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At least 685 records · Page 38Linked to original sources
PubMed1997

Cloning and functional analysis of new members of STAT induced STAT inhibitor (SSI) family: SSI-2 and SSI-3.

Upon the corresponding ligand's stimulation, the cytokine receptors activate several signal pathways: JAK-STAT pathway, Ras-MAP kinase pathway and so on. Recently, we demonstrated that one of the STAT3 (signal transducer and activator of transcription-3) target genes could suppress the function of STAT3 and designated as SSI-1(STAT induced STAT inhibitor-1). SSI-1 is thought to play a critical role in negative feedback control of JAK-STAT signaling pathway. In the present study, we identified two novel human genes which products have homologous region in their SH2 domain and its COOH-terminal region to mouse SSI-1. Northern blotting analysis and functional studies demonstrated that SSI-2 and SSI-3 mRNA were also induced by cytokine stimulation and their forced expression in mouse myeloid leukemia cell, M1, suppressed the apoptotic effect of LIF, like SSI-1. We also demonstrated the structure of human SSI-1.

Amino Acid Sequence↗
PubMed2005

Lessons from functional analysis of AChE covalent and noncovalent inhibitors for design of AD therapeutic agents.

Determination of the 3D-structure of acetylcholinesterase (AChE) of Torpedo californica over a decade ago, and more recently that of human enzyme together with extensive targeted mutagenesis of the mammalian AChEs led to a fine mapping of the multiple functional domains within the active center of the enzyme. Many of the contributions of this active center architecture to accommodation of noncovalent ligands could be deduced from the X-ray structures of the corresponding HuAChE complexes. Yet, Michaelis complexes leading to transient covalent adducts are not amenable to structural analysis. Since the rates of formation of the covalent adducts depend predominantly on the stabilities of the corresponding Michaelis complexes, it is essential to characterize the specific interactions contributing to stabilization of these complexes. Functional analysis of interactions with HuAChE enzymes allows for such characterization for carbamates, like pyridostigmine or rivastigmine, much in the same way as that for the noncovalent therapeutic ligands nivalin or aricept. In fact, the observed differences between the affinities toward carbamates and the noncovalent ligands seem to result from specific structural characteristics of the inhibitors rather than from the decomposition path of the particular complex. Replacements at the cation binding site (Trp86), hydrogen bond network (Glu202, Tyr133, Glu450), and hydrophobic pocket result in similar effects for the covalent as well as for the noncovalent inhibitors. Also, while the effects of perturbing the aromatic trapping of the catalytic His447 for pyridostigmine and nivalin were analogous to those for the substrate, the corresponding effects for rivastigmine and aricept were quite different. Thus, elucidation of the functional architecture of the HuAChE active center is bound to be of considerable utility in the current effort to design novel covalent AChE inhibitors as therapeutics for Alzheimer's disease (AD).

Acetylcholinesterase↗
PubMed1984

Non-renin dependent hypertension in renal allograft rejections. A structural and functional analysis.

A 25-year-old recipient of a cadaveric renal allograft underwent three acute rejection episodes within the first 40 days after transplant, the final episode necessitating nephrectomy. The saralasin acetate infusion test and plasma renin activity (as measured by radioimmunoassay of angiotensin I) were used as functional tests of the renin-angiotensin axis. Biopsy specimens of the allograft one hour after implantation and sections of the nephrectomy specimen were fixed and stained with hematoxylin-eosin for structural analysis. On three separate dates, during the final rejection episode, saralasin acetate infusion of up to 20 micrograms/kg/min failed to lower BP significantly. The final two trials were preceded by furosemide administration the previous day to reduce fluid volume. Plasma renin activity was low on all three dates. On nephrectomy, the allograft was noted grossly to be infarcted. Histologic examination revealed cortical necrosis, markedly narrowed or occluded intrarenal vessels, and extremely narrowed large vessels within the renal pelvis. The allograft renal artery was thickened and narrowed. From our structural and functional analysis we conclude that (1) hypertension, in this case, was probably volume dependent and was clearly renin independent; and (2) the low renin levels are explainable on the basis of extensive vascular occlusion producing renal infarction and resulting in a kidney incapable of producing significant amounts of renin (autonephrectomy).

Adult↗
PubMedDate not supplied

Functional analysis of the cooled rat testis.

Direct cooling of the testis results in the depletion of most germ cells in vivo. Germ cell-depleted testes are now commonly used to investigate spermatogenic regeneration and can serve as recipients for germ cell transplantation. The present study explored the effects of cooling rat testes on the depletion of endogenous germ cells, spermatogenic regeneration, and Sertoli cell function. Adult rat testes were cooled with iced Ringer's solution for 60 minutes, which results in the initiation of apoptotic germ cell loss within 8 hours. Pachytene spermatocytes at stages XII-I were the cells most sensitive to cooling. In 46%-67% of seminiferous tubule cross-sections, only Sertoli cells remained in the cooled testes 3-10 weeks after treatment. Germ cell loss was accompanied by a significant decrease in circulating inhibin B and an increase in follicle-stimulating hormone concentrations, which indicated a change in Sertoli cell function. Quantitative analysis of mRNA expression associated with apoptotic signals showed no significant uniform changes among the cooled testes, although some individuals had a distinct up-regulation of FAS mRNA at 24 hours. Attempts to use the cooled testes as recipient testes for mouse-to-rat germ cell transplantation were undertaken, but none of the mouse germ cells transplanted into the testes 15-34 days after cooling appeared to have undergone spermatogenesis 64-92 days after transplantation. These data suggest that modifications to Sertoli cell function resulting from testicular cooling create an environment that is unable to support spermatogenesis by donor germ cells.

Animals↗
PubMedDate not supplied

Role of bradykinin in inflammatory arthritis: identification and functional analysis of bradykinin receptors on human synovial fibroblasts.

Receptor type and function of bradykinin (BK) receptors on human synovial fibroblasts (HSF) was determined. Scatchard analysis of [3H]BK saturation binding to intact synovial cells revealed a single binding site, with a Kd of 3.8 +/- 0.6 nM. HSF express approximately 50,000 BK sites/cell. Specificity of [3H]BK binding was confirmed by the ability of several BK peptide agonists and antagonists to inhibit binding in a dose dependent manner. The rank order of potency for agonist inhibition of [3H]BK and the inability of selective antagonists of the B1-type to displace binding suggest that the BK receptor on HSF is a B2 subtype receptor. The addition of BK to HSF caused a time and concentration dependent increase in PGE2 production. This BK induced PGE2 production was blocked by specific B2 type BK antagonists and not by B1 antagonists. The results of this study identify B2 type BK receptors on synovial fibroblasts and suggest that BK may be a primary mediator in inflammatory arthritis.

Arthritis, Rheumatoid↗
PubMed1999

Revision of the functional analysis and structural features of immortalized dendritic cell lines derived from mice lacking both type I and type II interferon receptors.

Cell lines with dendritic morphology were obtained from several organs of mice lacking both type I and II interferon receptors after a retroviral immortalization procedure. Their surface antigen phenotype was analyzed by flow cytometry with monoclonal antibodies and their functional capabilities to induce antigen dependent specific immune response was also determined. Two representative cell lines called AG101 (skin-derived) and AG116 (brain-derived) were cloned and analyzed in more detail. Cytometric analysis showed that they constitutively expressed the cell surface markers CD45, CD1 1b, MHC class II, F4/80, N418, B7-2 and ICAM1. Despite both cell lines expressing Thy-1 only, the AG116 show CD4 but both were negative for CD8 and B220. The functional analysis showed that the cell lines were capable and very efficient at actively taking up, processing and presenting soluble antigens like Ovalbumin (OVA). The processed protein was presented by both cell lines to the OVA-peptide-specific T cell hybridoma BO97.105, which responded specifically with the production of IL-2. In addition AG101 and AG116 cells were able to induce in naive allogeneic T cells, a mixed lymphocyte reaction, determined by T cell proliferation and T cell dependent L-2 production. Moreover, the capability to prime naive syngeneic T cells was also demonstrated by loading AG101 and AG116 cells with soluble antigens, then co-culturing with naive T cells which yielded both T cell proliferation and IL-2 production. The cell lines priming capability was shown to be quite similar, as freshly isolated and cultured cutaneous dendritic cells from 129Sv/Lv mice (wtDCs) to prime naive T cells. In addition to a basal production of IL-6, the cell lines were found to increase their synthesis of IL-6 and IL-12 p40 after interaction with T cells in a similar way as mature wtDCs. Also it was determined that DC cell lines devoid of functional IFN system allow the replication of infectious agents like BDV and even are able to induce in vivo a specific humoral response against proteins of the BDV. Therefore, the cell lines AG101 and AG116 show structural and functional features of DCs. They are able to take up, process and present antigens as well as prime naive T cell in a similar manner as nontransformed DC. Therefore, these cell lines will be useful for studying the interactions between DC and the effectors cells of the immune response at the clonal level and in the absence of functional interferon receptors.

Animals↗
PubMed1994

Discovery and structure-function analysis of alpha-melanocyte-stimulating hormone antagonists.

Structure-function relationships of alpha-melanocyte-stimulating hormone (alpha-MSH, alpha-melanotropin) were investigated and novel alpha-MSH receptor antagonists were identified. Based on the alpha-MSH-[5-13] peptide sequence, a multi-use peptide library consisting of 31,360 structurally different candidates was generated, and approximately 40% of the peptides were individually screened for their ability to block receptor function. This led to the identification of antagonists with a range of potencies and revealed structural requirements necessary for receptor inactivation. The most potent antagonist Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val-NH2 has an IC50 value of 11 +/- 7 nM. Analysis revealed that D-Trp5 and Phe6 were crucial to its antagonistic properties which could be potentiated by D-Phe3. This study demonstrates that residues in positions 5-6, 7-9, and 10 of the alpha-MSH sequence constitute crucial determinants for potent antagonist activity.

Amino Acid Sequence↗
PubMed1991

Structural and functional analysis of the regulatory sequences of the ets-1 gene.

The ets-1 gene is a human homolog of the oncogene v-ets of avian acute leukemia virus E26. The ets-1 gene is preferentially expressed in lymphoid cells. To understand the regulation of the ets-1 gene expression the regulatory sequences of this gene were identified and isolated. The promoter is found to be functioning in the lymphoid cell line Daudi and the erythromyeloid cell line K562 but not in the promyelocytic cell line HL60. Sequence analysis indicates that the ets-1 promoter does not contain the TATA or CAT box. It contains multiple transcription initiation sites in a tight cluster. The promoter contains one binding site each for AP1 and AP2. It has one definitive and five presumptive sp1-binding sites. In addition, the ets-1 promoter contains one ets-1 protein-binding site. Functional analysis has shown that c-jun enhances the activity of the ets-1 promoter, whereas the combination of c-fos and c-jun expressions has no synergistic effect. The expression of exogenous AP2 and ets-1 also enhances the activity of the ets-1 promoter. These results suggest that the AP1, AP2 and ets-1 proteins have a positive regulatory effect. The presence of the ets-1 protein-binding site indicates the existence of an autoregulatory mechanism for the expression of the ets-1 gene. Our results suggested that the presence of a negative regulatory element upstream of the region where most of the positive regulators are located.

Base Sequence↗
PubMed2006

Functional analysis of CHX21: a putative sodium transporter in Arabidopsis.

The functional role of CHX21, a member of the Arabidopsis thaliana CHX cation transporter family, has been investigated in plants growing under "ideal" conditions and in the presence of elevated NaCl levels. In public databases, AtCHX21 (At2g31910) is annotated as a putative Na+/H+ antiporter. In this study, Southern analysis was used to identify a genotype that contained a single transposon insertion within its genome; using PCR, this insertion was shown to be within the CHX21 locus. No CHX21 transcript was detectable in Atchx21 (mutant) plants using RT-PCR. In the absence of salt stress, Atchx21 showed significant quantitative differences from the wild type (AtCHX21) in development with respect to characters such as rosette width and flowering time. In the presence of 50 mM NaCl, (i) roots of Atchx21 elongated more slowly than the wild type, (ii) the leaf sap Na+ concentration was significantly lower in Atchx21 compared with the wild type, and (iii) the concentra) in the xylem was lower compared with the wild type. The concentration of Na+ exported from the leaf in the phloem was unchanged. Thus, loading of Na+ into the root xylem could explain changes in leaf concentration of Na+. This hypothesis was supported by immunolocalization which demonstrated that the AtCHX21 transporter could only be detected in root endodermal cells. Immunogold labelling of ultra-thin sections, followed by transmission electron microscopy, demonstrated the localization of the protein in the plasma membrane. The data demonstrate that the CHX21 transporter may play a role in regulation of xylem Na+ concentration and, consequently, Na+ accumulation in the leaf.

Arabidopsis↗
PubMed2001

Functional analysis of leucine aminopeptidase in Caenorhabditis elegans.

To investigate the function of the enzyme leucine aminopeptidase in nematodes, a Caenorhabditis elegans leucine aminopeptidase gene identified in the genome sequence was functionally analysed by transfection of a leucine aminopeptidase beta-galactosidase reporter construct and characterisation of a null mutant. The leucine aminopeptidase transgene is expressed along the length of the gut, and immunolocalisation shows the enzyme in the buccal cavity, pharynx, anterior gut and rectum. It is constitutively expressed as seen by analysis of cDNAs constructed from mRNAs of nematodes taken at 2 h intervals through the life-cycle; and by western blot analysis of protein from the same set of nematodes. Leucine aminopeptidase null mutants had a slower growth rate and delayed onset of egg-laying. We suggest that in C. elegans, leucine aminopeptidase is a digestive enzyme.

Amino Acid Sequence↗
PubMed1977

Diagnostic effectiveness of biochemical liver-function tests, as evaluated by discriminant function analysis.

I evaluated the diagnostic value of routinely ordered liver-function tests in 175 biopsy-proven cases of hepatic disease by use of stepwise discriminant analysis. The tests studied-total and "direct" bilirubin, alkaline phosphatase, lactate dehydrogenase, and aspartate aminotransferase-correctly classified 45-73% of cases, depending on the homogeneity of the diagnostic groups. Aspartate aminotransferase and alkaline phosphatase were the best discriminators. When all tests were used in the most homogeneous groups (tumors, cirrhosis, and hepatitis), there was a stepwise improvement in diagnostic accuracy from 51 to 73%.

Alkaline Phosphatase↗
PubMed2002

X-ray crystal structure and functional analysis of vaccinia virus K3L reveals molecular determinants for PKR subversion and substrate recognition.

The vaccinia virus protein K3L subverts the mammalian antiviral defense mechanism by inhibiting the RNA-dependent protein kinase PKR. K3L is a structural mimic of PKR's natural substrate, the translation initiation factor eIF2alpha. To further our understanding of K3L inhibitory function and PKR substrate recognition, we have solved the 1.8 A X-ray crystal structure of K3L. The structure consists of a five-strand beta barrel with an intervening helix insert region similar in topology to the functionally divergent S1 domain. Mutational analysis identifies two proximal regions of the K3L structure as possessing specialized PKR binding and inhibitory function. Further analysis reveals that PKR dimerization composes a key switch that regulates both its catalytic activation and its molecular recognition of K3L and eIF2alpha.

Amino Acid Motifs↗
PubMed2005

Short- and long-range order in the positive electrode material, Li(NiMn)0.5O2: a joint X-ray and neutron diffraction, pair distribution function analysis and NMR study.

The local environments and short-range ordering of LiNi(0.5)Mn(0.5)O(2), a potential Li-ion battery positive electrode material, were investigated by using a combination of X-ray and neutron diffraction and isotopic substitution (NDIS) techniques, (6)Li Magic Angle Spinning (MAS) NMR spectroscopy, and for the first time, X-ray and neutron Pair Distribution Function (PDF) analysis, associated with Reverse Monte Carlo (RMC) calculations. Three samples were studied: (6)Li(NiMn)(0.5)O(2), (7)Li(NiMn)(0.5)O(2), and (7)Li(NiMn)(0.5)O(2) enriched with (62)Ni (denoted as (7)Li(ZERO)Ni(0.5)Mn(0.5)O(2)), so that the resulting scattering length of Ni atoms is null. LiNi(0.5)Mn(0.5)O(2) adopts the LiCoO(2) structure (space group Rm) and comprises separate lithium layers, transition metal layers (Ni, Mn), and oxygen layers. NMR experiments and Rietveld refinements show that there is approximately 10% of Ni/Li site exchange between the Li and transition metal layers. PDF analysis of the neutron data revealed considerable local distortions in the layers that were not captured in the Rietveld refinements performed using the Bragg diffraction data and the LiCoO(2) structure, resulting in different M-O bond lengths of 1.93 and 2.07 Angstroms for Mn-O and Ni/Li-O, respectively. Large clusters of 2400-3456 atoms were built to investigate cation ordering. The RMC method was then used to improve the fit between the calculated model and experimental PDF data. Both NMR and RMC results were consistent with a nonrandom distribution of Ni, Mn, and Li cations in the transition metal layers; both the Ni and Li atoms are, on average, close to more Mn ions than predicted based on a random distribution of these ions in the transition metal layers. Constraints from both experimental methods showed the presence of short-range order in the transition metal layers comprising LiMn(6) and LiMn(5)Ni clusters combined with Ni and Mn contacts resembling those found in the so-called "flower structure" or structures derived from ordered honeycomb arrays.

Journal Article↗
PubMed2000

Structure-function analysis of protease-activated receptor 4 tethered ligand peptides. Determinants of specificity and utility in assays of receptor function.

Thrombin activates protease-activated receptors (PARs) by specific cleavage of their amino-terminal exodomains to unmask a tethered ligand that binds intramolecularly to the body of the receptor to effect transmembrane signaling. Peptides that mimic such ligands are valuable as agonists for probing PAR function, but the tethered ligand peptide for PAR4, GYPGKF, lacks potency and is of limited utility. In a structure-activity analysis of PAR4 peptides, AYPGKF was approximately 10-fold more potent than GYPGKF and, unlike GYPGKF, elicited PAR4-mediated responses comparable in magnitude to those elicited by thrombin. AYPGKF was relatively specific for PAR4 in part due to the tyrosine at position 2; substitution of phenylalanine or p-fluorophenylalanine at this position produced peptides that activated both PAR1 and PAR4. Because human platelets express both PAR1 and PAR4, it might be desirable to inhibit both receptors. Identifying a single agonist for both receptors raises the possibility that a single antagonist for both receptors might be developed. The AYPGKF peptide is a useful new tool for probing PAR4 function. For example, AYPGKF activated and desensitized PAR4 in platelets and, like thrombin, triggered phosphoinositide hydrolysis but not inhibition of adenylyl cyclase in PAR4-expressing cells. The latter shows that, unlike PAR1, PAR4 couples to G(q) and not G(i).

Adenylate Cyclase Toxin↗
PubMed2002

Transient mismatch repair gene transfection for functional analysis of genetic hMLH1 and hMSH2 variants.

BACKGROUND: Germline mutations in the mismatch repair (MMR) genes hMLH1 and hMSH2 can cause hereditary non-polyposis colorectal cancer (HNPCC). However, the functional in vitro analysis of hMLH1 and hMSH2 mutations remains difficult. AIMS: To establish an in vitro method for the functional characterisation of hMLH1 and hMSH2 mutations. METHODS: hMLH1 and hMSH2 wild type (wt) genes and several mutated subclones were transiently transfected in mismatch repair deficient cell lines (HCT-116 and LOVO). Apoptosis, proliferation, and regulation of mRNA expression and protein expression of interacting proteins were analysed by Hoechst staining, AlamarBlue staining, real time polymerase chain reaction, and western blotting, respectively. RESULTS: The protein expression of hMLH1 and hMSH2 mutants was significantly decreased after transfection compared with wild type transfections. The hMLH1 and hMSH2 interacting proteins hPMS2 and hMSH6 became detectable only after transfection of the respective wild type genes. In parallel, hMSH6 mRNA levels were increased in hMSH2 wt transfected cells. However, hPMS2 mRNA levels were independent of the mutation status of its interacting partner hMLH1, indicating a post-transcriptional regulating pathway. In the hMLH1 deficient HCT-116 cell line apoptosis was not affected by transfection of any mismatch repair gene, whereas complementation of hMSH2 deficiency in LOVO cells increased apoptosis. Conversely, proliferative activity of HCT-116 was decreased by complementation with hMLH1wt and unaffected in hMSH2 deficient LOVO cells. CONCLUSION: These data show that the cellular role of the MMR genes and its mutations are assessable in a simple transient transfection system and show the influence of MMR gene regulation on major cell growth regulating mechanisms. This method is applicable for the functional definition of mutations in hMLH1 and hMSH2 genes observed in patients with suspected HNPCC.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Structure-function analysis of the active sites of complement receptor type 1.

Two functionally distinct but homologous sites in complement receptor type 1 (CR1) (CD35) were further characterized by homologous substitution mutagenesis of two CR1 derivatives, each containing one site. In both sites, reducing negative and/or increasing positive charge augmented interaction with iC3/C3b and C4b, supporting a role of ionic forces in the binding reaction. In one case, substitution of Asp at the end of complement control protein repeat (CCP) 2 with an Asn transformed the protein, with negligible cofactor activity and iC3 binding, into a mutant with activities similar to native CR1. Consequently, this protein, one-fourth the size of CR1, is a therapeutic candidate for a complement inhibitor. Another important observation is that the residues between two CCPs contribute to activity, probably because they influence positioning of one CCP relative to the next. The initial characterization of the third CCP of an active site led to identification of three peptides necessary for binding. In line with earlier findings for the first two CCPs, interactions with iC3/C3b are similar but not identical to those with C4b, implying overlapping but distinct binding domains. Moreover, changes in cofactor activity usually, but not always, parallel alterations in binding, indicating that these two activities are separable. We also mapped epitopes for a blocking and a function enhancing monoclonal antibody. Their effects can be explained by epitope location. The first antibody binds near functionally important residues. The second may shield inhibitory (negatively charged) residues. These results represent a comprehensive analysis of the active sites of CR1, which is built of modules found in more than 50 mammalian proteins.

Amino Acid Sequence↗
PubMed2006

[Partial lease squares approach to functional analysis].

We extend the partial least squares (PLS) approach to functional data represented in our models by sample paths of stochastic process with continuous time. Due to the infinite dimension, when functional data are used as a predictor for linear regression and classification models, the estimation problem is an ill-posed one. In this context, PLS offers a simple and efficient alternative to the methods based on the principal components of the stochastic process. We compare the results given by the PLS approach and other linear models using several datasets from economy, industry and medical fields.

Biometry↗