Preformation and epigenesis converge to specify primordial germ cell fate in the early Drosophila embryo

A critical step in animal development is the specification of primordial germ cells (PGCs), the precursors of the germline. Two seemingly mutually exclusive mechanisms are implemented across the animal kingdom: epigenesis and preformation. In epigenesis, PGC specification is non-autonomous and depends on extrinsic signaling pathways. The BMP pathway provides the key PGC specification signals in mammals. Preformation is autonomous and mediated by determinants localized within PGCs. In Drosophila, a classic example of preformation, constituents of the germ plasm localized at the embryonic posterior are thought to be both necessary and sufficient for proper determination of PGCs. Contrary to this longstanding model, here we show that these localized determinants are insufficient by themselves to direct PGC specification in blastoderm stage embryos. Instead, we find that the BMP signaling pathway is required at multiple steps during the specification process and functions in conjunction with components of the germ plasm to orchestrate PGC fate.     

The last exon of SNAP-23 regulates granule exocytosis from mast cells

SNAP-25 and its ubiquitous homolog SNAP-23 are members of the SNARE family of proteins that regulate membrane fusion during exocytosis. Although SNAP-23 has been shown to participate in a variety of intracellular transport processes, the structural domains of SNAP-23 that are required for its interaction with other SNAREs have not been determined. By employing deletion mutagenesis we found that deletion of the amino-terminal 18 amino acids of SNAP-23 (encoded in the first exon) dramatically inhibited binding of SNAP-23 to both the target SNARE syntaxin and the vesicle SNARE vesicle-associated membrane protein(VAMP). By contrast, deletion of the carboxyl-terminal 23 amino acids (encoded in the last exon) of SNAP-23 does not affect SNAP-23 binding to syntaxin but profoundly inhibits its binding to VAMP. To determine the functional relevance of the modular structure of SNAP-23, we overexpressed SNAP-23 in cells possessing the capacity to undergo regulated exocytosis. Expression of human SNAP-23 in a rat mast cell line significantly enhanced exocytosis, and this effect was not observed in transfectants expressing the carboxyl-terminal VAMP-binding mutant of SNAP-23. Despite considerable amino acid identity, we found that human SNAP-23 bound to SNAREs more efficiently than did rat SNAP-23. These data demonstrate that the introduction of a "better" SNARE binder into secretory cells augments exocytosis and defines the carboxyl terminus of SNAP-23 as an essential regulator of exocytosis in mast cells.     

Clathrin-dependent and -independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways

Sphingolipids (SLs) are plasma membrane constituents in eukaryotic cells which play important roles in a wide variety of cellular functions. However, little is known about the mechanisms of their internalization from the plasma membrane or subsequent intracellular targeting. We have begun to study these issues in human skin fibroblasts using fluorescent SL analogues. Using selective endocytic inhibitors and dominant negative constructs of dynamin and epidermal growth factor receptor pathway substrate clone 15, we found that analogues of lactosylceramide and globoside were internalized almost exclusively by a clathrin-independent ("caveolar-like") mechanism, whereas an analogue of sphingomyelin was taken up approximately equally by clathrin-dependent and -independent pathways. We also showed that the Golgi targeting of SL analogues internalized via the caveolar-like pathway was selectively perturbed by elevated intracellular cholesterol, demonstrating the existence of two discrete Golgi targeting pathways. Studies using SL-binding toxins internalized via clathrin-dependent or -independent mechanisms confirmed that endogenous SLs follow the same two pathways. These findings (a) provide a direct demonstration of differential SLs sorting into early endosomes in living cells, (b) provide a "vital marker" for endosomes derived from caveolar-like endocytosis, and (c) identify two independent pathways for lipid transport from the plasma membrane to the Golgi apparatus in human skin fibroblasts.     

Monomodal rigid-body registration and applications to the investigation of the effects of eicosapentaenoic acid intervention in neuropsychiatric disorders

The technique of monomodal rigid-body registration of serial magnetic resonance scans based on the sinc ((sin z)/z) interpolation function and its application to neuropsychiatric disorders, such as schizophrenia, depression and Huntington's disease, in relation to the assessment of the cerebral effects of intervention with the n-3 highly unsaturated fatty acid eicosapentaenoic acid are described. The evidence thus far indicates that researchers investigating the benefits of treatment with essential fatty acids in neuropsychiatric disorders should consider utilizing this technique.     

Sid4p-Cdc11p assembles the septation initiation network and its regulators at the S. pombe SPB

The Schizosaccharomyces pombe septation initiation network (SIN) triggers actomyosin ring constriction, septation, and cell division. It is organized at the spindle pole body (SPB) by the scaffold proteins Sid4p and Cdc11p. Here, we dissect the contributions of Sid4p and Cdc11p in anchoring SIN components and SIN regulators to the SPB. We find that Sid4p interacts with the SIN activator, Plo1p, in addition to Cdc11p and Dma1p. While the C terminus of Cdc11p is involved in binding Sid4p, its N-terminal half is involved in a wide variety of direct protein-protein interactions, including those with Spg1p, Sid2p, Cdc16p, and Cdk1p-Cdc13p. Given that the localizations of the remaining SIN components depend on Spg1p or Cdc16p, these data allow us to build a comprehensive model of SIN component organization at the SPB. FRAP experiments indicate that Sid4p and Cdc11p are stable SPB components, whereas signaling components of the SIN are dynamically associated with these structures. Our results suggest that the Sid4p-Cdc11p complex organizes a signaling hub on the SPB and that this hub coordinates cell and nuclear division.     

Simian virus-40 as a gene therapy vector

Simian virus-40 (SV40), an icosahedral papovavirus, has recently been modified to serve as a gene delivery vector. Recombinant SV40 vectors (rSV40) are good candidates for gene transfer, as they display some unique features: SV40 is a well-known virus, nonreplicative vectors are easy-to-make, and can be produced in titers of 10(12) IU/ml. They also efficiently transduce both resting and dividing cells, deliver persistent transgene expression to a wide range of cell types, and are nonimmunogenic. Present disadvantages of rSV40 vectors for gene therapy are a small cloning capacity and the possible risks related to random integration of the viral genome into the host genome. Considerable efforts have been devoted to modifing this virus and setting up protocols for viral production. Preliminary therapeutic results obtained both in tissue culture cells and in animal models for heritable and acquired diseases indicate that rSV40 vectors are promising gene transfer vehicles. This article reviews the work performed with SV40 viruses as recombinant vectors for gene transfer. A summary of the structure, genomic organization, and life cycle of wild-type SV40 viruses is presented. Furthermore, the strategies utilized for the development, production, and titering of rSV40 vectors are discussed. Last, the therapeutic applications developed to date are highlighted.     

Hemozoin formation in malaria: a two-step process involving histidine-rich proteins and lipids

Major blood stage antimalarial drugs like chloroquine and artemisinin target the heme detoxification process of the malaria parasite. Hemozoin formation reactions in vitro using the Plasmodium falciparum histidine-rich protein-2 (Pfhrp-2), lipids, and auto-catalysis are slow and could not explain the speed of detoxification needed for parasite survival. Here, we show that malarial hemozoin formation is a coordinated two component process involving both lipids and histidine-rich proteins. Hemozoin formation efficiency in vitro is 1-2% with Pfhrp-2 and 0.25-0.5% with lipids. We added lipids after 9h in a 12h Pfhrp-2 mediated reaction that resulted in sixfold increase in hemozoin formation. However, a lipid mediated reaction in which Pfhrp-2 was added after 9h produced only twofold increase in hemozoin production compared to the reaction with Pfhrp-2 alone. Synthetic peptides corresponding to the Pfhrp-2 heme binding sequences, based on repeats of AHHAAD, neither alone nor in combination with lipids were able to generate hemozoin in vitro. These results indicate that hemozoin formation in malaria parasite involves both the lipids and the scaffolding proteins. Histidine-rich proteins might facilitate hemozoin formation by binding with a large number of heme molecules, and facilitating the dimer formation involving iron-carboxylate bond between two heme molecules, and lipids may then subsequently assist the mechanism of long chain formation, held together by hydrogen bonds or through extensive networking of hydrogen bonds.     

Effects of an antiprogestin onapristone on the endometrium of bonnet monkeys: morphometric and ultrastructural studies

Our previous studies demonstrated the ability of low doses of antiprogestin ZK 98.299 (onapristone) to inhibit fertility in bonnet monkeys. In the present study cumulative effects of low doses of ZK 98.299 on the endometrial cytoarchitecture of bonnet monkeys were analyzed. Treatment with either the vehicle (n = 3) or onapristone at 2.5 mg (n = 4) or 5.0 mg (n = 3) was initiated on Day 5 of the first menstrual cycle and thereafter repeated every third day for four to seven consecutive cycles. The last treatment cycles were anovulatory in two animals treated with 2.5 mg and all animals treated with 5.0 mg. Endometrial biopsies were collected on Day 8 after the midcycle estradiol peak in ovulatory menstrual cycles and on Day 20 in anovulatory menstrual cycles during the last treatment cycle. Ultrathin sections of the fixed endometrium were stained with toluidine blue for morphometric analysis and uranyl acetate and lead citrate for ultrastructural analysis. The ZK 98.299-treated animals showed a dose-dependent endometrial atrophy as evident by a decrease in the height and diameter of the glands and early signs of compaction in the stroma. Ultrastructural analysis also revealed dose-dependent degenerative changes in the subcellular organelles such as the nucleus, mitochondria, endoplasmic reticulum, lysosomes, and Golgi apparatus. This suggests that long-term treatment with low doses of ZK 98.299 leads to the suppression of estrogen-dependent endometrial proliferation. However, this blockade operates independent of estradiol receptor (ER) and progesterone receptor (PR) concentrations as the expressions of these steroid receptors did not show any significant changes even after prolonged treatment. The study demonstrated an antiestrogenic effect of ZK 98.299 on endometrium after prolonged treatment in bonnet monkeys.     

Pneumocystis carinii BCK1 functions in a mitogen-activated protein kinase cascade regulating fungal cell-wall assembly

Pneumocystis pneumonia remains the most common AIDS-defining opportunistic infection in people with HIV. The process by which Pneumocystis carinii constructs its cell wall is not well known, although recent studies reveal that molecules such as beta-1-3-glucan synthetase (GSC1) and environmental pH-responsive genes such as PHR1 are important for cell-wall integrity. In closely related fungi, a specific mitogen-activated protein kinase (MAPK) cascade regulates cell-wall assembly in response to elevated temperature. The upstream mitogen-activated protein kinase kinase kinase (MAPKKK, or MEKK), BCK1, is an essential component in this pathway for maintaining cell-wall integrity and preventing fungal cell lysis. We have identified a P. carinii MEKK gene and have expressed it in Saccharomyces cerevisiae to gain insights into its function. The P. carinii MEKK, PCBCK1, corrects the temperature-sensitive cell lysis defect of bck1Delta yeast. Further, at elevated temperature PCBCK1 restored the signaling defect in bck1Delta yeast to maintain expression of the temperature-inducible beta-1-3-glucan synthetase gene, FKS2. PCBCK1, as a functional kinase, is capable of autophosphorylation and substrate phosphorylation. Since glucan machinery is not present in mammals, a better understanding of this pathway in P. carinii might aid in the development of novel medications which interfere with the integrity of the Pneumocystis cell wall.