Mouse zinc transporter 1 gene provides an essential function during early embryonic development

The SLC30 family of cation diffusion transporters includes at least nine members in mammals, most of which have been documented to play a role in zinc transport. The founding member of this family, Znt1, was discovered by virtue of its ability to efflux zinc from cells and to protect them from zinc toxicity. However, its physiological functions remain unknown. To address this issue, mice with targeted knockout of the Znt1 gene were generated by homologous recombination in embryonic stem cells. Heterozygous Znt1 mice were viable. In contrast, homozygous Znt1 mice died in utero soon after implantation due to a catastrophic failure of embryonic development. Although extraembryonic membranes formed around these embryos, the embryo proper failed to undergo morphogenesis past the egg cylinder stage and was amorphous by d9 of pregnancy. Expression of the Znt1 gene was detected predominantly in trophoblasts and in the maternal deciduum during the postimplantation period (d5 to d8). The failure of homozygous Znt1 embryos to develop could not be rescued by manipulating maternal dietary zinc (either excess or deficiency) during pregnancy. However, embryos in Znt1 heterozygous females were approximately 3 times more likely to develop abnormally when exposed to maternal dietary zinc deficiency during later pregnancy than were those in wildtype females. These studies suggest that Znt1 serves an essential function of transporting maternal zinc into the embryonic environment during the egg cylinder stage of development, and further suggest that Znt1 plays a role in zinc homeostasis in adult mice.     

The functions of Klarsicht and nuclear lamin in developmentally regulated nuclear migrations of photoreceptor cells in the Drosophila eye

Photoreceptor nuclei in the Drosophila eye undergo developmentally regulated migrations. Nuclear migration is known to require the perinuclear protein Klarsicht, but the function of Klarsicht has been obscure. Here, we show that Klarsicht is required for connecting the microtubule organizing center (MTOC) to the nucleus. In addition, in a genetic screen for klarsicht-interacting genes, we identified Lam Dm(0), which encodes nuclear lamin. We find that, like Klarsicht, lamin is required for photoreceptor nuclear migration and for nuclear attachment to the MTOC. Moreover, perinuclear localization of Klarsicht requires lamin. We propose that nuclear migration requires linkage of the MTOC to the nucleus through an interaction between microtubules, Klarsicht, and lamin. The Klarsicht/lamin interaction provides a framework for understanding the mechanistic basis of human laminopathies.     

Triggering of sarcoplasmic reticulum Ca2+ release and contraction by reverse mode Na+/Ca2+ exchange in trout atrial myocytes

Whole cell patch clamp and intracellular Ca(2+) transients in trout atrial cardiomyocytes were used to quantify calcium release from the sarcoplasmic reticulum (SR) and examine its dependency on the Ca(2+) trigger source. Short depolarization pulses (2-20 ms) elicited large caffeine-sensitive tail currents. The Ca(2+) carried by the caffeine-sensitive tail current after a 2-ms depolarization was 0.56 amol Ca(2+)/pF, giving an SR Ca(2+) release rate of 279 amol Ca(2+). pF(-1). s(-1) or 4.3 mM/s. Depolarizing cells for 10 ms to different membrane potentials resulted in a local maximum of SR Ca(2+) release, intracellular Ca(2+) transient, and cell shortening at 10 mV. Although 100 microM CdCl(2) abolished this local maximum, it had no effect on SR Ca(2+) release elicited by a depolarization to 110 or 150 mV, and the SR Ca(2+) release was proportional to the membrane potential in the range -50 to 150 mV with 100 microM CdCl(2). Increasing the intracellular Na(+) concentration ([Na(+)]) from 10 to 16 mM enhanced SR Ca(2+) release but reduced cell shortening at all membrane potentials examined. In the absence of TTX, SR Ca(2+) release was potentiated with 16 mM but not 10 mM pipette [Na(+)]. Comparison of the total sarcolemmal Ca(2+) entry and the Ca(2+) released from the SR gave a gain factor of 18.6 +/- 7.7. Nifedipine (Nif) at 10 microM inhibited L-type Ca(2+) current (I(Ca)) and reduced the time integral of the tail current by 61%. The gain of the Nif-sensitive SR Ca(2+) release was 16.0 +/- 4.7. A 2-ms depolarization still elicited a contraction in the presence of Nif that was abolished by addition of 10 mM NiCl(2). The gain of the Nif-insensitive but NiCl(2)-sensitive SR Ca(2+) release was 14.8 +/- 7.1. Thus both reverse-mode Na(+)/Ca(2+) exchange (NCX) and I(Ca) can elicit Ca(2+) release from the SR, but I(Ca) is more efficient than reverse-mode NCX in activating contraction. This difference may be due to extrusion of a larger fraction of the Ca(2+) released from the SR by reverse-mode NCX rather than a smaller gain for NCX-induced Ca(2+) release.     

Finite element analysis as a tool for parametric prosthetic foot design and evaluation. Technique development in the solid ankle cushioned heel (SACH) foot

In this study, we developed an approach for prosthetic foot design incorporating motion analysis, mechanical testing and computer analysis. Using computer modeling and finite element analysis, a three-dimensional (3D), numerical foot model of the solid ankle cushioned heel (SACH) foot was constructed and analyzed based upon loading conditions obtained from the gait analysis of an amputee and validated experimentally using mechanical testing. The model was then used to address effects of viscoelastic heel performance numerically. This is just one example of the type of parametric analysis and design enabled by this approach. More importantly, by incorporating the unique gait characteristics of the amputee, these parametric analyses may lead to prosthetic feet more appropriately representing a particular user's needs, comfort and activity level.     

Yolk proteolysis in rainbow trout oocytes after serum-free culture: evidence for a novel biochemical mechanism of atresia in oviparous vertebrates

Our recent studies show little evidence for increased granulosa cell apoptosis during atresia in teleost follicles, in direct contrast to the mammalian model. Histological evidence suggests that atresia in many oviparous vertebrates involves proteolytic degradation of the energy-rich yolk storage proteins within the oocyte. This study tests the hypothesis that physiological conditions that promote atresia (hormone withdrawal) lead to increased lysosomal protease activity in rainbow trout oocytes. We subjected rainbow trout ovarian follicles to conditions that promote atresia (serum-free culture) for up to 72 hr, and measured the activity of lysosomal proteases using routine enzymatic assays. Furthermore, we used high performance liquid chromatography to quantify the increase in free amino acids resulting from proteolysis of yolk proteins. Concomitantly, we evaluated the extent of follicular apoptosis during prolonged serum-free culture, using caspase-3-like activity and DNA fragmentation as indicators of apoptosis. Our results show a significant, time-dependent increase in cathepsin L-like, but not cathepsin D-like, activity levels during culture in serum-free medium; increased cathepsin L-like activity is confirmed by a significant increase in oocyte free amino acid content after 72 hr culture. In contrast, we detected only a transient increase in apoptosis during prolonged serum-free culture, as revealed through both radioactive 3'end-labeling of oligonucleosomal DNA fragments, and caspase-3-like activity. The results of this study provide the first evidence for a novel mechanism of follicular atresia in teleosts involving cathepsin-mediated yolk proteolysis.     

Development of recombinant vesicular stomatitis viruses that exploit defects in host defense to augment specific oncolytic activity

Vesicular stomatitis virus (VSV) is a negative-stranded RNA virus normally sensitive to the antiviral actions of alpha/beta interferon (IFN-alpha/beta). Recently, we reported that VSV replicates to high levels in many transformed cells due, in part, to susceptible cells harboring defects in the IFN system. These observations were exploited to demonstrate that VSV can be used as a viral oncolytic agent to eradicate malignant cells in vivo while leaving normal tissue relatively unaffected. To attempt to improve the specificity and efficacy of this system as a potential tool in gene therapy and against malignant disease, we have genetically engineered VSV that expresses the murine IFN-beta gene. The resultant virus (VSV-IFNbeta) was successfully propagated in cells not receptive to murine IFN-alpha/beta and expressed high levels of functional heterologous IFN-beta. In normal murine embryonic fibroblasts (MEFs), the growth of VSV-IFNbeta was greatly reduced and diminished cytopathic effect was observed due to the production of recombinant IFN-beta, which by functioning in a manner involving autocrine and paracrine mechanisms induced an antiviral effect, preventing virus growth. However, VSV-IFNbeta grew to high levels and induced the rapid apoptosis of transformed cells due to defective IFN pathways being prevalent and thus unable to initiate proficient IFN-mediated host defense. Importantly, VSV expressing the human IFN-beta gene (VSV-hIFNbeta) behaved comparably and, while nonlytic to normal human cells, readily killed their malignant counterparts. Similar to our in vitro observations, following intravenous and intranasal inoculation in mice, recombinant VSV (rVSV)-IFNbeta was also significantly attenuated compared to wild-type VSV or rVSV expressing green fluorescent protein. However, VSV-IFNbeta retained propitious oncolytic activity against metastatic lung disease in immunocompetent animals and was able to generate robust antitumor T-cell responses. Our data indicate that rVSV designed to exploit defects in mechanisms of host defense can provide the basis for new generations of effective, specific, and safer viral vectors for the treatment of malignant and other disease.     

Identification of a functionally critical protein kinase C phosphorylation residue of cardiac troponin T

Cardiac Troponin T (cTnT) is one prominent substrate through which protein kinase C (PKC) exerts its effect on cardiomyocyte function. To determine the specific functional effects of the cTnT PKC-dependent phosphorylation sites (Thr197, Ser201, Thr206, and Thr287) we first mutated these residues to glutamate (E) or alanine (A). cTnT was selectively mutated to generate single, double, triple, and quadruple mutants. Bacterially expressed mutants were evaluated in detergent-treated mouse left ventricular papillary muscle fiber bundles where the endogenous troponin was replaced with a recombinant troponin complex containing either cTnT phosphorylated by PKC-alpha or a mutant cTnT. We simultaneously determined isometric tension development and actomyosin Mg-ATPase activity of the exchanged fiber bundles as a function of Ca2+ concentration. Our systematic analysis of the functional role of the multiple PKC phosphorylation sites on cTnT identified a localized region that controls maximum tension, ATPase activity, and Ca2+ sensitivity of the myofilaments. An important and novel finding of our study was that Thr206 is a functionally critical cTnT PKC phosphorylation residue. Its exclusive phosphorylation by PKC-alpha or replacement by Glu (mimicking phosphorylation) significantly decreased maximum tension, actomyosin Mg-ATPase activity, myofilament Ca2+ sensitivity, and cooperativity. On the other hand the charge modification of the other three residues together (T197/S201/T287-E) had no functional effect. Fibers bundles containing phosphorylated cTnT-wt (but not the T197/S201/T206/T287-E) exhibited a significant decrease of tension cost as compared with cTnT-wt.     

Crystallization of beta-galactosidase does not reduce the range of activity of individual molecules

By use of a capillary electrophoresis-based procedure, it is possible to measure the activity of individual molecules of beta-galactosidase. Molecules from the crystallized enzyme as well as the original enzyme preparation used to grow the crystals both displayed a range of activity of 20-fold or greater. beta-Galactosidase molecules obtained from two different crystals had indistinguishable activity distributions of 31,600 +/- 1100 and 31,800 +/- 1100 reactions min(-1) (enzyme molecule)(-1). This activity was found to be significantly different from that of the enzyme used to grow the crystals, which showed an activity distribution of 38,500 +/- 900 reactions min(-1) (enzyme molecule)(-1).     

Effect of temperature and the F27W mutation on the Ca2+ activated structural transition of trout cardiac troponin C

The Ca(2+) sensitivity of cardiac contractile element is reduced at lower temperatures, in contrast to that in fast skeletal muscle. Cardiac troponin C (cTnC) replacement in mammalian skinned fibers showed that TnC plays a critical role in this phenomenon (Harrison and Bers, (1990), Am. J. Physiol. 258, C282-8). Understanding the differences in affinity and structure between cTnCs from cold-adapted ectothermic species and mammals may bring new insights into how the different isoforms provide different resistances to cold. We followed the Ca(2+) titration to the regulatory domain of rainbow trout cTnC by NMR (wild type at 7 and 30 degrees C and F27W mutant at 30 degrees C) and fluorescence (F27W mutant, at 7 and 30 degrees C) spectroscopies. Using NMR spectroscopy, we detected Ca(2+) binding to site I of trout cTnC at high concentrations. This places trout cTnC between mammalian cTnC, in which site I is completely inactive, and skeletal TnC, in which site I binds Ca(2+) during muscle activation, and which is not as much affected by lower temperatures. This binding was seen both at 7 and at 30 degrees C. Despite the low Ca(2+) affinity, trout TnC site I may increase the likelihood of an opening of the regulatory domain, thus increasing the affinity for TnI. This way, it may be responsible for trout cTnC's capacity to function at lower temperatures.