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K L Bennett

Publications and source records attributed to K L Bennett.

At least 19 recordsLinked to original sources
PubMed2001

A latent variable model of developmental instability in relation to men's sexual behaviour.

A single trait's fluctuating asymmetry (FA) is expected to be a poor measure of developmental instability. Hence, studies that examine associations between FA and outcomes expected to covary with developmental instability often have little power in detecting meaningful relationships. One way of increasing the power of detecting relationships between developmental instability and outcomes is through the use of multiple traits' FA. The way multiple traits have typically been used is in trait aggregates. Here, we illustrate another way of examining relationships with developmental instability using multiple traits' FA: through structural equation modelling. Covariances between measures of FA and an outcome variable are interpreted within the context of an explicit model of associations between variables, which is tested for fit and the parameters specified within the model are estimated. We used nine traits' FA as markers of a latent variable of men's developmental instability, which was associated with the number of sexual partners. The results indicate a sizeable correlation between developmental instability and men's sexual history, despite small correlations between individual traits' FA and sexual history.

Aggression↗
PubMed2000

Chemical cross-linking with thiol-cleavable reagents combined with differential mass spectrometric peptide mapping--a novel approach to assess intermolecular protein contacts.

The intermolecular contact regions between monomers of the homodimeric DNA binding protein ParR and the interaction between the glycoproteins CD28 and CD80 were investigated using a strategy that combined chemical cross-linking with differential MALDI-MS analyses. ParR dimers were modified in vitro with the thiol-cleavable cross-linker 3,3'-dithio-bis(succinimidylproprionate) (DTSSP), proteolytically digested with trypsin and analyzed by MALDI-MS peptide mapping. Comparison of the peptide maps obtained from digested cross-linked ParR dimers in the presence and absence of a thiol reagent strongly supported a "head-to-tail" arrangement of the monomers in the dimeric complex. Glycoprotein fusion constructs CD28-IgG and CD80-Fab were cross-linked in vitro by DTSSP, characterized by nonreducing SDS-PAGE, digested in situ with trypsin and analyzed by MALDI-MS peptide mapping (+/- thiol reagent). The data revealed the presence of an intermolecular cross-link between the receptor regions of the glycoprotein constructs, as well as a number of unexpected but nonetheless specific interactions between the fusion domains of CD28-IgG and the receptor domain of CD80-Fab. The strategy of chemical cross-linking combined with differential MALDI-MS peptide mapping (+ thiol reagent) enabled localization of the interface region(s) of the complexes studied and clearly demonstrates the utility of such an approach to obtain structural information on interacting noncovalent complexes.

Amino Acid Sequence↗
PubMed2000

Combinatorial RNA interference indicates GLH-4 can compensate for GLH-1; these two P granule components are critical for fertility in C. elegans.

We report that four putative germline RNA helicases, GLHs, are components of the germline-specific P granules in Caenorhabditis elegans. GLH-3 and GLH-4, newly discovered, belong to a multi-gene glh family. Although GLHs are homologous to Drosophila VASA, a polar granule component necessary for oogenesis and embryonic pattern formation, the GLHs are distinguished by containing multiple CCHC zinc fingers. RNA-mediated interference (RNAi) reveals the GLHs are critical for oogenesis. By RNAi at 20 degrees C, when either loss of GLH-1 or GLH-4 alone has no effect, loss of both GLH-1 and GLH-4 results in 97% sterility in the glh-1/4(RNAi) offspring of injected hermaphrodites. glh-1/4(RNAi) germlines are under-proliferated and are without oocytes. glh-1/4(RNAi) animals produce sperm; however, spermatogenesis is delayed and the sperm are defective. P granules are still present in glh-1/4(RNAi) sterile worms as revealed with antibodies against the remaining GLH-2 and GLH-3 proteins, indicating the GLHs function independently in P granule assembly. These studies reveal that C.elegans can use GLH-1 or GLH-4 to promote germline development.

Amino Acid Sequence↗
PubMed1999

Heparan sulfate composition of alternatively spliced CD44 fusion proteins.

Prior analyses of recombinant CD44 fusion proteins have indicated that combinatorial splicing of variant exons exerts distal effects on chondroitin sulfate content and structure, which may regulate the biological properties of the respective CD44 isoforms. The consequences of splicing of variant exons V4-7 on the heparan sulfate moieties were therefore examined, utilizing recombinant chimeras containing exons V3 and V8-10, engineered with or without exons V4-7 and expressed as Ig fusion proteins in COS cells. Splicing of exons V4-7, though they contain no consensus motifs for glycosaminoglycan assembly, resulted in markedly increased polymer sulfation levels of the heparan sulfates. The sulfate groups of both the CD44 V3-10 and V3,8-10 isoforms occurred as di- and tri-sulfated dissacharide units and were restricted to one N-sulfated block domain within the polymers. Compared to native human keratinocyte CD44, the recombinant heparan sulfates were relatively low in sulfate content. Our data indicate that variant exon V4-7 splicing exerts distal effects on the composition of this glycosaminoglycan. These effects may regulate those functions that are mediated through the heparan sulfate moieties, such as the binding of growth factors.

Alternative Splicing↗
PubMed1999

Characterization of the heparan sulfate and chondroitin sulfate assembly sites in CD44.

Isoforms of CD44 are differentially modified by the glycosaminoglycans (GAGs) chondroitin sulfate (CS), heparan sulfate (HS), and keratan sulfate. GAG assembly occurs at serines followed by glycines (SG), but not all SG are utilized. Seven SG motifs are distributed in five CD44 exons, and in this paper we identify the HS and CS assembly sites that are utilized in CD44. Not all the CD44 SG sites are modified. The SGSG motif in CD44 exon V3 is the only HS assembly site; this site is also modified with CS. HS and CS attachment at that site was eliminated by mutation of the serines in the V3 motif to alanine (AGAG). Exon E5 is the only other CD44 exon that supports GAG assembly and is modified with CS. Using a number of recombinant CD44 protein fragments we show herein that the eight amino acids located downstream of the SGSG site in V3 are responsible for the specific addition of HS to this site. If the eight amino acids located downstream from the first SG site in CD44 exon E5 are exchanged with those located downstream of the SGSG site in exon V3, the SG site in E5 becomes modified with HS and CS. Likewise if the eight amino acids found downstream from the first SG in E5 are placed downstream from the SGSG in V3, this site is modified with CS but not HS. We also show that these sequences cannot direct the modification of CD44 with HS from a distance. Constructs containing CD44 exon V3 in which the SGSG motif was mutated to AGAG were not modified with HS even though they contained other SG motifs. Thus, a number of sequence and structural requirements that dictate GAG synthesis on CD44 have been identified.

Amino Acid Sequence↗
PubMed1999

Generation of artificial proteoglycans containing glycosaminoglycan-modified CD44. Demonstration of the interaction between rantes and chondroitin sulfate.

All CD44 isoforms are modified with chondroitin sulfate (CS), while only those containing variably spliced exon V3 are modified with both CS and heparan sulfate (HS). The CS is added to a serine-glycine (SG) site in CD44 exon E5, while HS and CS are added to the SGSG site in exon V3. Site-directed mutagenesis and other molecular biology techniques were used to determine the minimal motifs responsible for the addition of CS and HS to CD44 (see accompanying paper (Greenfield, B., Wang, W.-C., Marquardt, H., Piepkorn, M., Wolff, E. A., Aruffo, A., and Bennett, K. L. (1999) J. Biol. Chem. 274, 2511-2517)). We have used this information to generate artificial proteoglycans containing the extracellular domain of the cell adhesion protein lymphocyte function-associated antigen-3 (LFA-3) (CD58) and CD44 motifs modified with CS or a combination of CS and HS. Analysis of the CD44-modified LFA-3 protein showed that it retains the ability to engage and trigger the function of its natural ligand CD2, resulting in T cell activation. In addition, the glycosaminoglycan-modified artificial proteoglycan is capable of binding the chemokine RANTES (regulated upon activation, normally T cell expressed and secreted) and delivering it to human T cells, resulting in enhanced T cell activation. These data demonstrate that artificial proteoglycans can be engineered with functional domains that have enhanced activity by codelivering glycosaminoglycan-binding molecules. The artificial proteoglycans were also used as a model system to explore the glycosaminoglycan binding properties of basic-fibroblast growth factor and the chemokine RANTES. While basic-fibroblast growth factor was shown to bind HS alone, this model revealed that RANTES binds not only HS, as has been demonstrated in the past, but also CS. Thus, artificial proteoglycans can be used for studying the glycosaminoglycan binding patterns of growth factors and chemokines and provide a means to manipulate the levels, types, and activity of glycosaminoglycan-binding proteins in vitro and in vivo.

Base Sequence↗
PubMed1999

Colocalization of basic fibroblast growth factor and CD44 isoforms containing the variably spliced exon v3 (CD44v3) in normal skin and in epidermal skin cancers.

Previous in vitro studies have shown CD44 isoforms containing the alternatively spliced exon v3 (CD44v3) to be modified with heparan sulphate (HS) and to bind HS-binding basic fibroblast growth factor (bFGF). Here, we demonstrate that exogenously added bFGF is also bound in vivo by CD44v3-positive keratinocytes in normal skin and by tumour cells in basal cell carcinoma and squamous cell carcinoma (SCC), two skin cancers of keratinocyte origin. bFGF binding and CD44v3 expression were colocalized in cultured human normal keratinocytes (HNK) and on the SCC cell line A431. By contrast, benign or malignant tumours of melanocyte origin failed to express CD44v3 and bound no bFGF. The bFGF binding to normal or transformed keratinocytes in vivo and in vitro was dependent on HS modification, as it was completely eliminated by pretreatment with heparitinase or by blocking with free heparin, whereas chondroitinase had no effect. In addition, specific removal of CD44v3 by antibody-induced shedding also diminished bFGF binding to keratinocytes. Furthermore, bFGF stimulated the proliferation of CD44v3-positive HNK and A431 in a dose-dependent fashion. This bFGF effect was again completely abolished by heparitinase or free heparin, but not by chondroitinase. In aggregate, our results suggest that a function of HS-modified CD44 isoforms such as CD44v3 in skin is to present the HS-binding growth factor bFGF, thereby stimulating the proliferation of normal or transformed keratinocytes.

Antigens, Neoplasm↗
PubMed1998

Epitope specificity of CD44 for monoclonal antibody-dependent facilitation of marrow engraftment in a canine model.

Primary graft rejection after marrow transplantation occurs more frequently in patients receiving HLA-haploidentical compared with HLA-identical sibling transplants. Both human and experimental animal data suggest that the cells responsible for this phenomenon are either host natural killer (NK) cells, T cells, or both. To investigate the mechanisms of graft rejection, we have developed a canine model of marrow transplantation, which uses DLA-nonidentical unrelated donors in the absence of postgrafting immunosuppression. In this model most animals rejected their marrow grafts after a preparative regimen of 9.2 Gy total body irradiation (TBI). However, engraftment of DLA-nonidentical marrow can be facilitated when the recipients are pretreated with monoclonal antibody (MoAb) S5, which recognizes CD44. In this report, we extended these observations by first cloning the canine CD44 and, next, mapping the epitope recognized by S5, which was located in a region conserved among human and canine CD44 and was distinct from the hyaluronan binding domain. However, in vitro binding of S5 caused a conformational change in CD44, which allowed increased hyaluronan binding. Then, we reexamined the in vivo model of marrow transplantation and compared results with MoAb S5 to those with two other anti-CD44 MoAbs, IM7 and S3. Only MoAb S5 significantly increased the engraftment rate of DLA-nonidentical unrelated marrow, whereas the two other anti-CD44 MoAbs were ineffective. The enhanced in vivo effect was not related to differences in the MoAbs' avidities, since both S5 and IM7 had equivalent binding to CD44, but most likely related to the specific epitope that S5 recognizes. Thus, this study shows that the effect of the anti-CD44 MoAb S5 in facilitating engraftment is epitope specific and if one is to use an anti-CD44 to facilitate engraftment of marrow in humans, one cannot assume that any anti-CD44 would work.

Amino Acid Sequence↗
PubMed1997

Interaction of the cytoplasmic tail of CTLA-4 (CD152) with a clathrin-associated protein is negatively regulated by tyrosine phosphorylation.

CTLA-4 (CD152), high-avidity receptor for CD80 and CD86, is a powerful regulator of T cell activation. While CTLA-4 functions at the cell surface, it is primarily localized in intracellular vesicles and cycles to the cell surface. The CTLA-4 cytoplasmic domain contains sequences that direct its intracellular localization and regulate its signaling. Here we demonstrate that effector molecules involved in receptor trafficking and signaling interact with distinct, but overlapping, sequences in the CTLA-4 cytoplasmic domain. Using the yeast two-hybrid method, we demonstrate association of the mu2 subunit of AP-2, the clathrin-associated complex found in plasma membrane-associated coated pits, with the cytoplasmic tail of CTLA-4, but not CD28. The mu1 subunit of AP-1, found in Golgi-associated coated pits, associated with neither CTLA-4 nor CD28. Sequences required for interaction of mu2 and CTLA-4 were localized to residues, 161TTGVY in CTLA-4; this sequence is N-terminal to, but overlaps with, a previously identified SH2 binding motif, 165YVKM, involved in CTLA-4 signaling. Mu2 interacted preferentially with CTLA-4 when residue 165Y was nonphosphorylated, whereas a PI3 kinase SH2 domain interacted preferentially when 165Y was phosphorylated. In co-transfection experiments, both tyrosine residues in the cytoplasmic tail of CTLA-4 (165Y and 182Y) were phosphorylated by the T lymphocyte-associated tyrosine kinase, p56lck. Thus, phosphorylation of CTLA-4 residue 165Y may reciprocally regulate signaling and trafficking of CTLA-4 by determining which effector molecules bind to its cytoplasmic tail.

Abatacept↗
PubMed1997

Deleted in colorectal carcinoma (DCC) binds heparin via its fifth fibronectin type III domain.

DCC (deleted in colorectal carcinoma) is a broadly expressed cell-surface receptor. Netrin-1 was recently identified as a DCC ligand in brain, but the possibility of other DCC ligands was suggested by the finding that an anti-DCC antibody (clone AF5) neutralized netrin-1-dependent commissural axon outgrowth without blocking DCC/netrin-1 interactions. Here we have searched for alternative cell-surface DCC ligands. A DCC-Ig fusion protein bound to neural and epithelial derived cell lines, indicating that these lines express ligand(s) for DCC. The cell-surface binding activity was mediated by the loop between beta-strands F and G of the fifth fibronectin type III repeat FNIII-D5. The loop included the sequence KNRR, which resembles heparin-binding motifs in other proteins. Heparinase and heparitinase treatment of cells reduced binding of DCC-Ig, suggesting that heparan sulfate proteoglycans are cell-surface DCC ligand(s). This was further supported by heparin blocking experiments and by binding of DCC-Ig to immobilized heparan sulfate. The interaction between DCC-Ig and heparan sulfate/heparin, both on the surface of cells and immobilized on plastic, was blocked by the same anti-DCC antibody that blocks netrin-1-dependent commissural axon outgrowth. Taken together, these findings suggest that the DCC-Ig/heparin interaction may contribute to the biological activity of DCC.

Amino Acid Sequence↗
PubMed1997

Chondroitin sulphate composition and structure in alternatively spliced CD44 fusion proteins.

Previous studies have indicated that CD44 isoforms, spliced with variant exons, are heterogeneously glycanated with chondroitin sulphate and heparan sulphate chains. Because such alternative splicing may regulate divergent biological effects of the specific isoforms, we analysed the consequences of this process on the composition and structure of the chondroitin-sulphate chains. Recombinant chimaeras were engineered with and without exons V3-10 or V3,8-10 and expressed as Ig fusion proteins in COS cells. In addition, the chondroitin sulphates of wild-type isoforms were contrasted with those of isoforms mutated with serine-to-alanine codon substitutions at a putative Ser-Gly-Ser-Gly glycosaminoglycan acceptor site within exon V3. The chondroitin sulphates contained both 4- and 6-sulphated galactosamine residues, although there was a high content of non-sulphated galactosamine-containing repeat units. Splicing of exons V4-7, which contain no Ser-Gly consensus motifs, resulted in increased glycanation with chondroitin-sulphate chains, as well as increased sulphation levels of the polymers. Comparison of wild-type and acceptor-site mutant isoforms showed that chondroitin-sulphate content declined by more than 60-80% in the mutant, indicating that assembly of chondroitin-sulphate chains occurs there, and a general decrease in the sulphation level of the remaining chains was observed. Undersulphation of the recombinant chondroitin sulphates was shown by parallel analyses with native human keratinocyte CD44 molecules and is most probably an artifact of transient expression in COS cells. Our data indicate that combinatorial exon splicing exerts complex and distal effects on glycanation patterns and structure, which presumably modulate those functions that may be mediated though the chondroitin-sulphate moieties, such as motility and matrix invasion.

Alternative Splicing↗
PubMed1997

Monitoring papain digestion of a monoclonal antibody by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) has been used to examine the Fab, F(ab')2 and deglycosylated Fc fragments obtained from the murine IgG1 B72.3 monoclonal antibody (MAb) by digestion with the sulfhydryl protease papain, in an attempt to determine the sites of cleavage and thus to clarify the mode of action of this enzyme on MAbs. ESI analysis of the Fab and F(ab')2 subunits indicated that the predominant site of papain cleavage occurred at C221 of the B72.3 MAb heavy chain. Reduction of the intra- and interchain disulfide bridges of these fragments by 1,4-dithiothreitol and subsequent electrospray analysis showed a loss of C221 from the C-terminal end of the Fd subunit. ESI analysis of the cleaved Fab fragment indicated that there was an apparent loss of amino acid residues from this fragment. Edman sequencing of the cleaved subunit revealed an intact light chain and the loss of QVQ from the N-terminal of the Fd subunit. Reduction of this subunit gave a Fd fragment approximately 32 Da greater than the predicted mass, which we have attributed to oxidation of the heavy chain methionine residues (M81 and M136). Removal of the carbohydrate portion from the Fc fragment by N-glycosidase F indicated that papain cleavage had occurred at C223 of the B72.3 MAb heavy chain. In addition, it was observed that the C-terminal lysine residue (K438) was absent from the deglycosylated Fc fragment, presumably due to carboxypeptidase B activity that occurs during the in vivo production of the B72.3 MAb in murine hosts. These data clearly illustrate the power of ESI-MS for determining small changes in mass on large proteins as well as providing a rapid and sensitive technique for assessing MAb fragments prior to use in radioimaging or radiotherapy.

Amino Acid Sequence↗
PubMed1996

Multiple potential germ-line helicases are components of the germ-line-specific P granules of Caenorhabditis elegans.

Two components of the germ-line-specific P granules of the nematode Caenorhabditis elgans have been identified using polyclonal antibodies specific for each. Both components are putative germ-line RNA helicases (GLHs) that contain CCHC zinc fingers of the type found in the RNA-binding nucleocapsid proteins of retroviruses. The predicted GLH-1 protein has four CCHC fingers; GLH-2 has six. Both GLH proteins localize in the P granules at all stage of germ-line development. However, the two glh genes display different patterns of RNA and protein accumulation in the germ lines of hermaphrodites and males. Injection of antisense glh-1 or glh-2 RNA into wild-type worms causes some offspring to develop into sterile adults, suggesting that either or both genes are required for normal germ-line development. As these very similar glh genes physically map within several hundred kilobases of one another, it seems likely that they represent a fairly recent gene duplication event.

Amino Acid Sequence↗
PubMed1996

Preventing intravasation in women undergoing hysteroscopic procedures.

Hysteroscopic procedures, which are an alternative to hysterectomy for surgical treatment of menorrhagia and uterine fibroids, place women at risk for intravasation of uterine distention fluid. Intravasation can produce fluid overload, pulmonary edema, congestive heart failure, and electrolyte imbalances. To examine risk factors for and evaluate nursing interventions to decrease the incidence of intravasation, the researchers compared mean arterial pressures (MAPs) and intrauterine pressures (IUPs) in two groups of women undergoing elective outpatient hysteroscopic procedures. The experimental group consisted of 20 women in whom fluid infusion pump pressures were maintained below the women's MAPs. The control group consisted of 20 women whose fluid infusion pump pressures were set at random. Distention fluid deficits and the total infused distention fluid volume differed significantly between the two groups, supporting the study hypothesis that maintaining equilibrium between women's IUPs and MAPs decreases the risk of uterine distention fluid absorption into the vasculature and fluid overload complications. Perioperative nurses need to monitor women's MAPs before and during hysteroscopic procedures and maintain fluid infusion pump pressures at or below women's MAPs to decrease the potential for intravasation.

Absorption↗
PubMed1996

Intracellular trafficking of CTLA-4 and focal localization towards sites of TCR engagement.

T lymphocyte receptor CTLA-4 binds costimulatory molecules CD80 (B7-1) and CD86 (B7-2) with high avidity and negatively regulates T cell activation. CTLA-4 functions at the cell surface, yet is primarily localized in intracellular vesicles. Here, we demonstrate cycling of CTLA-4 between intracellular stores and the cell surface. Intracellular vesicles containing CTLA-4 overlapped with endocytic compartment(s) and with perforin-containing secretory granules. Cell surface expression of CTLA-4 was rapidly increased by raising intracellular calcium levels. During T cell activation, intracellular and cell surface CTLA-4 became focused towards sites of TCR activation. Cycling and directional control of CTLA-4 expression may regulate its functional interaction with APCs bearing peptide-MHC complexes of appropriate specificity and avidity.

Abatacept↗
PubMed1996

Beta ig-h3, a transforming growth factor-beta-inducible gene, is overexpressed in atherosclerotic and restenotic human vascular lesions.

Transforming growth factor-beta (TGF-beta) plays an important role in vascular lesion formation and possibly the renarrowing process ("restenosis") that occurs after balloon angioplasty. Secreted in a latent form by most cells, TFG-beta requires enzymatic conversion before it is biologically active. TGF-beta-inducible gene h3 (beta ig-h3) is a novel molecule that is induced when cells are treated with TGF-beta1. This study examined the expression of beta ig-h3 in normal and diseased human vascular tissue. To determine the expression pattern of beta ig-h3 in human arteries, immunocytochemistry was performed on tissue sections from (1) normal internal mammary arteries, (2) the proximal left anterior descending coronary artery (with minimal intimal thickening) of 15 patients aged 18 to 40 years, (3) primary and restenotic coronary lesions from 7 patients, and (4) fresh directional atherectomy tissue from 11 patients. A polyclonal antibody consistently immunodetected beta ig-h3 protein in endothelial cells of all vascular tissue. In normal coronary arteries of young individuals, beta ig-h3 protein was absent from the intima and media but was found in the subendothelial smooth muscle cells of some arteries with modest intimal thickening. In diseased arteries beta ig-h3 protein was more abundant in the intima than the media. Restenotic coronary lesions tended to show higher levels of immunodetectable beta ig-h3 protein, especially in areas of dense fibrous connective tissue. Beta ig-h3 protein was immunodetected in the cytoplasm of plaque macrophages as well as smooth muscle and endothelial cells. By using in situ hybridization on fresh directional atherectomy specimens, we found beta ig-h3 mRNA to be overexpressed by plaque macrophages and smooth muscle cells. Nondiseased human internal mammary arteries also expressed beta ig-h3 mRNA in endothelial cells but not in the smooth muscle cells of the normal intima and media. These results document the expression of beta ig-h3 in diseased human arterial tissue and support the hypothesis that active TGF-beta plays a role in atherogenesis and restenosis.

Adolescent↗
PubMed1995

Functional and metabolic evidence for two different vanilloid (VN1 and VN2) receptors in perfused rat hindlimb.

Vanilloid spice principles, including capsaicin, stimulate vasoconstriction in the rat hindlimb perfused at constant flow and, depending on dose, either stimulate or inhibit oxygen consumption by this vascular bed. We now present metabolic and functional evidence for two different vanilloid (VN1 and VN2) receptor types. These receptors can be distinguished on the basis of their differing agonist affinity for capsaicin, their different calcium and oxygen dependencies for inducing vasoconstriction, and whether they stimulate, or inhibit, oxygen consumption. The higher affinity vanilloid receptor, VN1 can be distinguished on the basis of initiating vasoconstriction at low doses of capsaicin and simultaneously stimulating oxygen consumption. Its apparent biological function is dependent on the presence of oxygen and external calcium. In contrast, the lower affinity receptor, VN2 induces vasoconstriction associated with inhibition of oxygen consumption. Its vasoconstriction action can occur independently of either external calcium ions, or the presence of oxygen in the perfusate.

Animals↗