Birth and death rate estimates of cats and dogs in U.S. households and related factors

Studies report variable factors associated with dog and cat surpluses in the United States. Estimates of cat and dog birth and death rates help understand the problem. This study collected data through a commercial survey company, distributing questionnaires to 7,399 cat- and dog-owning households (HHs) in 1996. The study used an unequal probability sampling plan and reported estimates of means and variances as weighted averages. The study used estimates of HHs and companion animals for national projections. More than 9 million owned cats and dogs died during 1996-yielding crude death rates of 8.3 cat deaths/100 cats in HHs and 7.9 dog deaths/100 dogs in HHs. The study reported twice as many kitten as puppy litters, with an average litter size of 5.73 and 7.57, respectively. The study reported data on planned versus unplanned litters, reasons caregivers did not spay females, disposition of litters, and sources of animals added to HHs. These first national estimates indicate the magnitude of, and reasons for, animals leaving HHs. The crude birth rate was estimated to be 11.2 kittens/100 cats in HHs and 11.4 puppies/100 dogs in HHs.     

Recovery of Mycobacterium bovis from soft fresh cheese originating in Mexico

Recent outbreaks of human tuberculosis in the United States caused by Mycobacterium bovis have implicated cheese originating in Mexico as a source of these infections. A total of 203 samples of cheese originating in Mexico were cultured, and M. bovis was recovered from one specimen. Therefore, M. bovis can be recovered from cheese and may be a source of human infections.     

Identification of covalent modifications in P450 2E1 by 1,2-epoxy-3-butene in vitro

1,3-Butadiene is metabolized mainly by cytochrome P450 2E1 to several epoxides that are considered toxic and carcinogenic. The first step of BD metabolism is oxidation to 1,2-epoxy-3-butene (EB), a reactive metabolite. It has been shown that P450s can be inactivated by covalent binding of reactive metabolites to protein or heme. Molecular dosimetry studies have clearly shown that BD metabolism follows a supralinear dose response, suggestive of saturation of metabolic activation. In this study, potential binding sites of EB in human P450 2E1 were identified and modeled to test whether EB covalently binds to residues important for enzyme activity. Commercially available human P450 2E1 was reacted with EB, digested with trypsin and the resulting peptides were analyzed by Matrix-Assisted Laser Desorption/Ionization tandem Time-of-Flight mass spectrometry (MALDI-MS). The identity of EB modified peptides was confirmed by Matrix-Assisted Laser Desorption/Ionization tandem mass spectrometry (MALDI-MS/MS) sequencing. It was shown that EB binds to four histidine and two tyrosine residues. All modification sites were assigned by at least two adjacent and a minimum of eight peptide specific fragments. Protein modeling revealed that two of these covalent modifications (His(109), His(370)) are clearly associated with the active site, and that their Calpha atoms are located less than 9A from a known inhibitor binding site. In addition, the side chain of His(370) is within 4A of the heme group and its modification is expected to influence the orientation of the heme. The Calpha atom of Tyr(71) is within 14A of the potential inhibitor binding site and within 7A of the flap undergoing conformational change upon ligand binding, potentially placing Tyr(71) near the substrate as it enters and leaves the active site. The data support the hypothesis that EB can inactivate P450 2E1 by covalent modifications and thus add an additional regulatory mechanism for BD metabolism.     

The Molecular Mechanism of Eukaryotic Elongation Factor 2 Kinase Activation

Calmodulin (CaM)-dependent eukaryotic elongation factor 2 kinase (eEF-2K) impedes protein synthesis through phosphorylation of eukaryotic elongation factor 2 (eEF-2). It is subject to complex regulation by multiple upstream signaling pathways, through poorly described mechanisms. Precise integration of these signals is critical for eEF-2K to appropriately regulate protein translation rates. Here, an allosteric mechanism comprising two sequential conformations is described for eEF-2K activation. First, Ca²⁺/CaM binds eEF-2K with high affinity (K_d(CaM)^app = 24 ± 5 nm) to enhance its ability to autophosphorylate Thr-348 in the regulatory loop (R-loop) by > 10⁴-fold (k_auto = 2.6 ± 0.3 s⁻¹). Subsequent binding of phospho-Thr-348 to a conserved basic pocket in the kinase domain potentially drives a conformational transition of the R-loop, which is essential for efficient substrate phosphorylation. Ca²⁺/CaM binding activates autophosphorylated eEF-2K by allosterically enhancing k_cat^app for peptide substrate phosphorylation by 10³-fold. Thr-348 autophosphorylation results in a 25-fold increase in the specificity constant (k_cat^app/K_m(Pep-S)^app), with equal contributions from k_cat^app and K_m(Pep-S)^app, suggesting that peptide substrate binding is partly impeded in the unphosphorylated enzyme. In cells, Thr-348 autophosphorylation appears to control the catalytic output of active eEF-2K, contributing more than 5-fold to its ability to promote eEF-2 phosphorylation. Fundamentally, eEF-2K activation appears to be analogous to an amplifier, where output volume may be controlled by either toggling the power switch (switching on the kinase) or altering the volume control (modulating stability of the active R-loop conformation). Because upstream signaling events have the potential to modulate either allosteric step, this mechanism allows for exquisite control of eEF-2K output.     

Computer-assisted sperm analysis of canine spermatozoa motility measurements

Computer-assisted sperm analysis (CASA) allows for the determination of specific motion characteristics of sperm cells in vitro. This study was designed to develop a system for the use of CASA to objectively evaluate canine sperm motility, and specifically to determine whether motility characteristics vary between individual dogs. Ejaculates from 10 dogs were collected weekly. Sperm cells were extended in a glucose-free TALP medium, placed on slides and videotaped at 200x. Videotaped samples were then analyzed by the Hamilton-Thorn Motility Analyzer, with 100 cells evaluated per slide. Two slides were made from each ejaculate. Motility characteristics that were evaluated included lateral head displacement, beat cross frequency, path velocity, path linearity, path straightness, percentage of motile cells, and percentage of progressively motile cells. Sperm cell morphology was also evaluated. Canine spermatozoa maintained good overall motility (mean +/- SD, 73 +/- 9%) during the procedure. Mean sperm motility and morphology measurements differed significantly between dogs (P<0.01). There was no difference (P>0.05) between the mean measurements of different ejaculates for an individual dog, or for different slides made from the same ejaculate. Mean motility values for the 10 dogs are reported. There was a significant but not strong correlation (r=0.44) between the percentage of progressively motile sperm cells and the percentage of sperm cells with normal morphology.     

Hypothalamic gene expression following ghrelin therapy to gastrectomized rodents

We investigated whether ghrelin depletion (by gastrectomy surgery) and/or treatment/replacement with the gastric hormone ghrelin alters the expression of key hypothalamic genes involved in energy balance, in a manner consistent with ghrelin's pro-obesity effects. At 2 weeks after surgery mice were treated with ghrelin (12 nmol/mouse/day, sc) or vehicle for 8 weeks. Gastrectomy had little effect on the expression of these genes, with the exception of NPY mRNA in the arcuate nucleus that was increased. Ghrelin treatment (to gastrectomized and sham mice) increased the mRNA expression of orexigenic peptides NPY and AgRP while decreasing mRNA expression of the anorexigenic peptide POMC. Two weeks gavage treatment with the ghrelin mimetic, MK-0677, to rats increased NPY and POMC mRNA in the arcuate nucleus and MCH mRNA in the lateral hypothalamus. Thus, while predicted pro-obesity ghrelin signalling pathways were activated by ghrelin and ghrelin mimetics, these were largely unaffected by gastrectomy.