Cytodifferentiation of the crayfish spermatozoon: acrosome formation,transformation of mitochondria and development of microtubules

Summary During spermiogenesis in the crayfish, the acrosome, mitochondrial derivatives and the centrioles are retained within the admixed nucleoplasm and cytoplasm (spermioplasm). Fused nuclear and plasma membranes form the tegument that invests the spermioplasm. A well-defined system of small tubules that originate during spermiogenesis from densities surrounding the centrioles also defines the axes of the nuclear processes in the mature spermatozoon. These tubules are larger in diameter than the microtubules in adjacent interstitial cells and their development coincides with the formation and extension of the nuclear processes. The small tubules seem related to the changes in the cell accompanying nucleoplasmic streaming and to the growth and stabilization of form of the elongate, assymmetric nuclear processes.The mitochondria of spermatocytes are transformed into membranous lamellae that lie in the spermioplasm of the mature spermatozoon, and may by oxidative phosphorylation or some alternative pathway provide energy for metabolic activity and motility.The apical cap of the mature acrosome of the crayfish spermatozoon is enveloped by a sheath of PAS-positive material. The acrosomal process is attached to a dense crescent-shaped acrosome embedded in the spermioplasm. A fine granular substance at the base of the acrosome gives rise to beaded filaments that radiate into the central acrosomal concavity.This study was supported by Grants CA-04046, GM-08380 and GM-00582 from the United States Public Health Service.     

Angiopoietin: a TIE(d) balance in tumor angiogenesis

Angiopoietins (ANG-1 and ANG-2) and their TIE-2 receptor tyrosine kinase have wide-ranging effects on tumor malignancy that includes angiogenesis, inflammation, and vascular extravasation. These multifaceted pathways present a valuable opportunity in developing novel inhibition strategies for cancer treatment. However, the regulatory role of ANG-1 and ANG-2 in tumor angiogenesis remains controversial. There is a complex interplay between complementary yet conflicting roles of both the ANGs in shaping the outcome of angiogenesis. Embryonic vascular development suggests that ANG-1 is crucial in engaging interaction between endothelial and perivascular cells. However, recruitment of perivascular cells by ANG-1 has recently been implicated in its antiangiogenic effect on tumor growth. It is becoming clear that TIE-2 signaling may function in a paracrine and autocrine manner directly on tumor cells because the receptor has been increasingly found in tumor cells. In addition, alpha(5)beta(1) and alpha(v)beta(5) integrins were recently recognized as functional receptors for ANG-1 and ANG-2. Therefore, both the ligands may have wide-ranging functions in cellular activities that affect overall tumor development. Collectively, these TIE-2-dependent and TIE-2-independent activities may account for the conflicting findings of ANG-1 and ANG-2 in tumor angiogenesis. These uncertainties have impeded development of a clear strategy to target this important angiogenic pathway. A better understanding of the molecular basis of ANG-1 and ANG-2 activity in the pathophysiologic regulation of angiogenesis may set the stage for novel therapy targeting this pathway.     

Simplified ontologies allowing comparison of developmental mammalian gene expression

Model organisms represent an important resource for understanding the fundamental aspects of mammalian biology. Mapping of biological phenomena between model organisms is complex and if it is to be meaningful, a simplified representation can be a powerful means for comparison. The Developmental eVOC ontologies presented here are simplified orthogonal ontologies describing the temporal and spatial distribution of developmental human and mouse anatomy. We demonstrate the ontologies by identifying genes showing a bias for developmental brain expression in human and mouse.     

Modulation of TRPV1 by nonreceptor tyrosine kinase, c-Src kinase

The capsaicin receptor TRPV1 is a nonselective cation channel that is expressed in sensory neurons. In this study, we examined the role of the nonreceptor cellular tyrosine kinase c-Src kinase in the modulation of the rat TRPV1. Capsaicin-induced currents in identified colonic dorsal root ganglion neurons were blocked by the c-Src kinase inhibitor PP2 and enhanced by the tyrosine phosphatase inhibitor sodium orthovandate. PP2 also abolished currents in human embryonic kidney-293 cells transfected with rat TRPV1, whereas cotransfection of TRPV1 with v-Src resulted in fivefold increase in capsaicin-induced currents. In cells transfected with dominant-negative c-Src and TRPV1, capsaicin-induced currents were decreased by approximately fourfold. TRPV1 co-immunoprecipitated with Src kinase and was tyrosine phosphorylated. These studies demonstrate that TRPV1 is a potential target for cellular tyrosine kinase-dependent phosphorylation.     

Production of cloned pigs by whole-cell intracytoplasmic microinjection

Cloning by somatic cell nuclear transfer has been successfully achieved by both fusing of a donor cell with and injecting an isolated donor cell nucleus into an enucleated oocyte. However, each of the above methods involves extended manipulation of either the oocytes (fusion) or the donor cells (nucleus isolation). Additionally, cloning efficiency can be reduced by low fusion rate of the cell fusion method, and specialized micromanipulation equipment and exacting nucleus isolation techniques are required for the nucleus injection method. Here we report a whole-cell injection technique for nuclear transfer in pigs and the production of cloned piglets with comparable, if not higher, efficiency than the other two nuclear transfer procedures. First, we tested the feasibility of this technique with three types of frequently used donor cells (cumulus, mural granulosa, and fibroblasts) and obtained the optimal nuclear reprogramming conditions for these cells. We further improved our protocol by avoiding ultraviolet exposure during enucleation and achieved a 37% blastocyst rate. We then conducted whole-cell injection using skin fibroblasts from the ear of a sow transgenic for two genes, the porcine lactoferrin and the human factor IX, and produced four live-born cloned transgenic piglets from three recipients. The present study demonstrated the applicability of producing normal, cloned piglets by the simple and less labor-intensive whole-cell intracytoplasmic injection.     

Chemoresistance in human ovarian cancer: the role of apoptotic regulators

Ovarian cancer is among the most lethal of all malignancies in women. While chemotherapy is the preferred treatment modality, chemoresistance severely limits treatment success. Recent evidence suggests that deregulation of key pro- and anti-apoptotic pathways is a key factor in the onset and maintenance of chemoresistance. Furthermore, the discovery of novel interactions between these pathways suggests that chemoresistance may be multi-factorial. Ultimately, the decision of the cancer cell to live or die in response to a chemotherapeutic agent is a consequence of the overall apoptotic capacity of that cell. In this review, we discuss the biochemical pathways believed to promote cell survival and how they modulate chemosensitivity. We then conclude with some new research directions by which the fundamental mechanisms of chemoresistance can be elucidated.     

Angiopoietin 1 promotes tumor angiogenesis and tumor vessel plasticity of human cervical cancer in mice

We have previously shown that following psoralen photoactivation (PUVA treatment) human dermal fibroblasts undergo long-term growth arrest as well as morphological and functional changes reminiscent of cellular senescence [ 1 ]. In the absence of molecular data on what constitutes normal senescence, it has been difficult to decide whether these PUVA-induced changes reflect cellular senescence or rather a mimic thereof. We herein report that PUVA-induced growth arrest, the senescent phenotype with long-term induction of senescence-associated beta-galactosidase, as well as increased expression of matrix metalloprotease-1 are fully reversible at days 100 to 130 post PUVA treatment in four independently tested fibroblast strains. The late returning growth capacity in PUVA-treated fibroblasts is not due to immortalization, as shown by continued lack of telomerase activity, accelerated telomere shortening, and a decrease in overall growth rates in fibroblasts in their regrowing phase post PUVA treatment. Lack of anchorage-independent growth additionally suggests that the cells are also not tumorigenically transformed. Collectively, our data suggest that PUVA-induced changes do not fully reflect replicative senescence but rather represent a long-term transient phenocopy of senescence. The model reported here is particularly suited to elucidating mechanisms underlying long-term transient growth arrest, the related functional changes, and the release of cells thereof.