Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host''s oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.Download video file.^{(57M, mov)}     

Structural Basis for the Inverted Repeat Preferences of mariner Transposases

The inverted repeat (IR) sequences delimiting the left and right ends of many naturally active mariner DNA transposons are non-identical and have different affinities for their transposase. We have compared the preferences of two active mariner transposases, Mos1 and Mboumar-9, for their imperfect transposon IRs in each step of transposition: DNA binding, DNA cleavage, and DNA strand transfer. A 3.1 Å resolution crystal structure of the Mos1 paired-end complex containing the pre-cleaved left IR sequences reveals the molecular basis for the reduced affinity of the Mos1 transposase DNA-binding domain for the left IR as compared with the right IR. For both Mos1 and Mboumar-9, in vitro DNA transposition is most efficient when the preferred IR sequence is present at both transposon ends. We find that this is due to the higher efficiency of cleavage and strand transfer of the preferred transposon end. We show that the efficiency of Mboumar-9 transposition is improved almost 4-fold by changing the 3′ base of the preferred Mboumar-9 IR from guanine to adenine. This preference for adenine at the reactive 3′ end for both Mos1 and Mboumar-9 may be a general feature of mariner transposition.     

A QTL affecting daily feed intake maps to Chromosome 2 in pigs

Our understanding of the molecular genetic basis of several key performance traits in pigs has been significantly advanced through the quantitative trait loci (QTL) mapping approach. However, in contrast to growth and fatness traits, the genetic basis of feed intake traits has rarely been investigated through QTL mapping. Since feed intake is an important component of efficient pig production, the identification of QTL affecting feed intake may lead to the identification of genetic markers that can be used in selection programs. In this study a QTL analysis for feed intake, feeding behavior, and growth traits was performed in an F₂ population derived from a cross between Chinese Meishan and European Large White pigs. A QTL with a significant effect on daily feed intake (DFI) was identified on Sus scrofa Chromosome 2 (SSC2). A number of suggestive QTL with effects on daily gain, feed conversion, and feeding behavior traits were also located. The significant QTL lies close to a previously identified mutation in the insulin-like growth factor 2 gene (IGF2) that affects carcass composition traits, although the IGF2 mutation is not segregating in the populations analyzed in the current study. Therefore, a distinct causal variant may exist on the P arm of SSC2 with an effect on feed intake.     

Heat injuries in Canadian mass participation runs.

In two Canadian runs with a total of approximately 2900 participants 26 people collapsed with heat injury and were taken to hospital. All were relatively young (13 to 38 years old). A retrospective survey showed that all were novices to 10-km races and that many had collapsed even though they had consumed fluids before and during the run. A review of etiologic factors suggested that the extremes in the Canadian climate, which preclude heat acclimatization in the spring and early summer, may be an important influence on the incidence of heat injury.     

Control of cytoplasmic pH by Na+/H+ exchange in rat peritoneal macrophages activated with phorbol ester

The mechanisms underlying cytoplasmic pH (pHi) regulation in elicited rat peritoneal macrophages were investigated by electronic sizing and fluorescence determinations. Acid-loaded cells rapidly regained normal pHi by means of an amiloride-sensitive Na+/H+ exchange. When stimulated by 12-O-tetradecanoyl phorbol 13-acetate, macrophages displayed a biphasic pHi change: a marginal acidification followed by an alkalinization. The latter results from activation of Na+/H+ exchange, since it is Na+-dependent and prevented by amiloride. When the antiport is inhibited, the full magnitude of the initial acidification can be appreciated. This acidification is independent of the nature of the ionic composition of the medium and probably reflects accumulation of protons generated during the metabolic burst. Under physiological conditions, these protons are rapidly extruded by the Na+/H+ antiport.     

Finite element analysis of heel pad with insoles

To design optimal insoles for reduction of pedal tissue trauma, experimental measurements and computational analyses were performed. To characterize the mechanical properties of the tissues, indentation tests were performed. Pedal tissue geometry and morphology were obtained from magnetic resonance scan of the subject's foot. Axisymmetrical finite element models of the heel of the foot were created with 1/4 of body weight load applied. The stress, strain and strain energy density (SED) fields produced in the pedal tissues were computed. The effects of various insole designs and materials on the resulting stress, strain, and SED in the soft pedal tissues were analyzed. The results showed: (a) Flat insoles made of soft material provide some reductions in the maximum stress, strain and SED produced in the pedal tissues. These maximum values were computed near the calcaneus. (b) Flat insoles, with conical/cylindrical reliefs, provided more reductions in these maximum values than without reliefs. (c) Custom insoles, contoured to match the pedal geometry provide most reductions in the maximum stress, strain and SED. Also note, the maximum stress, strain and SED computed near the calcaneus were found to be about 10 times the corresponding peak values computed on the skin surface. Based on the FEA analysis, it can be concluded that changing insole design and using different material can significantly redistribute the stress/strain inside the heel pad as well as on the skin surface.     

Inhibition of gingipains by their profragments as the mechanism protecting Porphyromonas gingivalis against premature activation of secreted proteases

Background

Arginine-specific (RgpB and RgpA) and lysine-specific (Kgp) gingipains are secretory cysteine proteinases of Porphyromonas gingivalis that act as important virulence factors for the organism. They are translated as zymogens with both N- and C-terminal extensions, which are proteolytically cleaved during secretion. In this report, we describe and characterize inhibition of the gingipains by their N-terminal prodomains to maintain latency during their export through the cellular compartments.

Methods

Recombinant forms of various prodomains (PD) were analyzed for their interaction with mature gingipains. The kinetics of their inhibition of proteolytic activity along with the formation of stable inhibitory complexes with native gingipains was studied by gel filtration, native PAGE and substrate hydrolysis.

Results

PD_RgpB and PD_RgpA formed tight complexes with arginine-specific gingipains (Ki in the range from 6.2 nM to 0.85 nM). In contrast, PD_Kgp showed no inhibitory activity. A conserved Arg-102 residue in PD_RgpB and PD_RgpA was recognized as the P1 residue. Mutation of Arg-102 to Lys reduced inhibitory potency of PD_RgpB by one order of magnitude while its substitutions with Ala, Gln or Gly totally abolished the PD inhibitory activity. Covalent modification of the catalytic cysteine with tosyl-l-Lys-chloromethylketone (TLCK) or H-D-Phe-Arg-chloromethylketone did not affect formation of the stable complex.

Conclusion

Latency of arginine-specific progingipains is efficiently exerted by N-terminal prodomains thus protecting the periplasm from potentially damaging effect of prematurely activated gingipains.

General significance

Blocking progingipain activation may offer an attractive strategy to attenuate P. gingivalis pathogenicity.     

Decrease in myosin light chain kinase activity of rabbit fast muscle by chronic stimulation

Analysis of myosin light chain kinase (MLCK) activity in tibialis anterior muscles of the rabbit revealed that chronic stimulation at a frequency of 10 Hz for 24 h per day reduced the enzyme activity in a timedependent manner. Since fast twitch muscle contains significantly more myosin light chain kinase than slow twitch muscle, the observed reductions are consistent with the type of fast-to-slow transformation observed for other type-specific muscle characteristics. The present data also indicate that the stimulation-induced decrease in MLCK activity precedes the fast-to-slow conversion of the myosin molecule as judged by pyrophosphate-polyacrylamide gel electrophoresis.