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K L Wilson

Publications and source records attributed to K L Wilson.

At least 19 recordsLinked to original sources
PubMed1999

Stable five- and six-coordinated silicate anions in aqueous solution.

Addition of aliphatic polyols to aqueous silicate solutions is shown to yield high concentrations of stable polyolate complexes containing five- or six-coordinated silicon. Coordinating polyols require at least four hydroxy groups, two of which must be in threo configuration, and coordinate to silicon via hydroxy oxygens at chain positions on either side of the threo pair. The remarkable ease by which these simple sugar-like molecules react to form hypervalent silicon complexes in aqueous solution supports a long-standing supposition that such species play a significant role in the biological uptake and transport of silicon and in mineral diagenesis.

Anions↗
PubMed1999

Roles of LAP2 proteins in nuclear assembly and DNA replication: truncated LAP2beta proteins alter lamina assembly, envelope formation, nuclear size, and DNA replication efficiency in Xenopus laevis extracts.

Humans express three major splicing isoforms of LAP2, a lamin- and chromatin-binding nuclear protein. LAP2beta and gamma are integral membrane proteins, whereas alpha is intranuclear. When truncated recombinant human LAP2beta proteins were added to cell-free Xenopus laevis nuclear assembly reactions at high concentrations, a domain common to all LAP2 isoforms (residues 1-187) inhibited membrane binding to chromatin, whereas the chromatin- and lamin-binding region (residues 1-408) inhibited chromatin expansion. At lower concentrations of the common domain, membranes attached to chromatin with a unique scalloped morphology, but these nuclei neither accumulated lamins nor replicated. At lower concentrations of the chromatin- and lamin-binding region, nuclear envelopes and lamins assembled, but nuclei failed to enlarge and replicated on average 2. 5-fold better than controls. This enhancement was not due to rereplication, as shown by density substitution experiments, suggesting the hypothesis that LAP2beta is a downstream effector of lamina assembly in promoting replication competence. Overall, our findings suggest that LAP2 proteins mediate membrane-chromatin attachment and lamina assembly, and may promote replication by influencing chromatin structure.

Amino Acid Sequence↗
PubMed1999

Heat shock protein 70 (Hsp70) protects postimplantation murine embryos from the embryolethal effects of hyperthermia.

Previous work has shown that there is a positive correlation between the induction of Hsp70 and its transient nuclear localization and the acquisition and loss of induced thermotolerance in postimplantation rat embryos. To determine whether Hsp70 is sufficient to induce thermotolerance in postimplantation mammalian embryos, we used a transgenic mouse in which the normally strictly inducible Hsp70 is constitutively expressed in the embryo under the control of a beta-actin promoter. Day 8.0 mouse embryos heterozygous for the Hsp70 transgene were not protected from the embryotoxic effects of hyperthermia (43 degrees C); however, homozygous embryos, expressing approximately twice as much Hsp70 as heterozygous embryos, were partially protected (increased embryo viability) from the embryolethal effects of hyperthermia. Although the viability of transgenic embryos was significantly increased compared with that of nontransgenic embryos, this protection did not extend to embryo growth and development. To determine whether the failure to achieve a more robust protection was related to the expression of insufficient Hsp70 in transgenic embryos, we undertook experiments to determine whether the level of Hsp70 correlated with the level of thermotolerance induced by various lengths of a 41 degrees C heat shock. A 41 degrees C, 5-minute heat shock failed to induce Hsp70 or thermotolerance, a 41 degrees C, 15-minute heat shock induced Hsp70 and a significant level of thermotolerance, while a 41 degrees C, 60-minute heat shock induced an even higher level of Hsp70 as well as a higher level of thermotolerance. Quantitation of Hsp70 levels indicated that thermotolerance was associated with levels of Hsp70 of 820 pg/microg embryo protein or greater. Subsequent quantitation of the amount of Hsp70 expressed in homozygous transgenic embryos indicated a level of 577 pg/microg embryo protein, that is, a level below that associated with induced thermotolerance. Overall, results presented indicate that Hsp70 does play a direct role in the induction of thermotolerance in postimplantation mouse embryos; however, the level of thermotolerance is dependent on the level of Hsp70 expressed.

Animals↗
PubMed1999

Demonstration of Tritrichomonas foetus in the external genitalia and of specific antibodies in preputial secretions of naturally infected bulls.

Portions of penis and prepuce were collected from 24 bulls with current or recent Tritrichomonas foetus infection. Epididymides were collected from seven of the bulls, and seminal vesicles and prostate were collected from four. Following immunohistochemical staining with two monoclonal antibodies (34.7C4.4 and TF1.15) prepared against T. foetus surface antigens, trichomonads were identified in sections from 15 of the bulls. Organisms were most often located in penile crypts in the midshaft and caudal regions and less often in preputial crypts. Trichomonads were not observed in sections from other genitalia or in subepithelial tissue. T. foetus antigen, however, was present in the cytoplasm of some epithelial cells and the cytoplasm of some mononuclear cells in subepithelial lymphoid aggregates and follicles. Preputial smegma was collected from 16 T. foetus-infected bulls and from 16 control bulls with negative T. foetus cultures. Preputial antibody levels to TF1.17, a surface antigen of T. foetus, were determined by an enzyme-linked immunosorbent assay. Preputial secretions from infected bulls contained specific antibody of each isotype and subisotype tested. IgG1 responses were the greatest, IgM and IgA responses were approximately equal, and IgG2 responses were low. Each isotype and subisotype response in infected bulls was significantly greater than that in the controls. These results confirm previous speculation concerning anatomical sites of infection and suggest that parasite antigen can be taken up and processed locally, resulting in deposition of specific IgG1, IgG2, IgA, and IgM antibodies in the preputial cavity.

Animals↗
PubMed1998

Ro 32-3555, an orally active collagenase selective inhibitor, prevents structural damage in the STR/ORT mouse model of osteoarthritis.

OBJECTIVE: To determine the efficacy of a selective inhibitor of collagenases in an animal model of osteoarthritis (OA). METHODS: Ro 32-3555, an orally active collagenase selective inhibitor, was administered to STR/ORT mice. Microfocal x-ray-generated images of the hind limbs were visually scored for joint space narrowing, osteophyte formation, and calcification of tendons. Histologic sections of the knees were scored for cartilage changes including loss of surface matrix, fibrillation, and eburnation. RESULTS: Significant inhibition of joint space narrowing and osteophyte formation was achieved in groups of animals treated with 10-50 mg/kg(-1) of Ro 32-3555. These effects were confirmed histologically in the same groups of animals: histologic analysis revealed that Ro 32-3555 protected cartilage from degradative changes. CONCLUSION: Ro 32-3555, a collagenase selective inhibitor, inhibits both the cartilage and bone changes in this mouse model of OA, and thus shows great potential as a treatment of OA in humans.

Animals↗
PubMed1998

Growing or just getting along? Technical and adaptive competence in coping among adolescents.

This study examined coping among African American adolescents with learning disabilities. Ninety-seven African American adolescents and their mother or primary caregiver participated in the study. The study centered on a new conceptual distinction between technical competence in coping and adaptive competence in coping. Technical competence referred to short-term, reactive attempts at coping based on individuals' abilities to find techniques for reducing their feelings of distress. Adaptive competence referred to longer-term, developmental processes of adaptive change that resulted in more global benefits for the individual. Past literature was reassessed on the basis of this conceptual distinction, and a new model of technical and adaptive competence in coping was proposed based on developmental theory. Perceptions of coping efficacy and the incidence of behavioral problems were regressed on measures of technical and adaptive competence in coping. Results were explored first as a general test of the model on the total sample, and second as a comparative analysis between gender subsamples. Total sample findings were consistent with hypothesized results. Technical competence was a better predictor of feelings of efficacy and adaptive competence was a better predictor of behavioral problems. Gender subsample differences were significant and supported a picture of gender-typed approaches to coping.

Adaptation, Psychological↗
PubMed1997

Ionophore-releasable lumenal Ca2+ stores are not required for nuclear envelope assembly or nuclear protein import in Xenopus egg extracts.

The nuclear envelope of higher eukaryotes disassembles early in mitosis and reassembles later around the daughter chromosomes. Previous in vitro work supported the hypothesis that the release of lumenal Ca2+ stores via inositol 1,4,5-trisphosphate-gated Ca2+ channels is required for nuclear assembly in Xenopus egg extracts. Other work suggested that lumenal Ca2+ stores are required for nuclear protein import in mammalian cells in vivo, but not in vitro. Here, we rigorously tested the role of lumenal Ca2+ stores in nuclear assembly and nuclear protein import using Xenopus egg extracts. Lumenal Ca2+ stores were depleted by pretreating the extracts with Ca2+ ionophores (ionomycin, A23187) or inhibitors of Ca(2+)-sequestering pumps (thapsigargin, cyclopiazonic acid). Extracts depleted of lumenal Ca2+ stores assembled nuclei around demembranated sperm chromatin. These nuclei were morphologically indistinguishable from control nuclei when viewed by light or electron microscopy. Nuclei lacking lumenal Ca2+ stores excluded membrane-impermeant fluorescent dextrans, indicating the formation of a sealed nuclear envelope, and they accumulated a fluorescent nucleophilic protein, nucleoplasmin, indicating that nuclear pore complexes were functional. DNA replication occurred in the lumenal-Ca(2+)-depleted nuclei, though less efficiently than control nuclei. Our demonstration that in vitro nuclear import does not depend on lumenal Ca2+ stores confirms a previous unpublished observation by Greber and Gerace, and suggests that import defects seen in ionophore-treated living cells are not directly due to the loss of lumenal Ca2+. Finally, we concluded that, contrary to our expectations, lumenal Ca2+ stores are not required for nuclear envelope assembly in Xenopus egg extracts.

Animals↗
PubMed1997

Nuclear assembly.

We review old and new insights into the structure of the nuclear envelope and the components responsible for its dynamic reassembly during mitosis. New information is coming to light about several of the proteins that mediate nuclear reassembly. These proteins include the lamins and their emerging relationship with proteins such as otefin and the MAN antigens: peripheral proteins that might participate in lamina structure. There are four identified proteins localized to the inner nuclear membrane: the lamina-associated proteins LAP1 and LAP2, emerin, and the lamin B receptor (LBR). LBR can interact independently with lamin B and a chromodomain protein, Hp1, and appears to be a central player in targeting nuclear membranes to chromatin. Intermediates in the assembly of nuclear pore complexes (NPCs) can now be studied biochemically and visualized by high resolution scanning electron microscopy. We discuss the possibility that the filament-forming proteins Tpr/p270, NuMA, and perhaps actin may have roles in nuclear assembly.

Animals↗
PubMed1997

Nuclear envelope assembly in Xenopus extracts visualized by scanning EM reveals a transport-dependent 'envelope smoothing' event.

We analyzed the pathway of nuclear envelope assembly in Xenopus egg extracts using field emission in-lens scanning electron microscopy. The binding, fusion, and flattening of vesicles onto the chromatin surface were visualized in detail. The first nuclear pore complexes assembled in flattened patches of nuclear envelope, before the chromatin was fully enclosed by membranes. Confirming previous transmission electron microscope observations, two morphologically distinct types of vesicles contributed to the nuclear membranes: ribosome-carrying ('rough') vesicles, many of which bound directly to chromatin, and 'smooth' vesicles, which appeared to associate primarily with other nuclear vesicles or membrane patches. The presence of ribosomes, an outer nuclear membrane marker, on many chromatin-binding vesicles suggested that chromatin-attachment proteins integral to the inner membrane were present on vesicles that also carried markers of the outer membrane and endoplasmic reticulum. Chromatin-associated vesicles also carried pore membrane proteins, since pore complexes formed when these vesicles were incubated with cytosol. A change in nuclear envelope morphology termed 'envelope smoothing' occurred 5-15 minutes after enclosure. Nuclear envelopes that were assembled in extracts depleted of wheat-germ-agglutinin-binding nucleoporins, and therefore unable to form functional pore complexes, remained wrinkled, suggesting that 'smoothing' required active nuclear transport. Lamins accumulated with time when nuclei were enclosed and had functional pore complexes, whereas lamins were not detected on nuclei that lacked functional pore complexes. Very low levels of lamins were detected on nuclear intermediates whose surfaces were substantially covered with patches of pore-complex-containing envelope, suggesting that pore complexes might be functional before enclosure.

Animals↗
PubMed1997

Dimples, pores, star-rings, and thin rings on growing nuclear envelopes: evidence for structural intermediates in nuclear pore complex assembly.

We used field emission in-lens scanning electron microscopy to examine newly-assembled, growing nuclear envelopes in Xenopus egg extracts. Scattered among nuclear pore complexes were rare 'dimples' (outer membrane depressions, 5-35 nm diameter), more abundant holes (pores) with a variety of edge geometries (35-45 nm diameter; 3.3% of structures), pores containing one to eight triangular 'star-ring' subunits (2.1% of total), and more complicated structures. Neither mature complexes, nor these novel structures, formed when wheat germ agglutinin (which binds O-glycosylated nucleoporins) was added at high concentrations (>500 microg/ml) directly to the assembly reaction; low concentrations (10 microg/ml) had no effect. However at intermediate concentrations (50-100 microg/ml), wheat germ agglutinin caused a dramatic, sugar-reversible accumulation of 'empty' pores, and other structures; this effect correlated with the lectin-induced precipitation of a variable proportion of each major Xenopus wheat-germ-agglutinin-binding nucleoporin. Another inhibitor, dibromo-BAPTA (5,5'-dibromo-1,2-bis[o-aminophenoxylethane-N,N,N',N'-tetraacetic acid), had different effects depending on its time of addition to the assembly reaction. When 1 mM dibromo-BAPTA was added at time zero, no pore-related structures formed. However, when dibromo-BAPTA was added to growing nuclei 40-45 minutes after initiating assembly, star-rings and other structures accumulated, suggesting that dibromo-BAPTA can inhibit multiple stages in pore complex assembly. We propose that assembly begins with the formation and stabilization of a hole (pore) through the nuclear envelope, and that dimples, pores, star-rings, and thin rings are structural intermediates in nuclear pore complex assembly.

Animals↗
PubMed1997

ARF is not required for nuclear vesicle fusion or mitotic membrane disassembly in vitro: evidence for a non-ARF GTPase in fusion.

Xenopus laevis egg extracts are a well-characterized system for studying nuclear envelope dynamics in vitro. ADP-ribosylation factor (ARF), a 21 kDa GTPase involved in vesicular transport, inhibits nuclear vesicle fusion in vitro when membranes are preincubated with ARF and GTP gamma S (Boman et al., Nature 358, 512-514 (1992)). To test the hypothesis that ARF was required for nuclear envelope assembly or disassembly, we examined these events in cytosol from which ARF was depleted by size fractionation or inhibited with brefeldin A (BFA). In ARF-depleted extracts, vesicles bound chromatin and fused to enclose the chromatin, but the resulting enclosed nuclei lacked pore complexes and remained small. Further growth was not stimulated by adding ARF1, suggesting that fractionation removed other proteins required for pore complex assembly and nuclear growth. Nuclei assembled in ARF-depleted extracts, and rat liver nuclei, disassembled normally in mitotic ARF-depleted reactions. BFA, which inhibits ARF binding to membranes, had no effect on nuclear assembly or disassembly. We concluded that ARF is not essential for nuclear membrane dynamics. Nuclear vesicle fusion was still inhibited by GTP gamma S in ARF-depleted reactions and in reactions containing BFA, strongly suggesting that there is another unidentified GTPase that is either required for vesicle fusion or capable of inhibiting fusion in the presence of GTP gamma S.

ADP-Ribosylation Factor 1↗
PubMed1996

Purification and mass spectrometric analysis of ADP-ribosylation factor proteins from Xenopus egg cytosol.

The GTP analog GTP gamma S potently inhibits nuclear envelope assembly in cell-free Xenopus egg extracts. GTP gamma S does not affect vesicle binding to chromatin but blocks vesicle fusion. Fusion inhibition by GTP gamma S is mediated by a soluble factor, initially named GSF (GTP gamma S-dependent soluble factor). We previously showed that vesicles pretreated with GTP gamma S plus recombinant mammalian ARF1 were inhibited for fusion, suggesting that "GSF activity" was due to the ARF (ADP-ribosylation factor) family of small GTP-binding proteins. To ask if any soluble proteins other than ARF also inhibited vesicle fusion in the pretreatment assay, we purified GSF activity from Xenopus egg cytosol. At all steps in the purification, fractions containing ARF, but no other fractions, showed GSF activity. The purified GSF was identified as Xenopus ARF by immunoblotting and peptide sequence analysis. Reverse phase HPLC and mass spectrometry revealed that GSF contained at least three distinct ARF proteins, all of which copurified through three chromatography steps. The most abundant isoform was identified as ARF1 (62% of the total GSF), because its experimentally determined mass of 20 791 Da matched within experimental error that predicted by the sequence of the Xenopus ARF1 cDNA, which is reported here. The second-most abundant isoform (25% of GSF activity) was identified as ARF3. We concluded that ARF is most likely the only soluble protein that inhibits nuclear vesicle fusion after pretreatment with GTP gamma S.

ADP-Ribosylation Factor 1↗
PubMed1995

Inhibition of nuclear vesicle fusion by antibodies that block activation of inositol 1,4,5-trisphosphate receptors.

Inositol 1,4,5-trisphosphate (IP3) receptors are ligand-gated channels that release intracellular Ca2+ stores in response to the second messenger, IP3. We investigated the potential role of IP3 receptors during nuclear envelope assembly in vitro, using Xenopus egg extracts. Previous work suggested that Ca2+ mobilization is required for nuclear vesicle fusion and implicated IP3 receptor activity. To test the involvement of IP3 receptors using selective reagents, we obtained three distinct polyclonal antibodies to the type 1 IP3 receptor. Pretreatment of membranes with two of the antibodies inhibited IP3-stimulated CA2+ release in vitro and also inhibited nuclear vesicle fusion. One inhibitory serum was directed against 420 residues within the "coupling" domain, which includes several potential regulatory sites. The other inhibitory serum was directed against 95 residues near the C terminus and identifies an inhibitory epitope(s) in this region. The antibodies had no effect on receptor affinity for IP3. Because nuclear vesicle fusion was inhibited by antibodies that block Ca2+ flux, but not by control and preimmune antibodies, we concluded that the activation of IP3 receptors is required for fusion. The signal that activates the channel during fusion is unknown.

Animals↗
PubMed1995

Immunohistochemical detection of Tritrichomonas foetus in formalin-fixed, paraffin-embedded sections of bovine placenta and fetal lung.

An immunohistochemical technique using a monoclonal antibody was evaluated as a diagnostic tool to specifically label Tritrichomonas foetus in formalin-fixed, paraffin-embedded sections of placenta and fetal lung from bovine abortions. Trichomonads were demonstrated in tissues from each of 12 abortions due to T. foetus and none of 15 abortions due to other or unidentified causes. Moderate to marked background staining occurred only in severely autolyzed tissues from T. foetus-infected fetuses. The antibody faintly labeled 1 of 3 other species of trichomonads (Trichomonas gallinae) but did not label other protozoa, bacteria, or fungi tested.

Abortion, Veterinary↗
PubMed1994

A new role for IP3 receptors: Ca2+ release during nuclear vesicle fusion.

During nuclear assembly, vesicles derived from the mitotic disassembly of the nuclear membranes reform the nuclear envelope. The vesicles first bind to chromosomes, specifically recognize other nuclear vesicles and then fuse to enclose the chromosomes. The proteins that mediate these events are largely unknown. Using reconstituted extracts of Xenopus eggs, we found that nuclear vesicle fusion required elevated (microM) concentrations of free Ca2+ [Sullivan KMC. Busa WB. Wilson KL. (1993) Cell, 73, 1411-1422]. Our data suggest that Ca2+ is released from the vesicle lumen by the activation of IP3 receptors (ligand-gated Ca2+ channels). We propose that the role of IP3 receptors during nuclear assembly may be analogous to that of voltage-gated Ca2+ channels during regulated secretion: to provide a microdomain of high cytosolic Ca2+ that triggers fusion. In this article, we will briefly describe current ideas about nuclear assembly and disassembly, and summarize the evidence that IP3 receptors are required for nuclear vesicle fusion. We will discuss parallels between our results and the role of voltage-gated Ca2+ channels, and Ca2+, in regulated exocytosis. Finally, we will address the question of how IP3 receptors are activated during nuclear vesicle fusion: is there a signal that stimulates IP3 production, or is the channel activated directly?

Animals↗
PubMed1993

Calcium mobilization is required for nuclear vesicle fusion in vitro: implications for membrane traffic and IP3 receptor function.

We studied the fusion of nuclear vesicles bound to chromatin in Xenopus egg extracts. Fusion was inhibited by 5 mM BAPTA, a Ca2+ buffer that suppresses cytosolic [Ca2+] gradients. The BAPTA-inhibited step in fusion was biochemically distinct from, and occurred later than, the GTP gamma S-sensitive step mediated by the monomeric GTPase, ADP-ribosylation factor. Exogenous inositol 1,4,5-trisphosphate (IP3), which triggers Ca2+ release from lumenal stores via IP3 receptors, stimulated fusion in the presence of BAPTA. This rescue was specific, because inositol 1,3,4-trisphosphate had no effect. Heparin, a potent antagonist of IP3 receptors, independently blocked fusion in an IP3-reversible manner. We suggest that phosphoinositide signaling may regulate nuclear vesicle fusion.

Animals↗