Saccharomyces cerevisiae Cells with Defective Spindle Pole Body Outer Plaques Accomplish Nuclear Migration via Half-Bridge–organized Microtubules

Cnm67p, a novel yeast protein, localizes to the microtubule organizing center, the spindle pole body (SPB). Deletion of CNM67 (YNL225c) frequently results in spindle misorientation and impaired nuclear migration, leading to the generation of bi- and multinucleated cells (40%). Electron microscopy indicated that CNM67 is required for proper formation of the SPB outer plaque, a structure that nucleates cytoplasmic (astral) microtubules. Interestingly, cytoplasmic microtubules that are essential for spindle orientation and nuclear migration are still present in cnm67Δ1 cells that lack a detectable outer plaque. These microtubules are attached to the SPB half- bridge throughout the cell cycle. This interaction presumably allows for low-efficiency nuclear migration and thus provides a rescue mechanism in the absence of a functional outer plaque. Although CNM67 is not strictly required for mitosis, it is essential for sporulation. Time-lapse microscopy of cnm67Δ1 cells with green fluorescent protein (GFP)-labeled nuclei indicated that CNM67 is dispensable for nuclear migration (congression) and nuclear fusion during conjugation. This is in agreement with previous data, indicating that cytoplasmic microtubules are organized by the half-bridge during mating.

Thrombospondin-1 Induces Tyrosine Phosphorylation of Adherens Junction Proteins and Regulates an Endothelial Paracellular Pathway

Thrombospondin-1 (TSP) induces endothelial cell (EC) actin reorganization and focal adhesion disassembly and influences multiple EC functions. To determine whether TSP might regulate EC–EC interactions, we studied the effect of exogenous TSP on the movement of albumin across postconfluent EC monolayers. TSP increased transendothelial albumin flux in a dose-dependent manner at concentrations ≥1 μg/ml (2.2 nM). Increases in albumin flux were observed as early as 1 h after exposure to 30 μg/ml (71 nM) TSP. Inhibition of tyrosine kinases with herbimycin A or genistein protected against the TSP-induced barrier dysfunction by >80% and >50%, respectively. TSP-exposed monolayers exhibited actin reorganization and intercellular gap formation, whereas pretreatment with herbimycin A protected against this effect. Increased staining of phosphotyrosine-containing proteins was observed in plaque-like structures and at the intercellular boundaries of TSP-treated cells. In the presence of protein tyrosine phosphatase inhibition, TSP induced dose- and time-dependent increments in levels of phosphotyrosine-containing proteins; these TSP dose and time requirements were compatible with those defined for EC barrier dysfunction. Phosphoproteins that were identified include the adherens junction proteins focal adhesion kinase, paxillin, γ-catenin, and p120Cas. These combined data indicate that TSP can modulate endothelial barrier function, in part, through tyrosine phosphorylation of EC proteins.

High-Voltage Electron Tomography of Spindle Pole Bodies and Early Mitotic Spindles in the Yeast Saccharomyces cerevisiaeV⃞

The spindle pole body (SPB) is the major microtubule-organizing center of budding yeast and is the functional equivalent of the centrosome in higher eukaryotic cells. We used fast-frozen, freeze-substituted cells in conjunction with high-voltage electron tomography to study the fine structure of the SPB and the events of early spindle formation. Individual structures were imaged at 5–10 nm resolution in three dimensions, significantly better than can be achieved by serial section electron microscopy. The SPB is organized in distinct but coupled layers, two of which show ordered two-dimensional packing. The SPB central plaque is anchored in the nuclear envelope with hook-like structures. The minus ends of nuclear microtubules (MTs) are capped and are tethered to the SPB inner plaque, whereas the majority of MT plus ends show a distinct flaring. Unbudded cells containing a single SPB retain 16 MTs, enough to attach to each of the expected 16 chromosomes. Their median length is ∼150 nm. MTs growing from duplicated but not separated SPBs have a median length of ∼130 nm and interdigitate over the bridge that connects the SPBs. As a bipolar spindle is formed, the median MT length increases to ∼300 nm and then decreases to ∼30 nm in late anaphase. Three-dimensional models confirm that there is no conventional metaphase and that anaphase A occurs. These studies complement and extend what is known about the three-dimensional structure of the yeast mitotic spindle and further our understanding of the organization of the SPB in intact cells.

A Genetic Analysis of Interactions with Spc110p Reveals Distinct Functions of Spc97p and Spc98p, Components of the Yeast γ-Tubulin Complex

The spindle pole body (SPB) in Saccharomyces cerevisiae functions as the microtubule-organizing center. Spc110p is an essential structural component of the SPB and spans between the central and inner plaques of this multilamellar organelle. The amino terminus of Spc110p faces the inner plaque, the substructure from which spindle microtubules radiate. We have undertaken a synthetic lethal screen to identify mutations that enhance the phenotype of the temperature-sensitive spc110–221 allele, which encodes mutations in the amino terminus. The screen identified mutations in SPC97 and SPC98, two genes encoding components of the Tub4p complex in yeast. The spc98–63 allele is synthetic lethal only with spc110 alleles that encode mutations in the N terminus of Spc110p. In contrast, the spc97 alleles are synthetic lethal with spc110 alleles that encode mutations in either the N terminus or the C terminus. Using the two-hybrid assay, we show that the interactions of Spc110p with Spc97p and Spc98p are not equivalent. The N terminus of Spc110p displays a robust interaction with Spc98p in two different two-hybrid assays, while the interaction between Spc97p and Spc110p is not detectable in one strain and gives a weak signal in the other. Extra copies of SPC98 enhance the interaction between Spc97p and Spc110p, while extra copies of SPC97 interfere with the interaction between Spc98p and Spc110p. By testing the interactions between mutant proteins, we show that the lethal phenotype in spc98–63 spc110–221 cells is caused by the failure of Spc98–63p to interact with Spc110–221p. In contrast, the lethal phenotype in spc97–62 spc110–221 cells can be attributed to a decreased interaction between Spc97–62p and Spc98p. Together, these studies provide evidence that Spc110p directly links the Tub4p complex to the SPB. Moreover, an interaction between Spc98p and the amino-terminal region of Spc110p is a critical component of the linkage, whereas the interaction between Spc97p and Spc110p is dependent on Spc98p.

A Mutational Analysis Identifies Three Functional Regions of the Spindle Pole Component Spc110p in Saccharomyces cerevisiae

The central coiled coil of the essential spindle pole component Spc110p spans the distance between the central and inner plaques of the Saccharomyces cerevisiae spindle pole body (SPB). The carboxy terminus of Spc110p, which binds calmodulin, resides at the central plaque, and the amino terminus resides at the inner plaque from which nuclear microtubules originate. To dissect the functions of Spc110p, we created temperature-sensitive mutations in the amino and carboxy termini. Analysis of the temperature-sensitive spc110 mutations and intragenic complementation analysis of the spc110 alleles defined three functional regions of Spc110p. Region I is located at the amino terminus. Region II is located at the carboxy-terminal end of the coiled coil, and region III is the previously defined calmodulin-binding site. Overexpression of SPC98 suppresses the temperature sensitivity conferred by mutations in region I but not the phenotypes conferred by mutations in the other two regions, suggesting that the amino terminus of Spc110p is involved in an interaction with the γ-tubulin complex composed of Spc97p, Spc98p, and Tub4p. Mutations in region II lead to loss of SPB integrity during mitosis, suggesting that this region is required for the stable attachment of Spc110p to the central plaque. Our results strongly argue that Spc110p links the γ-tubulin complex to the central plaque of the SPB.

Modulation of the baboon (Papio anubis) uterine endometrium by chorionic gonadotrophin during the period of uterine receptivity

This study was undertaken to determine the modulation of uterine function by chorionic gonadotrophin (CG) in a nonhuman primate. Infusion of recombinant human CG (hCG) between days 6 and 10 post ovulation initiated the endoreplication of the uterine surface epithelium to form distinct epithelial plaques. These plaque cells stained intensely for cytokeratin and the proliferating cell nuclear antigen. The stromal fibroblasts below the epithelial plaques stained positively for α-smooth muscle actin (αSMA). Expression of αSMA is associated with the initiation of decidualization in the baboon endometrium. Synthesis of the glandular secretory protein glycodelin, as assessed by Western blot analysis, was markedly up-regulated by hCG, and this increase was confirmed by immunocytochemistry, Northern blot analysis, and reverse transcriptase-PCR. To determine whether hCG directly modulated these uterine responses, we treated ovariectomized baboons sequentially with estradiol and progesterone to mimic the hormonal profile of the normal menstrual cycle. Infusion of hCG into the oviduct of steroid-hormone-treated ovariectomized baboons induced the expression of αSMA in the stromal cells and glycodelin in the glandular epithelium. The epithelial plaque reaction, however, was not readily evident. These studies demonstrate a physiological effect of CG on the uterine endometrium in vivo and suggest that the primate blastocyst signal, like the blastocyst signals of other species, modulates the uterine environment prior to implantation.

Spc29p is a component of the Spc110p subcomplex and is essential for spindle pole body duplication

In yeast, microtubules are organized by the spindle pole body (SPB). The SPB is a disk-like multilayered structure that is embedded in the nuclear envelope via its central plaque, whereas the outer and inner plaques are exposed to the cytoplasm and nucleoplasm, respectively. How the SPB assembles is poorly understood. We show that the inner/central plaque is composed of a stable SPB subcomplex, containing the γ-tubulin complex-binding protein Spc110p, calmodulin, Spc42p, and Spc29p. Spc29p acts as a linker between the central plaque component Spc42p and the inner plaque protein Spc110p. Evidence is provided that the calmodulin-binding site of Spc110p influences the binding of Spc29p to Spc110p. Spc42p also was identified as a component of a cytoplasmic SPB subcomplex containing Spc94p/Nud1p, Cnm67p, and Spc42p. Spc29p and Spc42p may be part of a critical interface of nucleoplasmic and cytoplasmic assembled SPB subcomplexes that form during SPB duplication. In agreement with this, overexpressed Spc29p was found to be a nuclear protein, whereas Spc42p is cytoplasmic. In addition, an essential function of SPC29 during SPB assembly is indicated by the SPB duplication defect of conditional lethal spc29(ts) cells and by the genetic interaction of SPC29 with CDC31 and KAR1, two genes that are involved in SPB duplication.

Phosphate is a specific signal for induction of osteopontin gene expression

Osteopontin is a phosphorylated glycoprotein secreted to the mineralizing extracellular matrix by osteoblasts during bone development. It is believed to facilitate the attachment of osteoblasts and osteoclasts to the extracellular matrix, allowing them to perform their respective functions during osteogenesis. Several other functions have been suggested for this protein, and its up-regulation is associated with various disease states related to calcification, including arterial plaque formation and the formation of kidney stones. Although expression of this gene has been demonstrated in multiple tissues, its regulation is not well understood. Our previous studies on the roles of the retinoblastoma protein (pRB) and p300/CBP in the regulation of osteoblast differentiation revealed a link between osteopontin induction and the synthesis of alkaline phosphatase. In this paper, we describe results specifically linking induction of osteopontin to the enzymatic activity of alkaline phosphatase in the medium, which results in the generation of free phosphate. This elevation of free phosphate in the medium is sufficient to signal induction of osteopontin RNA and protein. The strong and specific induction of osteopontin in direct response to increased phosphate levels provides a mechanism to explain how expression of this product is normally regulated in bone and suggests how it may become up-regulated in damaged tissue.

Opposite roles of apolipoprotein E in normal brains and in Alzheimer’s disease

We have characterized the interaction between apolipoprotein E (apoE) and amyloid β peptide (Aβ) in the soluble fraction of the cerebral cortex of Alzheimer’s disease (AD) and control subjects. Western blot analysis with specific antibodies identified in both groups a complex composed of the full-length apoE and Aβ peptides ending at residues 40 and 42. The apoE–Aβ soluble aggregate is less stable in AD brains than in controls, when treated with the anionic detergent SDS. The complex is present in significantly higher quantity in control than in AD brains, whereas in the insoluble fraction an inverse correlation has previously been reported. Moreover, in the AD subjects the Aβ bound to apoE is more sensitive to protease digestion than is the unbound Aβ. Taken together, our results indicate that in normal brains apoE efficiently binds and sequesters Aβ, preventing its aggregation. In AD, the impaired apoE–Aβ binding leads to the critical accumulation of Aβ, facilitating plaque formation.

Anti-DNA antibodies are a major component of the intrathecal B cell response in multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of unknown cause that afflicts the central nervous system. MS is typified by a highly clonally restricted antigen-driven antibody response that is confined largely to the central nervous system. The major antigenic targets of this response and the role of antibody in disease pathogenesis remain unclear. To help resolve these issues, we cloned the IgG repertoire directly from active plaque and periplaque regions in MS brain and from B cells recovered from the cerebrospinal fluid of a patient with MS with subacute disease. We found that high-affinity anti-DNA antibodies are a major component of the intrathecal IgG response in the patients with MS that we studied. Furthermore, we show DNA-specific monoclonal antibodies rescued from two subjects with MS as well as a DNA-specific antibody rescued from an individual suffering from systemic lupus erythematosus bound efficiently to the surface of neuronal cells and oligodendrocytes. For two of these antibodies, cell-surface recognition was DNA dependent. Our findings indicate that anti-DNA antibodies may promote important neuropathologic mechanisms in chronic inflammatory disorders, such as MS and systemic lupus erythematosus.

Acceleration of atherogenesis by COX-1-dependent prostanoid formation in low density lipoprotein receptor knockout mice

The cyclooxygenase (COX) product, prostacyclin (PGI2), inhibits platelet activation and vascular smooth-muscle cell migration and proliferation. Biochemically selective inhibition of COX-2 reduces PGI2 biosynthesis substantially in humans. Because deletion of the PGI2 receptor accelerates atherogenesis in the fat-fed low density lipoprotein receptor knockout mouse, we wished to determine whether selective inhibition of COX-2 would accelerate atherogenesis in this model. To address this hypothesis, we used dosing with nimesulide, which inhibited COX-2 ex vivo, depressed urinary 2,3 dinor 6-keto PGF1α by approximately 60% but had no effect on thromboxane formation by platelets, which only express COX-1. By contrast, the isoform nonspecific inhibitor, indomethacin, suppressed platelet function and thromboxane formation ex vivo and in vivo, coincident with effects on PGI2 biosynthesis indistinguishable from nimesulide. Indomethacin reduced the extent of atherosclerosis by 55 ± 4%, whereas nimesulide failed to increase the rate of atherogenesis. Despite their divergent effects on atherogenesis, both drugs depressed two indices of systemic inflammation, soluble intracellular adhesion molecule-1, and monocyte chemoattractant protein-1 to a similar but incomplete degree. Neither drug altered serum lipids and the marked increase in vascular expression of COX-2 during atherogenesis. Accelerated progression of atherosclerosis is unlikely during chronic intake of specific COX-2 inhibitors. Furthermore, evidence that COX-1-derived prostanoids contribute to atherogenesis suggests that controlled evaluation of the effects of nonsteroidal anti-inflammatory drugs and/or aspirin on plaque progression in humans is timely.

Local gene transfer of tissue factor pathway inhibitor regulates intimal hyperplasia in atherosclerotic arteries

Tissue factor (TF), the initiator of blood coagulation and thrombosis, is up-regulated after vascular injury and in atherosclerotic states. Systemic administration of recombinant TF pathway inhibitor (TFPI) has been reported to decrease intimal hyperplasia after vascular injury and also to suppress systemic mechanisms of blood coagulation and thrombosis. Here we report that, in heritable hyperlipidemic Watanabe rabbits, adenoviral gene transfer of TFPI to balloon-injured atherosclerotic arteries reduced the extent of intimal hyperplasia by 43% (P < 0.05) compared with a control vector used at identical titer (1 × 1010 plaque-forming units/ml). Platelet aggregation and coagulation studies performed 7 days after local gene transfer of TFPI failed to show any impairment in systemic hemostasis. At time of sacrifice, 4 weeks after vascular injury, the 10 Ad-TFPI treated carotid arteries were free of thrombi, whereas two control-treated arteries were occluded (P, not significant). These findings suggest that TFPI overexpressed in atherosclerotic arteries can regulate hyperplastic response to injury in the absence of changes in the hemostatic system, establishing a role for local TF regulation as target for gene transfer-based antirestenosis therapies.

Comparative assessment of virulence of recombinant vaccinia viruses expressing IL-2 and IL-15 in immunodeficient mice

IL-2 and -15 belong to the four α-helix bundle family of cytokines and display a spectrum of overlapping immune functions because of shared signal transducing receptor components of the IL-2 receptor complex. However, recent evidence suggests a nonredundant unique role for IL-15 in the establishment and perhaps maintenance of peripheral natural killer (NK) cell populations in vivo. To explore the contribution of locally released IL-15 on peripheral NK-cell-mediated innate immune responses, we generated a recombinant vaccinia virus that expresses IL-15 and evaluated the course of vaccinial disease in athymic nude mice. Coexpression of IL-15 resulted in the attenuation of virulence of vaccinia virus, and mice inoculated with 105 plaque-forming units or less resolved the infection successfully. In contrast, mice inoculated with a similar dose of the control vaccinia virus failed to eliminate the virus and died of generalized vaccinial disease. Enhanced expression of IL-12 and IFN-γ as well as induction of chemokines were evident in the mice inoculated with IL-15-expressing vaccinia virus in addition to an increase in NK cells in the spleen. However, in this model system, the degree of attenuation in viral virulence attained with coexpression of IL-15 was much less than that achieved with coexpression of IL-2, suggesting that the peripheral NK-cell-mediated events are more responsive to IL-2 than to IL-15.

Generalized transduction in Streptomyces coelicolor

We report the isolation of generalized transducing phages for Streptomyces species able to transduce chromosomal markers or plasmids between derivatives of Streptomyces coelicolor, the principal genetic model system for this important bacterial genus. We describe four apparently distinct phages (DAH2, DAH4, DAH5, and DAH6) that are capable of transducing multiple chromosomal markers at frequencies ranging from 10−5 to 10−9 per plaque-forming unit. The phages contain DNA ranging in size from 93 to 121 kb and mediate linked transfer of genetic loci at neighboring chromosomal sites sufficiently close to be packaged within the same phage particle. The key to our ability to demonstrate transduction by these phages was the establishment of conditions expected to severely reduce superinfection killing during the selection of transductants. The host range of these phages, as measured by the ability to form plaques, extends to species as distantly related as Streptomyces avermitilis and Streptomyces verticillus, which are among the most commercially important species of this genus. Transduction of plasmid DNA between S. coelicolor and S. verticillus was observed at frequencies of ≈10−4 transductants per colony-forming unit.

Oncolytic herpes simplex virus vector with enhanced MHC class I presentation and tumor cell killing

Oncolytic herpes simplex virus type 1 (HSV-1) vectors are promising therapeutic agents for cancer. Their efficacy depends on the extent of both intratumoral viral replication and induction of a host antitumor immune response. To enhance these properties while employing ample safeguards, two conditionally replicating HSV-1 vectors, termed G47Δ and R47Δ, have been constructed by deleting the α47 gene and the promoter region of US11 from γ34.5-deficient HSV-1 vectors, G207 and R3616, respectively. Because the α47 gene product is responsible for inhibiting the transporter associated with antigen presentation (TAP), its absence led to increased MHC class I expression in infected human cells. Moreover, some G47Δ-infected human melanoma cells exhibited enhanced stimulation of matched antitumor T cell activity. The deletion also places the late US11 gene under control of the immediate-early α47 promoter, which suppresses the reduced growth properties of γ34.5-deficient mutants. G47Δ and R47Δ showed enhanced viral growth in a variety of cell lines, leading to higher virus yields and enhanced cytopathic effect in tumor cells. G47Δ was significantly more efficacious in vivo than its parent G207 at inhibiting tumor growth in both immune-competent and immune-deficient animal models. Yet, when inoculated into the brains of HSV-1-sensitive A/J mice at 2 × 106 plaque forming units, G47Δ was as safe as G207. These results suggest that G47Δ may have enhanced antitumor activity in humans.