Characterisation of mouse monoclonal antibodies against rhesus macaque killer immunoglobulin-like receptors KIR3D

Killer immunoglobulin-like receptors (KIRs) represent a highly polymorphic and diverse gene family in rhesus macaques. Analyses of the respective gene products have been hampered until now due to non-availability of specific monoclonal antibodies and failure of cross-reactivity of anti-human KIR antibodies. We utilised one activating (KIR3DSW08) and two inhibitory (KIR3DLW03 and KIR3DL05) rhesus macaque KIR-Fc fusion proteins for generation of monoclonal antibodies in mice. Besides broadly reacting ones, we obtained anti-rhesus macaque KIR antibodies with intermediate and with single specificity. These monoclonal antibodies were tested for binding to a panel of rhesus macaque KIR proteins after heterologous expression on transiently transfected cells. Epitope mapping identified two polymorphic regions that are located next to each other in the mature KIR proteins. The availability of monoclonal antibodies against rhesus macaque KIR proteins will enable future studies on KIR at the protein level in rhesus macaques as important animal models of human infectious diseases.     

Solution structure of Mycobacterium tuberculosis NmtR in the apo state: insights into Ni(II)-mediated allostery

Mycobacterium tuberculosis is an obligate human respiratory pathogen that encodes approximately 10 arsenic repressor (ArsR) family regulatory proteins that allow the organism to respond to a wide range of changes in its immediate microenvironment. How individual ArsR repressors have evolved to respond to selective stimuli is of intrinsic interest. The Ni(II)/Co(II)-specific repressor NmtR and related actinomycete nickel sensors harbor a conserved N-terminal α-NH(2)-Gly2-His3-Gly4 sequence. Here, we present the solution structure of homodimeric apo-NmtR and show that the core of the molecule adopts a typical winged-helix ArsR repressor (α1-α2-α3-αR-β1-β2-α5) "open conformation" that is similar to that of the related zinc sensor Staphylococcus aureus CzrA, but harboring long, flexible N-terminal (residues 2-16) and C-terminal (residues 109-120) extensions. Binding of Ni(II) to the regulatory sites induces strong paramagnetic broadening of the α5 helical region and the extreme N-terminal tail to residue 10. Ratiometric pulse chase amidination mass spectrometry reveals that the rate of amidination of the α-amino group of Gly2 is strongly attenuated in the Ni(II) complex relative to the apo state and noncognate Zn(II) complex. Ni(II) binding also induces dynamic disorder on the microsecond to millisecond time scale of key DNA interacting regions that likely contributes to the negative regulation of DNA binding by Ni(II). Molecular dynamics simulations and quantum chemical calculations reveal that NmtR readily accommodates a distal Ni(II) hexacoordination model involving the α-amine and His3 of the N-terminal region and α5 residues Asp91', His93', His104, and His107, which collectively define a new metal sensing site configuration in ArsR family regulators.     

UCP2 induced by natural birth regulates neuronal differentiation of the hippocampus and related adult behavior

Mitochondrial uncoupling protein 2 (UCP2) is induced by cellular stress and is involved in regulation of fuel utilization, mitochondrial bioenergetics, cell proliferation, neuroprotection and synaptogenesis in the adult brain. Here we show that natural birth in mice triggers UCP2 expression in hippocampal neurons. Chemical inhibition or genetic ablation of UCP2 lead to diminished neuronal number and size, dendritic growth and synaptogenezis in vitro and impaired complex behaviors in the adult. These data reveal a critical role for Ucp2 expression in the development of hippocampal neurons and circuits and hippocampus-related adult behaviors.     

Human fibroblast stromelysin catalytic domain: expression, purification, and characterization of a C-terminally truncated form.

Stromelysin-1 is a member of a tissue metalloproteinase family whose members are all capable of degrading extracellular matrix components. A truncated form of human fibroblast prostromelysin 1 lacking the C-terminal, hemopexin-like domain has been expressed in Escherichia coli and purified to homogeneity. Treatment of this short form of prostromelysin with (aminophenyl)mercuric acetate resulted in activation and loss of the propeptide in a manner identical with the wild-type, full-length protein. Kinetic comparisons using Nle11-substance P as a substrate showed that the wild-type stromelysin and the truncated form of the enzyme had similar kcat and Km values. Likewise, both enzymes displayed similar Ki values for a hydroxamate-containing peptide inhibitor. Taken together, these results indicate that the C-terminal portion of stromelysin is not required for proper folding of the catalytic domain, maintenance of the enzyme in a latent form, activation with an organomercurial, cleavage of a peptide substrate, or interaction with an inhibitor. Moreover, the active short form of stromelysin displayed a reduction in the C-terminal heterogeneity, a characteristic degradation of the full-length stromelysin, and thereby provides a more suitable protein for future structural studies.     

Ticks (Acari: Ixodidae) of the state of Amazonas,Brazil

The tick fauna of Brazil is currently composed by 72 species. The state of Amazonas is the largest of Brazil, with an area of ≈ 19% of the Brazilian land. Besides its vast geographic area, only 19 tick species have been reported for Amazonas. Herein, lots containing ticks from the state of Amazonas were examined in three major tick collections from Brazil. A total of 5933 tick specimens were examined and recorded, comprising 2693 males, 1247 females, 1509 nymphs, and 484 larvae. These ticks were identified into the following 22 species: Amblyomma cajennense sensu lato, Amblyomma calcaratum, Amblyomma coelebs, Amblyomma dissimile, Amblyomma dubitatum, Amblyomma geayi, Amblyomma goeldii, Amblyomma humerale, Amblyomma latepunctatun, Amblyomma longirostre, Amblyomma naponense, Amblyomma oblongoguttatum, Amblyomma ovale, Amblyomma rotundatum, Amblyomma scalpturatum, Amblyomma varium, Dermacentor nitens, Haemaphysalis juxtakochi, Ixodes cf. Ixodes fuscipes, Ixodes luciae, Rhipicephalus microplus, Rhipicephalus sanguineus sensu lato. Ticks were collected from 17 (27.4%) out of the 62 municipalities that currently compose the state of Amazonas. The following four species are reported for the first time in the state of Amazonas: A. coelebs, A. dubitatum, H. juxtakochi, and Ixodes cf. I. fuscipes. The only tick species previously reported for Amazonas and not found in the present study is Amblyomma parvum. This study provides a great expansion of geographical and host records of ticks for the state of Amazonas, which is now considered to have a tick fauna composed by 23 species. It is noteworthy that we report 1391 Amblyomma nymphs that were identified to 13 different species.     

New species of Eimeria Schneider, 1875 and Isospora Schneider, 1881 (Apicomplexa: Eimeriidae) from the short-crested flycatcher Myiarchus ferox (Gmelin) (Passeriformes: Tyrannidae) in South America

In the current study, two new coccidian species (Protozoa: Apicomplexa: Eimeriidae) obtained from short-crested flycatcher Myiarchus ferox (Gmelin) are reported from Brazil. Isospora feroxis n. sp. has o?cysts which are spheroidal to sub-spheroidal, 18.7 × 18.0 μm, with a smooth and bi-layered wall, c. 1.2 μm. The micropyle and o?cyst residuum are absent, but two polar granules are present. Its sporocysts are broadly ellipsoidal and 11.7 × 8.5 μm. Stieda and substieda bodies are present. A sporocyst residuum is present and the sporozoites have a refractile body and nucleus. O?cysts of Eimeria sicki n. sp. are spheroidal to sub-spheroidal, 30.3 × 28.5 μm, with a smooth and bi-layered wall, c. 1.3 μm. The micropyle, o?cyst residuum and polar granule are absent. Its sporocysts are ellipsoidal, 18.4 × 10.0 μm. Stieda and substieda bodies are present. A sporocyst residuum is present and sporozoites have a refractile body and nucleus.     

Sperm sex-sorting in the Asian elephant (Elephas maximus)

In captive Asian elephants, there is a strong need for production of female offspring to enhance reproduction, counter premature aging processes in female animals and reduce challenging management situations derived from husbandry of several bulls in one institution. Artificial insemination of flow cytometrically sex-sorted spermatozoa offers the possibility to predetermine the sex of offspring with high accuracy. The aims of this study were to determine a suitable semen extender and basic parameters for flow cytometrical sex-sorting of Asian elephant spermatozoa. In total 18 semen samples were collected by manual rectal stimulation from one bull. Sperm quality parameters and sex sortability of spermatozoa were evaluated after dilution in three semen extenders (MES-HEPES-skim milk, MES-HEPES, TRIS-citric acid) and DNA staining. MES-HEPES-skim milk was the only semen extender found suitable to sex Asian elephant spermatozoa. From 18 ejaculates collected, 12 were successfully sorted with a purity of 94.5+/-0.7% at an average sort rate of 1945.5+/-187.5 spermatozoa per second. Sperm integrity, progressive and total motility were 42.6+/-3.9%, 48.1+/-3.3%, 59.4+/-3.8% after DNA labelling, and 64.8+/-3.2%, 58.0+/-5.0%, 70.8+/-4.4% after sorting, respectively. After liquid storage of sorted spermatozoa for 12h at 4 degrees C, sperm integrity, progressive and total motility were 46.4+/-5.2%, 32.2+/-4.2% and 58.2+/-3.9%, respectively. The obtained results provide a promising base to inseminate Asian elephants with sexed semen.     

MALDI-TOF "fingerprint" phospholipid mass spectra allow the differentiation between ruminantia and feloideae spermatozoa

The detailed comparative analysis of sperm lipids could essentially contribute to a better understanding of membrane function in the context of fertilization and, moreover, of sperm preservation. The application of sensitive analytical methods is particularly necessary for endangered species as the available amount of spermatozoa (and, accordingly, extractable lipids) is strongly limited. It will be shown that matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a fast, simple and sensitive method for the determination of the phospholipid composition of spermatozoa from several ruminantia (cattle, roe deer, Klipspringer) and feloideae species (domestic cat, Siberian tiger, fosa). Characteristic “fingerprints” are obtained from the positive ion spectra that allow the differentiation between both animal groups. In contrast to the lipid extracts of ruminantia spermatozoa which predominantly contain ether lipids including essential amounts of plasmalogens, the more complex phospholipid composition of feloideae spermatozoa is clearly dominated by diacyl phospholipids and contains only marginal amounts of plasmalogens. It will also be shown that the lipid compositions of ejaculated, electroejaculated and cauda epididymal spermatozoa of the same species are very similar and give comparable data. Therefore, the analysis of ejaculated spermatozoa is not an absolute must.     

Ovarian superstimulation, transrectal ultrasound-guided oocyte recovery, and IVF in rhinoceros

Numerous reports on reproductive pathology in all rhinoceros species illustrate the abundance of female infertility in captive populations. In infertile rhinoceroses, oocyte collection and embryo production could represent the best remaining option for these animals to reproduce and to contribute to the genetic pool. We report here on superstimulation, repeated oocyte recovery, and attempted in vitro fertilization (IVF) in white and black rhinoceroses. Four anestrous rhinoceroses (two white, two black) with unknown follicular status were treated with gonadotropin-releasing hormone analogue, deslorelin acetate, for 6 to 7 d. Number and size of follicles in superstimulated females was significantly higher and larger compared with those in nonstimulated anestrous females (n = 9). Ovum pick-up was achieved by transrectal ultrasound-guided follicle aspiration. Up to 15 follicles were aspirated per ovary. During six ovum pick-ups, a total of 29 cumulus-oocyte complexes (COCs) were harvested with a range of 2 to 9 COCs per collection. No postsurgical complications were noted on the rhinoceros ovaries using this minimally invasive approach. Various in vitro maturation (IVM) and IVF protocols were tested on the collected COCs. Despite the low total number of COCs available for IVM and IVF in this study, we can report the first rhinoceros embryo ever produced in vitro. The production of a 4-cell embryo demonstrated the potential of transrectal ultrasound-guided oocyte recovery as a valuable tool for in vitro production of rhinoceros embryos from otherwise infertile females.