The association between subclass-specific IgG Fc N-glycosylation profiles and hypertension in the Uygur, Kazak, Kirgiz, and Tajik populations.

Hypertension results from the interaction of genetic and acquired factors. IgG occurs in the form of different subclasses, of which the effector functions show significant variation. The detailed differences between the glycosylation profiles of the individual IgG subclasses may be lost in a profiling method for total IgG N-glycosylation. In this study, subclass-specific IgG Fc glycosylation profile was investigated in the four northwestern Chinese minority populations, namely, Uygur (UIG), Kazak (KZK), Kirgiz (KGZ), and Tajik (TJK), composed of 274 hypertensive patients and 356 healthy controls. The results showed that ten directly measured IgG N-glycan traits (i.e., IgG1G0F, IgG2G0F, IgG2G1FN, IgG2G1FS, IgG2G2S, IgG4G0F, IgG4G1FS, IgG4G1S, IgG4G2FS, and IgG4G2N) representing galactosylation and sialylation are significantly associated with hypertension, with IgG4 consistently showing weaker associations of its sialylation, across the four ethnic groups. We observed a modest improvement on the AUC of ROC curve when the IgG Fc N-glycan traits are added into the glycan-based model (difference between AUCs, 0.044, 95% CI: 0.016-0.072, P = 0.002). The AUC of the diagnostic model indicated that the subclass-specific IgG Fc N-glycan profiles provide more information reinforcing current models utilizing age, gender, BMI, and ethnicity, and demonstrate the potential of subclass-specific IgG Fc N-glycosylation profiles to serve as a biomarker for hypertension. Further research is however required to determine the additive value of subclass-specific IgG Fc N-glycosylation on top of biomarkers, which are currently used.

Light absorption engineering of hydrogenated nanocrystalline silicon by femtosecond laser.

The light absorption coefficient of hydrogenated nanocrystalline silicon has been engineered to have a Gaussian distribution by means of absorption modification using a femtosecond laser. The absorption-modified sample exhibits a significant absorption enhancement of up to ∼700%, and the strong absorption does not depend on the incident light. We propose a model responsible for this interesting behavior. In addition, we present an optical limiter constructed through this absorption engineering method.

Tunable nonlinear absorption of hydrogenated nanocrystalline silicon.

Nonlinear absorption (NLA) of hydrogenated nanocrystalline silicon (nc-Si:H) has been investigated through the open aperture Z-scan method for the photon energy of the incident irradiance slightly less than the bandgap of the sample. NLA responses have been observed to be highly sensitive to the wavelength and intensity of the incident irradiance as well as to the bandgap of the sample, indicating greatly tunable NLA of nc-Si:H. The band tail of nc-Si:H appears to play a crucial role in such NLA responses.

Epitope-specific antibodies to the beta(1C) integrin cytoplasmic domain variant.

The beta(1C) integrin is an alternatively spliced variant of the beta(1) subunit that contains a unique 48-amino-acid sequence in its cytoplasmic domain. We have shown previously that beta(1C) is a potent inhibitor of cell proliferation and that in vivo its expression is downregulated in prostate and breast carcinoma. In this study, we describe a panel of specific monoclonal antibodies that react with the beta(1C) cytodomain. We show by immunoblot analysis that the newly generated monoclonal antibodies specifically recognize the beta(1C) cytodomain expressed as glutathione S-transferase fusion protein. The specificity of the antibodies to beta(1C) was confirmed in competition studies by immunoblotting using beta(1C)-specific synthetic peptides. These monoclonal antibodies reacted, in enzyme-linked immunosorbent assays, with the beta(1C) 785-808 peptide but failed to bind the beta(1C) 778-794, beta(1C) 805-825, or beta(1A) 765-798 peptides. Thus, the epitope recognized by the antibodies is located within the Q(795)-F(804) beta(1C) cytoplasmic sequence; this region overlaps the previously described Q(795)-Q(802) domain necessary for beta(1C) to inhibit cell proliferation. To our knowledge, these are the first monoclonal antibodies specific for a beta(1) cytoplasmic isoform. The monoclonal antibodies described here will be useful tools for dissecting functional differences, among beta(1) integrin variants, as well as for the study of the role of beta(1C) in prostate and breast epithelial cell proliferation.

Substrate specificity of alpha(v)beta(3) integrin-mediated cell migration and phosphatidylinositol 3-kinase/AKT pathway activation.

The alpha(v)beta(3) integrin has been shown to bind several ligands, including osteopontin and vitronectin. Its role in modulating cell migration and downstream signaling pathways in response to specific extracellular matrix ligands has been investigated in this study. Highly invasive prostate cancer PC3 cells that constitutively express alpha(v)beta(3) adhere and migrate on osteopontin and vitronectin in an alpha(v)beta(3)-dependent manner. However, exogenous expression of alpha(v)beta(3) in noninvasive prostate cancer LNCaP (beta(3)-LNCaP) cells mediates adhesion and migration on vitronectin but not on osteopontin. Activation of alpha(v)beta(3) by epidermal growth factor stimulation is required to mediate adhesion to osteopontin but is not sufficient to support migration on this substrate. We show that alpha(v)beta(3)-mediated cell migration requires activation of the phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (PKB/AKT) pathway since wortmannin, a PI 3-kinase inhibitor, prevents PC3 cell migration on both osteopontin and vitronectin; furthermore, alpha(v)beta(3) engagement by osteopontin and vitronectin activates the PI 3-kinase/AKT pathway. Migration of beta(3)-LNCaP cells on vitronectin also occurs through activation of the PI 3-kinase pathway; however, AKT phosphorylation is not increased upon engagement by osteopontin. Furthermore, phosphorylation of focal adhesion kinase (FAK), known to support cell migration in beta(3)-LNCaP cells, is detected on both substrates. Thus, in PC3 cells, alpha(v)beta(3) mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin and osteopontin; in beta(3)-LNCaP cells, alpha(v)beta(3) mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin, whereas adhesion to osteopontin does not support alpha(v)beta(3)-mediated cell migration and PI 3-kinase/AKT pathway activation. We conclude therefore that alpha(v)beta(3) exists in multiple functional states that can bind either selectively vitronectin or both vitronectin and osteopontin and that can differentially activate cell migration and intracellular signaling pathways in a ligand-specific manner.

Prostatic carcinoma cell migration via alpha(v)beta3 integrin is modulated by a focal adhesion kinase pathway.

The highly invasive human prostate cancer PC3 cell line was found to express the alpha(v)beta3 integrin; in contrast, the noninvasive LNCaP prostate cancer cell line did not express alpha(v)beta3. PC3 cells adhered to and migrated on vitronectin (VN), an alpha(v)beta3 ligand expressed in mature bone where prostate cancer cells preferentially metastasize. In contrast, LNCaP cells did not adhere to or migrate on VN. Analysis of primary human prostate cancer cells isolated from 16 surgical specimens, showed that these cells expressed alpha(v)beta3, whereas normal prostate epithelial cells did not. In addition, only primary prostate cancer cells adhered to and migrated on VN. The role of alpha(v)beta3 in mediating prostate epithelial cell migration was confirmed using LNCaP cell transfectants expressing beta3 (beta3-LNCaP). Exogenous expression of alpha(v)beta3 induced LNCaP cells to adhere to and migrate on VN. In response to alpha(v)beta3 engagement, increased tyrosine phosphorylation of focal adhesion kinase (FAK), a signaling molecule activated by integrins and able to modulate cell migration, was detected. Transfection of FAK-related nonkinase, known to compete with FAK for its correct localization and phosphorylation, caused inhibition of beta3-LNCaP cell migration, specifically on VN. These data indicate that de novo expression of alpha(v)beta3 integrin in prostate cancer cells generates a migratory phenotype that is modulated by a FAK signaling pathway. This study points to alpha(v)beta3 as potential target in prostate cancer cell invasion and metastasis.

p27(kip1) acts as a downstream effector of and is coexpressed with the beta1C integrin in prostatic adenocarcinoma.

Integrins are a large family of transmembrane receptors that, in addition to mediating cell adhesion, modulate cell proliferation. The beta1C integrin is an alternatively spliced variant of the beta1 subfamily that contains a unique 48-amino acid sequence in its cytoplasmic domain. We have shown previously that in vitro beta1C inhibits cell proliferation and that in vivo beta1C is expressed in nonproliferative, differentiated epithelium and is selectively downregulated in prostatic adenocarcinoma. Here we show, by immunohistochemistry and immunoblotting analysis, that beta1C is coexpressed in human prostate epithelial cells with the cell-cycle inhibitor p27(kip1), the loss of which correlates with poor prognosis in prostate cancer. In the 37 specimens analyzed, beta1C and p27(kip1) are concurrently expressed in 93% of benign and 84%-91% of tumor prostate cells. Forced expression of beta1C in vitro is accompanied by an increase in p27(kip1) levels, by inhibition of cyclin A-dependent kinase activity, and by increased association of p27(kip1) with cyclin A. beta1C inhibitory effect on cell proliferation is completely prevented by p27(kip1) antisense, but not mismatch oligonucleotides. beta1C expression does not affect either cyclin A or E levels, or cyclin E-associated kinase activity, nor the mitogen-activated protein (MAP) kinase pathway. These findings show a unique mechanism of cell growth inhibition by integrins and point to beta1C as an upstream regulator of p27(kip1) expression and, therefore, a potential target for tumor suppression in prostate cancer.

Modulation of cell proliferation by the integrin cytoplasmic domain.

Integrin adhesion receptors modulate cell functions, including cell proliferation and survival. The beta 1C integrin, an alternatively spliced form of beta 1A, containing a unique cytoplasmic domain sequence, inhibits cell growth in vitro. In vivo, the expression of beta 1C correlates with a benign, nonproliferative phenotype in epithelial cells. The studies discussed in this article indicate that modulation of cell proliferation, in normal or pathological conditions, might be achieved by the regulated expression of variant integrin subunits.

Factor VII G331D: a variant molecule involving replacement of a residue in the substrate-binding region of the catalytic domain.

An individual identified as having a dysfunctional factor VII was studied to seek underlying genetic defects. A heterozygous mutation in the factor VII gene exon 8 was identified as substitution of A for G at nucleotide position 10,909 [Gly331(GGC) to Asp (GAC)]. An abolished MspI restriction site was used to confirm heterozygosity for the defect. The mutation occurs within the substrate-binding pocket at a locus on the surface of the factor VII molecule containing a protein-protein interactive site for substrates, providing an explanation for the observed dysfunctional procoagulant activity.

The novel structural motif Gln795-Gln802 in the integrin beta 1C cytoplasmic domain regulates cell proliferation.

Alternative splicing of the integrin beta 1 subunit mRNA generates a variant form, beta 1C, with a unique cytoplasmic domain that differs from beta 1 for a 48-amino acid COOH-terminal sequence. The potential role of this unique sequence in modulating cellular functions was investigated using Chinese hamster ovary (CHO)1 cells transiently transfected with cDNAs coding for human integrin beta 1C or beta 1 subunits or mutants containing truncated forms of the beta 1C cytoplasmic domain. A differential effect of beta 1C and beta 1 on cell proliferation was observed. Expression of wild type beta 1 was associated with a 6-10-fold increase in cell proliferation in response to serum, as measured by [3H]thymidine incorporation. In contrast, only a 2-fold increase in cell proliferation was observed in transfectants expressing comparable levels of beta 1C. Cells expressing the beta 1C mutant truncated at Leu794 and lacking the last 31 amino acids of the cytoplasmic domain showed a 12-fold proliferation increase in response to serum. However, three beta 1C deletion mutants, lacking the COOH-terminal 23, 13, and 8 amino acids, which all contained residues Gln795-Gln802 of the variant cytoplasmic domain responded to serum stimulation with a 2-fold increase in [3H]thymidine uptake. The effect of beta 1C expression on cell proliferation was not associated with changes in exposure of integrin functional epitopes, as judged by the finding that CHO transfectants expressing beta 1C, full-length or deletion mutants, or beta 1 equally adhered to a functionally inhibitory monoclonal antibody against human beta 1 integrin. Expression of beta 1C inversely correlated with the mitogenic potential of vascular cells. Absent on growing cultured endothelial cells, surface expression of beta 1C was induced in growth-arrested, tumor necrosis factor-stimulated endothelial cells. These findings suggest that integrin alternative splicing may provide an accessory mechanism to modulate cell type-specific growth regulatory pathways during vascular cell injury in vivo.

Apoptosis is antagonized by large T antigens in the pathway to immortalization by polyomaviruses.

The viability of rat embryo cells immortalized by thermosensitive mutants of SV40 or polyoma Large T antigen is impaired at the non-permissive temperature thus demonstrating that the immortal phenotype is dominantly maintained by Large T antigens. We have observed that exposing these cells to the restrictive temperature not only induces growth arrest but also causes apoptotic cell death. We present evidence supporting the model that polyomaviruses may indeed establish immortality by antagonizing the lethal effects of tumor suppressor genes via physical interactions between their products and Large T antigens. In the case of SV40-immortalized cells REtsAF, shift-up to 39.5 degrees C dissociates Large T antigen/p53 complexes releasing wild-type p53 molecules capable of inducing apoptotic cell death. In polyomavirus-immortalized cells, apoptosis may result from an alternative pathway mediated by other unidentified negatively acting molecules.

Prothrombin Padua I: incomplete activation due to an amino acid substitution at a factor Xa cleavage site.

An individual and an affected brother previously identified as having the variant prothrombin Padua I were studied in order to identify underlying genetic defects. A heterozygous mutation in the prothrombin gene exon 8 was identified as substitution of A for G at nucleotide position 7,312 (Arg271 (CGT) to His (CAT)). An abolished RsaI restriction site was used to confirm heterozygosity for the defect. Lack of the requisite cleavage of the His271-Thr272 bond in prothrombin Padua I could prevent release of fragment 2 and block the conversion of the intermediate meizothrombin des fragment 1 to alpha-thrombin, providing an explanation of reduced potential for clotting activity and for the observed mild bleeding tendency.

SV40 induced cellular immortalization: phenotypic changes associated with the loss of proliferative capacity in a conditionally immortalized cell line.

Immortalization of rodent embryo fibroblasts by SV40 is dominantly maintained by the large T antigen. The aim of this work is to characterize some of the events associated with the loss of proliferative capacity in a rat cell line, called REtsAF, which is conditionally immortalized by the tsA58 allele of SV40 large T antigen. DNA replication is arrested less than 24 h after the shift to the restrictive temperature (39 degrees C). This arrest occurs without specificity relative to the cell cycle stage, which suggests that a function essential throughout the cell cycle is affected. A two-dimensional SDS polyacrylamide gel electrophoresis analysis of proteins shows that, although the global rate of protein synthesis is only slightly affected at 39 degrees C, the rate of accumulation of specific proteins is either increased or decreased. Finally we present biochemical and electron microscopy data showing that alterations of the mitochondria occur upon shift to 39 degrees C.