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PubMed2003

Biochemical and functional characterization of the human zona pellucida.

A successful interaction between spermatozoa and the zona pellucida is critical for fertilization. This biological step reflects multiple sperm functions, including the acquisition and completion of capacitation, recognition and binding to specific zona pellucida receptors, and induction of the physiological acrosome reaction. The recognition of carbohydrate sequences by complimentary receptors has been demonstrated in gamete interaction in different animal species. It has been proposed that, in the human, sperm binding to the zona pellucida requires a 'selectin-like' interaction. The hemizona assay (a unique internally controlled bioassay that evaluates tight binding of human spermatozoa to the homologous zona pellucida) and advanced methods of carbohydrate analysis have been used to test this hypothesis. Compelling evidence exists to demonstrate that oligosaccharide recognition is also required for specific, tight human gamete binding. The induction of the acrosome reaction using the physiological inducers, i.e. the zona pellucida and progesterone, was also examined. It has also been demonstrated that there is a priming effect of the steroid on the acrosome reaction inducing capacity of the zona pellucida. These studies may allow for a better understanding of human gamete interaction in physiological and pathological situations.

Acrosome↗
PubMed1998

Phenotypic and functional characterization of a panel of cytotoxic murine NK cell clones that are heterogeneous in their enhancement of Ig secretion in vitro.

NK cells not only function as cytotoxic effector cells, but also have immunoregulatory roles including the enhancement of Ig secretion. To have a stable and uniform population of NK cells to study their role in Ig secretion, we generated murine NK clones. Thus, culture of splenocytes from mice that were homozygous for a mutation in the p53 tumor suppressor gene (p53-KO) with IL-2 and poly(IC) resulted in a long-term NK line, from which four stable clones were derived. This approach also yielded a long-term NK line from splenocytes of normal C57BL/6 mice. Identification of the clones as members of the NK lineage was based on large granular morphology, expression of NK-TR and absence of TCR gene rearrangement. Flow cytometry revealed that all clones expressed IL-2R alpha and beta, chains and B220, but no CD3, NK1.1, DX5 or Ly-49. RT-PCR analysis showed heterogeneity in NK1.1 gene expression, and demonstrated expression of perforin and several granzymes in all clones. Three out of four clones lysed YAC-1, but not P815 target cells, corresponding to a pattern of NK specificity. All NK clones enhanced Ig secretion in an in vitro model for T cell-independent type 2 antigens, albeit to varying degrees. We found no correlation between the degree of helper activity of the NK clones and the level of their cytotoxic activity on YAC-1 targets. Thus, we established murine NK clones, and show that they mediate both cytotoxicity and enhancement of Ig secretion.

Animals↗
PubMed1995

Identification and functional characterization of mouse CD29 with a mAb.

The beta 1 integrin subfamily, alternatively called very late activation antigen (VLA), has been implicated in various cellular functions. In this study, we generated a mAb against the mouse beta 1 subunit (CD29) to examine the functional property of mouse VLA proteins. After immunization with affinity-purified mouse VLA-4 (alpha 4 beta 1), a hamster mAb, HM beta 1-1, was established by screening mAb that reacted with alpha 4-negative neuroblastoma C1300. The antigen defined by HM beta 1-1 was widely distributed in various mouse cell lines and HM beta 1-1 immunoprecipitated a 110-120 kDa protein common to VLA-1 and VLA-6, indicating that HM beta 1-1 recognizes the beta 1 subunit of mouse integrins. We then examined the inhibitory effect of HM beta 1-1 on VLA-dependent cell adhesion and activation. HM beta 1-1 blocked the adhesion of mouse tumor cell lines to extracellular matrix proteins including collagen, laminin and fibronectin. Moreover, splenic T cell proliferation induced by anti-CD3 mAb and allogeneic mixed lymphocyte response were strongly inhibited by HM beta 1-1 in combination with an anti-LFA-1 mAb. We conclude that HM beta 1-1 reactive with mouse CD29 can inhibit VLA-dependent cellular functions and, thus, would be useful for studying the physiological role of beta 1 integrins in vivo.

Animals↗
PubMed2001

Functional characterization of the C-terminus of the human ether-à-go-go-related gene K(+) channel (HERG).

1. In the present study the functional role of the C-terminus of the human ether-à-go-go-related gene K(+) channel HERG was investigated using a series of C-terminal deletion constructs expressed in Xenopus oocytes. 2. Constructs with deletions of 311 or more amino acid residues failed to form functional channels. Truncation by 215 amino acid residues or fewer had no discernable effects on channel activity. Truncation by 236 or 278 amino acid residues accelerated deactivation, and caused a faster recovery from inactivation. 3. In high extracellular K(+), channel deactivation of HERG results from the binding of the N-terminus to a site within the pore. This slows channel deactivation by a knock-off mechanism. Here, it was shown that C-terminal deletions also abolished this effect of high extracellular K(+). Mutants containing deletions in both the N- and C-termini deactivated with rates similar to those observed in individual deletion mutants. 4. In contrast, experiments with double-deletion constructs showed additive effects of the N- and C-termini on the voltage dependence of activation, and on the kinetics of inactivation and recovery from inactivation. The reduction of inactivation in these mutants contributed to an increase in peak current amplitude. 5. These results indicate that residues within the C-terminus of HERG play a role in channel expression as well as in most aspects of channel gating. The regulation of channel deactivation is likely to be mediated by an interaction with the N-terminus, but the regulation of the voltage dependence of activation, and of rate processes associated with inactivation, does not require the N-terminus.

Animals↗
PubMed2004

Functional characterization of mouse alpha4beta2 nicotinic acetylcholine receptors stably expressed in HEK293T cells.

Mouse alpha4beta2 nicotinic acetylcholine receptors (nAchRs) were stably expressed in HEK293T cells. The function of this stable cell line, termed mmalpha4beta2, was assessed using an aequorin-based luminescence method that measures agonist-evoked changes in intracellular calcium. Agonist-elicited changes in intracellular calcium were due primarily to direct entry of calcium through the alpha4beta2 channel, although release of calcium from intracellular stores contributed approximately 28% of the agonist-evoked response. Agonist pharmacologies were very similar between the mmalpha4beta2 cells and most cell lines that stably express human alpha4beta2 nAchRs. Based on agonist profiles and sensitivity to the antagonist dihydro-beta-erythroidine (DHbetaE), the predominant alpha4beta2 nAchR expressed in the mmalpha4beta2 cells exhibits a pharmacology that most resembles the DHbetaE-sensitive component of 86Rb+ efflux from mouse brain synaptosomes. However, when evaluated with the aequorin assay, the mmalpha4beta2 nAchR was found to be atypically sensitive to blockade by the presumed alpha7-selective antagonist methyllycaconitine (MLA), exhibiting an IC50 value of 31 +/- 0.1 nm. Similar IC50 values have been reported for the MLA inhibition of nicotine-stimulated dopamine release, a response that is mediated by beta2-subunit-containing nAchRs and not alpha7-subunit-containing nAchRs. Consequently, at low nanomolar concentrations, MLA may not be as selective for alpha7-containing nAchRs as previously thought.

Acetylcholine↗
PubMed1995

Feline immunodeficiency virus reverse transcriptase: expression, functional characterization, and reconstitution of the 66- and 51-kilodalton subunits.

The two subunits of the feline immunodeficiency virus (FIV) reverse transcriptase (RT) were cloned and functionally expressed in Escherichia coli. The recombinant proteins are enzymatically active as homodimers (p66 and p51) as well as a heterodimer p66/p51. The biochemical properties of the FIV RT are very similar to those of the counterpart of the human immunodeficiency virus type 1 in being an RNA-dependent and DNA-dependent DNA polymerase. When a double-stranded DNA containing a small gap of 26 nucleotides was tested, we found a new activity of the FIV RT p66/p51 heterodimer--the cat viral enzyme could perform strand displacement DNA synthesis of approximately 300 bases. The FIV RT homodimer p66 alone could carry out limited strand displacement DNA synthesis, but this activity was stimulated by the p51 subunit at a molar ratio of one molecule of p66 to five molecules of p51. On the other hand, the homodimeric p51 itself was unable to fill a small gap of 26 nucleotides in a double-stranded DNA substrate and was not active by itself in strand displacement DNA synthesis. These data are in agreement with an earlier finding of strand displacement DNA synthesis by human immunodeficiency virus type 1 RT (M. Hottiger, V.N. Podust, R.L. Thimmig, C.S. McHenry, and U. Hübscher. J. Biol. Chem. 269:986-991, 1994). Our data therefore suggest a general and important function of lentiviral p51 subunits in strand displacement DNA synthesis which appears to be required in later stages of the lentiviral replication cycle, when DNA-dependent DNA synthesis occurs on double-stranded DNA.

Amino Acid Sequence↗
PubMed2006

[Isolation, identification and functional characterization of human CD4+ CD25+ Treg cells from human peripheral blood].

AIM: To isolate CD4(+) CD25(+) Treg cells from human peripheral blood, and study their functional characteristics. METHODS: The expressions of Foxp3 in human CD4(+) CD25(+) Treg cells were test by RT-PCR. The regulatory properties of CD4(+) CD25(+) Treg cells were assessed by co-culturing with CD4(+) CD25(-) T cells and CD8(+) T cells, or adding exogenous IL-2. The detection of intracellular cytokine production of IL-4, IL-10 and IFN-gamma was done by flow cytometry. RESULTS: CD4(+) CD25(+) Treg cells highly expressed Foxp3 and mainly synthesized IL-10 that suppressed the proliferation of CD4(+) CD25(-) T and CD8(+) T cells. Its suppressive function was reversed by high concentration of IL-2 and (or) IL-4. CONCLUSION: CD4(+) CD25(+) Treg cells exhibited a subpopulation of immunoregulatory T cells with suppressive function, which could be reversed by high concentration of IL-2.

Blood Cells↗
PubMed1985

Functional characterization of human T lymphocyte subsets distinguished by monoclonal anti-leu-8.

Previous studies have shown that monoclonal anti-Leu-8 antibody identifies functionally distinct subpopulations within both the Leu-2 (T8+) and Leu-3 (T4+) lineages of human T lymphocytes. We now report in detail on the tissue distribution of the Leu-8 antigen and on extensive functional studies of T cells subsets distinguished by their expression or lack of expression of this marker. Leu-8 is present on a wide variety of hematologic cells, including granulocytes, T and B lymphocytes, monocytes, and null or NK cells. Within lymph nodes and tonsils, Leu-8 is absent from both B and T cells within germinal centers but is present on nearly all paracortical lymphocytes. Leu-8 is present on most but not all EBV-transformed B cell lines, reflecting its presence on a subset of normal peripheral blood B cells. None of six malignant T cell lines tested were Leu-8+, whereas most circulating T cells are Leu-8+. Although standard immunoprecipitation techniques failed to demonstrate any specific bands on SDS polyacrylamide gels, the antigenic determinant recognized by anti-Leu-8 is protein or protein-associated, because brief treatment of target cells with pronase abrogated binding of anti-Leu-8. Both Leu-3+8+ and Leu-3+8- cells proliferated in response to several soluble antigens and to autologous and allogeneic non-T cells. Nonetheless, nearly all of the helper T cells for PWM- and AMLR-induced PFC were contained within the Leu3+8- subset. Optimal suppression of the PWM-induced PFC response required both Leu-2+8+ and Leu-2+8- cells, and irradiation of either subset with 3000 R abrogated the capacity of the recombined subsets to effect suppression. In contrast to help for B cell differentiation, both Leu-3+8+ and Leu-3+8- cells were capable of amplifying the development of allospecific T killer cells; precursor and effector T killer cells could be found within both Leu-2+8+ and Leu-2+8- subpopulations. The correlation between Leu-8 phenotype and selected immune functions of T cells (and B cells; see companion paper) indicates that anti-Leu-8 distinguishes important immunoregulatory T and B lymphocyte subsets in man.

Animals↗
PubMed2005

Functional characterization of the Candida albicans CRZ1 gene encoding a calcineurin-regulated transcription factor.

Calcineurin is a phosphoprotein phosphatase devoted to the transduction of Ca(2+)-signals in eukaryotes. In the human pathogen Candida albicans, calcineurin function is required for cell morphogenesis, azole tolerance, membrane stress responses, survival in serum and virulence in mice. Molecular mechanisms as well as targets downstream C. albicans calcineurin involved in all these processes are still uncharacterized. Here we report the characterization of a C. albicans Crz1 calcineurin-regulated transcription factor using a Saccharomyces cerevisiae crz1Delta defective strain as heterologous host. CaCrz1p fulfils the function of its S. cerevisiae homolog protein to control the expression of several Ca(2+)/calcineurin-responsive genes acting on the CDRE sequence in promoters. In the model yeast, CaCrz1p activity and localization are regulated by calcineurin. Deletion of CRZ1 gene renders C. albicans hypersensitive to alkaline cations and membrane stress conditions, including that elicited by SDS and antifungal azoles. Our findings indicate that CaCrz1p is member of a calcium-regulated pathway required for the maintenance of membrane integrity.

Binding Sites↗
PubMed2003

Cloning and functional characterization of the multidrug resistance-associated protein (MRP1/ABCC1) from the cynomolgus monkey.

The multidrug resistance-associated protein 1 (ABCC1) gene from human (hMRP1), dog (canMRP1), and mouse (muMRP1) all encode proteins that efficiently transport the endogenous MRP1 substrate glutathione-S-leukotriene C(4) and confer resistance to anticancer agents, including vincristine and etoposide. hMRP1 also confers resistance to anthracyclines, whereas this is not true of canMRP1 or muMRP1. To determine whether MRP1 from another animal species used in toxicological studies would be more functionally similar to hMRP1, we cloned and characterized two alleles of the MRP1 homologue from the cynomolgus monkey Macaca fascicularis (monMRP1). The monMRP1 cDNAs encode proteins of 1531 residues that are 98, 90, and 88% identical to hMRP1, canMRP1, and muMRP1, respectively. Stable overexpression of both monMRP1 alleles and hMRP1 in transformed human embryonic kidney cells was achieved using an episomal expression vector. Transporters encoded by both monMRP1 alleles were functionally very similar to hMRP1. monMRP1 conferred an increased resistance to vincristine and etoposide and transported glutathione-S-leukotriene C(4) into membrane vesicles. In addition, MRP1-mediated drug resistance was effectively reversed in monMRP1 and hMRP1 transfectants by LY402913, a new MRP1-selective inhibitor in the class of tricyclic isoxazoles. However, monMRP1 transporters conferred a reduced level of resistance to the anthracyclines doxorubicin, daunorubicin, and epirubicin relative to hMRP1, although resistance levels were significant relative to vector control cells. These functional differences between human and monkey MRP1 transporters will need to be considered when designing pharmacokinetic and toxicological studies for the preclinical evaluation of MRP1 modulators.

Adenosine Triphosphate↗
PubMed2006

Functional characterization of HIV-1 Nef mutants in the context of viral infection.

Nef is an important pathogenesis factor of HIV-1 with a multitude of effector functions. We have designed a broad panel of isogenic viruses encoding defined mutants of HIV-1(SF2) Nef and analyzed their biological activity in the context of productive HIV-1 infection. Analysis of subcellular localization, virion incorporation, downregulation of cell surface CD4 and MHC-I, enhancement of virion infectivity and facilitation of HIV replication in primary human T lymphocytes mostly confirmed the mapping of Nef determinants previously reported upon isolated expression of Nef. However, reduced activity in downregulation of CD4, infectivity enhancement and virion incorporation of a Nef variant (Delta12-39) lacking an amphipatic helix required for binding of a cellular kinase complex and the association of Nef with MHC-I/AP-1 suggested a novel role of this N-terminal motif. The SH3 binding motif of Nef was partially required for infectivity enhancement and replication but not for receptor downmodulation. In contrast to previous results obtained using other Nef alleles, non-myristoylated SF2-Nef was only partly defective when expressed during HIV infection and was present in HIV-1 particles. Importantly, incorporation of Nef into HIV-1 virions was not required for any of the tested Nef activities. Altogether, this study provides a broad characterization and mapping of multiple Nef activities in HIV-infected cells. The results emphasize that multiple activities govern Nef's effects on HIV replication and argue against a role of virion incorporation for Nef's activity as pathogenicity factor.

CD4 Antigens↗
PubMed1993

Physical and functional characterization of the ColS8 plasmid.

The restriction map of the 5.1-kb colicinogenic plasmid ColS8 is reported. Transposon-insertion mutagenesis has been carried out to investigate the location of the various functional regions of this plasmid. Twenty-six independent ColS8::Tn1 insertions and six different deletion mutant plasmids were isolated, and the locations of the insertions and deletions were determined. The mapping of the transposon-insertion sites, together with characterization of the phenotypes of these mutants, permitted the localization of the regions of DNA involved in colicin production, colicin S8 immunity and mobilization by other plasmids. There is a polar effect in some mutants in which single insertions result in the non-expression of all three functions. In minicell preparations, the ColS8 plasmid directed the synthesis of colicin S8 (60 kDa) and a 14-kDa immunity protein. One deletion mutant with a colicin-less phenotype can synthesize colicin S8 in minicells, which placed the DNA region involved in colicin release between the colicin production and immunity regions. Both the 60-kDa and 14-kDa proteins are expressed from pColS8 in maxicell preparations.

Colicins↗
PubMed1998

Purification and functional characterization of an 85-kDa gelsolin from the ascidians Microcosmus sulcatus and Phallusia mammilata.

From the pharyngeal baskets of the ascidians Microcosmus sulcatus and Phallusia mammilata we have purified an 85-kDa protein that is characterized as a member of the gelsolin family. These proteins from both species show the same behaviour in functional assays. The ascidian gelsolin binds two actin monomers in a highly cooperative manner. This complex formation is Ca(2+)-dependent, but not completely reversible, as on removal of Ca2+ one actin monomer dissociates leaving a 1:1 complex between gelsolin and G-actin. The properties of F-actin severing and G-actin nucleation depend on the presence of free Ca2+ in a micromolar range, with half maximum activation at about 3 x 10(-6) M. The protein becomes inactivated when Ca2+ concentrations of 0.5 mM are exceeded. Fragmentation of F-actin by the ascidian gelsolin is comparably fast to that of vertebrate gelsolin. A steady state of actin fragmentation is reached within 2-4 s. Promotion of G-actin nucleation is also comparable to that of vertebrate gelsolin. Regarding functional aspects, the ascidian gelsolin is more closely related to vertebrate gelsolin than to an arthropod gelsolin from crayfish tail muscle.

Actins↗
PubMed1991

Functional characterization of RNA-dependent DNA polymerase and RNase H activities of a recombinant HIV reverse transcriptase.

The DNA polymerase and RNase H activities of HIV reverse transcriptase are both essential for HIV replication. Although the two activities are both catalyzed by a single polypeptide, they are physically separate; i.e., the DNA polymerase resides in the N-terminal domain whereas the RNase H is localized in the C-terminal domain. The present study was undertaken to characterize the enzymatic properties of these two activities and to determine whether the two catalytic sites are also functionally distinct. We have observed that EGTA specifically stimulates, whereas CaCl2 selectively inhibits, the RNA-dependent DNA polymerase activity but that neither compound has any effect on the RNase H activity of a recombinant HIV reverse transcriptase. The stimulation of the DNA polymerase activity by EGTA is dependent on the Mg2+ concentration; the greatest stimulation is observed at low Mg2+ concentrations. Similarly, the inhibition of DNA polymerase activity by Ca2+ is influenced by Mg2+ concentration. Ca2+ inhibition can be reversed by increasing Mg2+ concentrations, suggesting the possibility that CaCl2 inhibits the reverse transcriptase activity by competing for a metal-binding site on the enzyme. The pyrophosphate analogue phosphonoformate selectively inhibits the polymerase activity but not the RNase H activity of HIV reverse transcriptase. In contrast, the RNase H activity can be selectively inhibited by deoxyadenosine 5'-monophosphate, whereas the DNA polymerase activity is not inhibited. These results suggest that the DNA polymerase and RNase activities are not only physically separate but that they are also functionally distinct.

Blotting, Western↗
PubMed2005

Functional characterization of two human olfactory receptors expressed in the baculovirus Sf9 insect cell system.

Olfactory receptors (ORs) are the largest member of the G-protein-coupled receptors which mediate early olfactory perception in discriminating among thousands of odorant molecules. Assigning odorous ligands to ORs is a prerequisite to gaining an understanding of the mechanisms of odorant recognition. The functional expression of ORs represents a critical step in addressing this issue. Due to limitations in heterologous expression, very few mammal ORs have been characterized, and so far only one is from human origin. Consequently, OR function still remains poorly understood, especially in humans, whose genome encodes a restricted chemosensory repertoire compared with most mammal species. In this study, we have designed cassette baculovirus vectors to coexpress human OR 17-209 or OR 17-210 with either G(alpha olf) or G(alpha16) proteins in Sf9 cells. Each OR was found to be expressed at the cell surface and colocalized with both G(alpha) proteins. Using Ca2+ imaging, we showed that OR 17-209 and OR 17-210 proteins are activated by esters and ketones respectively. Odorant-induced calcium response was increased when ORs were coexpressed with G(alpha16) protein, whereas coexpression with G(alpha olf) abolished calcium signaling. This strategy has been found to overcome most of the limitations encountered when expressing an OR protein and has permitted odorant screening of functional ORs. Our approach could thus be of interest for further expression and ligand assignment of other orphan receptor proteins.

Animals↗
PubMed2002

Structural and functional characterization of the upstream regulatory region of the human gene encoding prostate apoptosis response factor-4.

Prostate apoptosis response factor-4 (Par-4) is critical to cell growth and apoptosis. Induction of Par-4 expression has been shown to be required for apoptosis in a diversity of cellular systems, including neurons. Neuronal populations in individuals with degenerative disorders show elevated levels of Par-4 protein in advance of cellular and functional loss. To understand the regulation of par-4 expression, we isolated and characterized 5.7 kb of the human par-4 promoter. We demonstrated that the isolated promoter was functional. Similar to the endogenous par-4 gene, par-4 expression could be induced upon apoptotic insult with thapsigargin following introduction of the promoter DNA into human A375 cells. Also, increased levels of the atypical protein kinase C, zetaPKC, was shown to negatively regulate expression from the ectopic par-4 promoter. A 550 bp sequence immediately upstream to the 5'-untranslated region of the gene was found to be responsible for par-4 promoter induction to apoptosis by thapsigargin.

5' Flanking Region↗
PubMedDate not supplied

Recent progresses in the phenotypic and functional characterization of human cytolytic T lymphocytes.

Over the past several years, a number of major advances have been made in our understanding of the phenotypic and functional heterogeneity of human T lymphocytes. Most of these advances have resulted from: 1. the availability of monoclonal antibodies directed to T cell surface antigens; 2. the development of sensitive microassays to measure lymphocyte function in vitro; 3. the refining of T cell cloning technology. The most relevant studies in delineating how human T cell functions have been based on the analysis of T cell clones with specific cytolytic activity. These studies allowed the recent molecular definition of structures serving as specific antigen receptor on T cells as well as the understanding of the role of accessory molecules involved in specific receptor activity such as T3, T8 and T4 antigens. The development of microculture conditions allowing clonal proliferation of all human T lymphocytes, whether resting or activated, has provided a major tool for the precise definition of the frequency and subset distribution of T cells with different functional capabilities. Studies along this line have shown that precursors of cytolytic T lymphocytes (CTL-P) represent about one third of peripheral blood human T lymphocytes; in addition, all T8+ were CTL-P, whereas CTL-P were relatively rare among normal T4+ lymphocytes. As much as 20% cytolytic T lymphocytes were found to be capable to release interleukin-2 and B cell growth factor, thus indicating that cytolytic and helper function may be associated at the single cell level.

Antibodies, Monoclonal↗