Ovum transport in the rat oviductal ampulla in the absence of muscle contractility

Ovum transport in mammalian oviducts involves two main effectors: ciliary motility and muscle contractility. To study the relative contribution of cilia to ovum transport in the rat, we blocked smooth muscle activity with isoproterenol, a beta-adrenergic agonist, and measured transport rates of surrogate ova in situ. Transport rates before isoproterenol administration were 0.04 mm/s in the cephalic ampulla and 0.03 mm/s in the caudal ampulla; rates were unchanged after administration of isoproterenol. To determine if isoproterenol affected ciliary activity, we measured ciliary beat frequency with laser-scattering spectroscopy over the effective isoproterenol dosage. Isoproterenol did not cause a significant change in ciliary beat frequency. Our results show that in the rat oviductal ampulla, ciliary motion is capable of transporting ova in the absence of muscle contractility.     

Multifactorial diversity sustains microbial community stability

Maintenance of a high degree of biodiversity in homogeneous environments is poorly understood. A complex cheese starter culture with a long history of use was characterized as a model system to study simple microbial communities. Eight distinct genetic lineages were identified, encompassing two species: Lactococcus lactis and Leuconostoc mesenteroides. The genetic lineages were found to be collections of strains with variable plasmid content and phage sensitivities. Kill-the-winner hypothesis explaining the suppression of the fittest strains by density-dependent phage predation was operational at the strain level. This prevents the eradication of entire genetic lineages from the community during propagation regimes (back-slopping), stabilizing the genetic heterogeneity in the starter culture against environmental uncertainty.     

The Active and the Inactive Form of the hAT1 Receptor Have an Identical Ligand-Binding Environment: An MPA Study on a Constitutively Active Angiotensin II Receptor Mutant

Several models of activation mechanisms were proposed for G protein-coupled receptors (GPCRs), yet no direct methods exist for their elucidation. The availability of constitutively active mutants has given an opportunity to study active receptor conformations within acceptable limits using models such as the angiotensin II type 1 (AT₁)¹ receptor mutant N111G-hAT₁ which displays an important constitutive activity. Recently, by using methionine proximity assay, we showed for the hAT₁ receptor that TMD III, VI, and VII form the ligand-binding pocket of the C-terminal amino acid of an antagonistic AngII analogue. In the present contribution, we investigated whether the same residues would also constitute the ligand-binding contacts in constitutively activated mutant (CAM) receptors. For this purpose, the same Met mutagenesis strategy was carried out on the N111G double mutants. Analysis of 43 receptors mutants in the N111G-hAT₁ series, photolabeled and CNBr digested, showed that there were only subtle structural changes between the wt-receptor and its constitutively active form.     

Genetic portrait of Lisboa immigrant population from Cabo Verde with mitochondrial DNA analysis

Journal of Genetics -     

A comprehensive evaluation of cattle introgression into US federal bison herds

Genetic introgression, especially from interspecies hybridization, is a significant threat to species conservation worldwide. In this study, 11 US federal bison populations were comprehensively examined for evidence of both mitochondrial and nuclear domestic cattle (Bos taurus) introgression. Mitochondrial introgression was examined using established polymerase chain reaction methods and confirmed through analysis of D-loop sequences. Nuclear introgression was assessed in 14 chromosomal regions through examination of microsatellite electromorph and sequence differences between bison and domestic cattle. Only one population was identified with domestic cattle mitochondrial DNA introgression. In contrast, evidence of nuclear introgression was found in 7 (63.6%) of the examined populations. Historic accounts of bison transfers among populations were corroborated with evidence of introgressed DNA transmission. While neither nuclear nor mitochondrial domestic cattle introgression was detected in bison from Grand Teton National Park, Sully's Hill National Game Preserve, Wind Cave National Park, or Yellowstone National Park, adequate sample sizes were available only from the last 2 populations to allow for statistical confidence (>90%) in nuclear introgression detection limits. The identification of genetically unique and undisturbed populations is critical to species conservation efforts, and this study serves as a model for the genetic evaluation of interspecies introgression.     

Functional characterization of both MAP kinases of the human malaria parasite Plasmodium falciparum by reverse genetics

The kinome of the human malaria parasite Plasmodium falciparum includes two genes encoding mitogen-activated protein kinase (MAPK) homologues, pfmap-1 and pfmap-2, but no clear orthologue of the MAPK kinase (MAPKK) family, raising the question of the mode of activation and function of the plasmodial MAPKs. Functional studies in the rodent malaria model Plasmodium berghei recently showed the map-2 gene to be dispensable for asexual growth and gametocytogenesis, but essential for male gametogenesis in the mosquito vector. Here, we demonstrate by using a reverse genetics approach that the map-2 gene is essential for completion of the asexual cycle of P. falciparum, an unexpected result in view of the non-essentiality of the orthologous gene for P. berghei erythrocytic schizogony. This validates Pfmap-2 as a potential target for chemotherapeutic intervention. In contrast, the other P. falciparum MAPK, Pfmap-1, is required neither for in vitro schizogony and gametocytogenesis in erythrocytes, nor for gametogenesis and sporogony in the mosquito vector. However, Pfmap-2 protein levels are elevated in pfmap-1(-) parasites, suggesting that Pfmap-1 fulfils an important function in asexual parasites that necessitates compensatory adaptation in parasites lacking this enzyme.     

Expression of the HPV16 E7 gene generates proliferation in stratified squamous cell cultures which is independent of endogenous p53 levels.

Monolayer cultures of human foreskin and ectocervical epithelial cells were infected with retroviral vectors expressing HPV16 oncogenes, selected for G418 resistance, and cultured organotypically so that they reformed the fully differentiated, stratified squamous tissues from which they were originally derived. Expression of HPV16 E7 prevented cell cycle withdrawal in the suprabasal layers of these stratified cultures but had no effect on terminal differentiation. Cultures expressing E7 alone and those coexpressing E6 and E7 were identical in terms of suprabasal proliferation and terminal differentiation, but they differed in expression of the endogenous tumor suppressor protein p53. Immunohistochemically detectable p53 protein localized to the proliferative compartment in normal and E7-containing cultures but was undetectable in those cultures which coexpressed E6 and E7. This result suggests that E7-induced suprabasal proliferation is independent of the steady-state level of p53.     

A technique for transcervical intrauterine insemination of ewes

In commercial artificial insemination (AI) of sheep, fresh extended semen is deposited into the vagina or cervical os, or fresh extended or frozen semen is placed laparoscopically into the uterus. Transcervical intrauterine insemination of the ewe is not used commercially. In this study, methods of restraint and instrumentation for AI were evaluated and modified to produce a transcervical intrauterine technique suitable for commercial application. Four methods of restraint, four vaginal specula, three forceps and four instruments suitable for transcervical passage were compared. From these comparisons a technique was developed in which the ewes were positioned in dorsal recumbency with their hindquarters elevated. The vagina was dilated using a duck-billed speculum, the cervix was grasped and retracted using forceps, and an inseminating instrument was introduced into the cervical opening and manipulated through the cervical canal. The technique was repeated on 89 mature, multiparous ewes: the difficulty in locating the cervical opening, the force required to retract the cervix and the time required to penetrate into the uterus were recorded. Uterine penetration was achieved in 82% of the ewes. This technique has the potential to be applied in commercial artificial insemination programs of sheep.