocytes were 0.02+/-0.01 and 0.03+/-0.01 min-1 (+/-standard error, degrees of freedom=7 - 8) at 20 - 23 degrees C. 4 The Hill coefficients for +/-[3H]epibatidine binding to the native brain, alpha4beta2 receptors expressed in oocytes, and alpha4beta2 receptors expressed in CV-1 cells (using recombinant adenovirus) were 0.69 - 0.70 suggesting a heterogeneous receptor population. Fits of the +/-[3H]-epibatidine concentration-binding data to a two-site model gave KD s of 8 - 30 and 560 - 1,200 pM. The high-affinity sites comprised 73 - 74% of the native brain and oocyte alpha4beta2 receptor population, 85% of the CV-1 alpha4beta2 receptor population. 5 The expression of rat alpha4beta2 receptors in CV-1 cells using vaccinia viral infection-transfection resulted in a more homogeneous receptor population (Hill coefficient of 1. 0+/-0.2). Fits of the +/-[3H]-epibatidine binding data to a single-site model gave a KD of 40+/-3 pM. 6 DHbetaE (IC50=260-470 nM) and the novel nicotine analogue NDNI (IC50=7-10 microM) inhibited 30 pM+/-[3H]-epibatidine binding to the native brain and heterologously expressed alpha4beta2 receptors equally well. 7 The results show that alpha4beta2-containing nicotinic receptors in the rat brain and heterologously expressed rat alpha4beta2 receptors have similar affinities for +/-[3H]-epibatidine, DHbetaE, and NDNI.

Two mutations linked to nocturnal frontal lobe epilepsy cause use-dependent potentiation of the nicotinic ACh response.

1. We constructed rat homologues (S252F and +L264) of two human alpha4 nicotinic mutations - alpha4(S248F) and alpha4(777ins3) - that have been linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and co-expressed them with wild-type rat beta2 subunits in Xenopus oocytes. 2. The S252F and +L264 mutations had three common effects on the ACh response. First, they caused use-dependent potentiation of the response during a train of brief 100 nM ACh pulses. Second, they delayed the rise times of the 5-15 nM (+L264) and 30 nM (S252F) ACh responses. Third, they reduced extracellular Ca2+-induced increases in the 30 microM ACh response. 3. Beside these shared effects, the S252F mutation also reduced the channel burst duration measured from voltage-jump relaxations, enhanced steady-state desensitization and reduced the single-channel conductance. In contrast, the +L264 mutation prolonged the channel burst duration, did not affect desensitization and slightly increased single-channel conductance. Neither mutation affected the number of surface receptors measured by antibody binding but the S252F mutation reduced the maximum ACh response. 4. The ACh concentration dependence of use-dependent potentiation and the delay in the rising phase of the mutant ACh response suggest that these effects are caused by a slow unblocking of the closed mutant receptors. Use-dependent potentiation of the mutant response during a series of high-frequency cholinergic inputs to the presynaptic terminal could trigger ADNFLE seizures by suddenly increasing nicotinic-mediated transmitter release.

The alpha4 subunit of rat alpha4beta2 nicotinic receptors is phosphorylated in vivo.

The intracellular domains of the alpha4 and beta2 neuronal nicotinic subunits between transmembrane segments 3 and 4 contain a number of predicted phosphorylation sites but there is no direct evidence that any of these sites are actually phosphorylated in vivo. We expressed rat alpha4beta2 nicotinic receptors in Xenopus oocytes, labeled them by an overnight incubation in [32P]orthophosphate, and analyzed the immunoprecipitated receptors by autoradiography and Western blotting. Our results show that the oocytes contained three kinds of alpha4 subunits with apparent weights of 69, 79, and 89 kDa. The 89 kDa alpha4 subunit was the most heavily phosphorylated.

A recombinant adenovirus that directs secretion of biologically active kappa-bungarotoxin from mammalian cells.

A novel Cre-lox system was used to construct an adenovirus encoding kappa-bungarotoxin (kappa-Bgt), modified to be secreted by attachment of a bovine prolactin signal sequence at the N-terminus of the toxin. Western blot of medium from HEK-293 cells infected with the virus demonstrated that recombinant kappa-Bgt (R-kappa-Bgt) was secreted. The biological activity of the secreted R-kappa-Bgt was investigated in Xenopus oocytes that expressed neuronal nicotinic acetylcholine receptor (nAChR) subtypes alpha3beta2 and alpha2beta2. The recombinant toxin inhibited the response of alpha3beta2 type AChRs to ACh, but did not inhibit the response of alpha2beta2 type AChRs. These data demonstrated that the recombinant adenovirus directs the secretion of biologically active kappa-Bgt from a mammalian cell line. Because adenovirus can be used to infect post-mitotic cells, recombinant adenoviruses encoding biologically active peptides may be of use as delivery vehicles for in vivo experiments where repeated application of the purified peptide is unfeasible.

Analysis of the Anaplasma marginale genome by pulsed-field electrophoresis.

Anaplasma marginale is a rickettsial parasite of bovine erythrocytes causing world-wide economic losses in livestock production. Despite its importance, little is known about this rickettsia at a molecular level because it has not been cultured in vitro, and there is no small-animal model. Although several genes have been cloned and sequenced, the gross genome structure of the organism has not yet been well characterized. We separated intact bovine erythrocytes from leucocytes, and determined the genome size of A. marginale by use of restriction endonuclease cleavage and pulsed-field gel electrophoresis (PFGE). A value of 56 mol% G+C was obtained for this genome by spectral analysis. Undigested A. marginale DNA failed to migrate under several different electrophoretic conditions, indicating a circular genome. Digestions of intact A. marginale DNA were performed using restriction endonucleases NotI, SfiI and PacI. Complete digestion with SfiI resulted in 12 distinct bands ranging in size from 14 to 170 kbp. Total size determined by addition of SfiI-digested fragments was approximately 1200 kbp. PacI cleaved the A. marginale genome from three different isolates into just three fragments, of 598, 557 and 97 kbp. Incomplete digestion produced a band measuring 1250 kbp. These results indicate that A. marginale has a circular genome between 1200 and 1260 kbp, with a G+C content of 56 mol%.

Evidence for kinetoplast and nuclear DNA homogeneity in Trypanosoma evansi isolates.

The kinetoplast DNA minicircles from 13 stocks of trypanosomes designated as Trypanosoma evansi were digested with various restriction enzymes. We also examined the distribution of restriction site polymorphisms in the nuclear DNA of 9 of these stocks, using 7 different variable surface glycoprotein (VSG) and non-VSG probes. Restricted kinetoplast DNA (kDNA) fragments of some of these strains were cloned into M13 or PUC 18 vectors and sequenced. The restriction and sequence mapping showed that most of T. evansi isolates belonged to the A1 and A2 types of Borst and to two new closely related types A3 and A4. A notable exception was RoTat 4/1 derived from a Sudanese stock which was found to display a characteristic brucei-like minicircle heterogeneity. The T. evansi minicircles analysed are not only homogeneous in sequence but also the region similar to the conserved region in Trypanosoma brucei and Trypanosoma equiperdum is flanked on its 5' end by a palindromic repeat of part of the conserved region. The highly conserved sequence GGGCGGT which appears to correspond to the initiation of synthesis of one of the Okazaki fragments contains an additional G and is located as in T. brucei and T. equiperdum about 73 bp 5' from the ORI. The nuclear DNA analysis confirms the kDNA study in that all the T. evansi stocks are members of a very homogeneous group in terms of sequence divergence. Moreover, our analysis also confirms that T. evansi is more closely related to the West African T. b. brucei and T. b. gambiense than to other African trypanosomes.

Specific DNA probe for the sensitive detection of Trypanosoma evansi.

Trypanosoma evansi is the parasitic protozoon that causes "Surra", a wasting disease of domestic animals. Detection of T. evansi plays an important role in epidemiology and animal health. DNA probes were constructed from T. evansi genomic DNA and kinetoplast DNA for sensitive detection of the parasite in infected blood. A 6.5 kb DNA insert of pMUT ec6 plasmid derived from the genomic DNA of T. evansi Npl isolate, selected from 575 recombinant E. coli exhibited the strongest nucleic acid hybridization signal to the T. evansi DNA. Using as the DNA probe, pMUT ec6 could detect as little as 60 pg T. evansi DNA and it did not hybridize to the DNA of cattle, waterbuffalo and two related blood parasites. A simple detection procedure by spotting 10 microliters infected blood onto nylon membrane could sense as little as 1000 parasites. The kinetoplast DNA was cloned in E. coli and found to show a comparable sensitivity to that of the pMUT ec6. However, the kinetoplast DNA exhibited variation in copy number among parasite isolates thus pMUT ec6 should be the DNA probe of choice for sensitive detection of T. evansi.