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PubMed2005

Quantitative high-pressure pair distribution function analysis.

The collection of scattering data at high pressure and temperature is now relatively straightforward thanks to developments at high-brightness synchrotron radiation facilities. Reliable data from powders, that are suitable for structure determination and Rietveld refinement, are routinely collected up to about 30 GPa in either a large-volume high-pressure apparatus or diamond anvil cell. In those cases where the total elastic scattering is of interest, as it is in the case of nano-crystalline and glassy materials, technical developments, including the use of focused high-energy X-rays (>80 keV), are advantageous. Recently completed experiments on nano-crystalline materials at the 1-ID beamline at the Advanced Photon Source suggest that quantitative data, suitable for pair distribution function analysis, can be obtained.

Journal Article↗

PubMed1992

Functional analysis of Box II mutations in yeast site-specific recombinases Flp and R. Significance of amino acid conservation within the Int family and the yeast sub-family.

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that share approximately 30% amino acid matches. They exhibit a common reaction mechanism that appears to be conserved within the larger Integrase family of site-specific recombinases. Two regions of the proteins, designated as Box I and Box II, harbor, in addition to amino acid conservation, a significantly high degree of nucleotide sequence homology within their coding segments. Box II also contains two amino acids, a histidine and an arginine, that are invariant throughout the Int family. We have performed functional analysis of Flp and R variants carrying point mutations within the Box II segment. Several positions within Box II can tolerate substitutions with no effect, or only modest effects on recombination. Alterations of the Int family residues, His305 and Arg308, in the R protein lead to the arrest of recombination at the strand cleavage or the strand exchange step. This is very similar to previously observed "step-arrest" phenotypes in Flp variants altered at these positions and has strong implications for the catalytic mechanism of recombination. Flp and R variants at His305 and His309 can be complemented in half-site strand transfer by a corresponding Tyr343 to phenylalanine variant. In contrast to Arg308 Flp variants, which are efficiently complemented in half-site strand transfer by Flp(Y343F), no strong complementation has been observed between Arg308 variants of R and R (Y343F).

DNA Mutational Analysis↗

PubMed1987

Functional analysis of a clonal expansion of Leu 11 positive NK active lymphoid cells.

A female patient with an unusual lymphoproliferative disease associated with marked neutropenia has been observed for 36 months. The expanded cell population consists of large lymphocytes, many of which contain large azurophilic granules with acid phosphatase activity. These cells were T3, T8, T11 and Leu 11 positive but lacked the M1, T10, IL-2 receptor and HLA.DR antigens. The majority of these cells (60-70%) were also Leu 7 (HNK-1) positive. Strong natural killer (NK) activity was found in both the Leu 7 positive and negative cell populations. This cytotoxic activity was inhibited by monoclonal antibodies known to inhibit NK activity but was unaffected by antibodies which block T cell and T/NK cell cytotoxicity. Further functional analysis indicated that these cells suppressed normal T cell responses to mitogens, MLC responses and PWM induced B cell immunoglobulin synthesis. No effect on bone marrow progenitor cell growth was demonstrated. Antibody dependent cellular cytotoxic (ADCC) activity was barely detectable despite the presence of the Leu 11 antigen. Southern blot DNA analysis demonstrated clonal rearrangement of the T cell receptor beta gene thereby confirming that this variant of T gamma lymphoproliferative disease was a neoplastic condition.

Adult↗

PubMed1997

Electronic measurements of relative tongue-palate contact time. Development and testing for orthodontic functional analysis.

The importance of the tongue to the form of the jaws and dental arches has long been accepted. Clear-cut differences in arch width and arch height are observed between mouth and nasal breathing. Course measurements, e.g. duration of tongue contact with the gum, are not feasible with traditional measuring methods. The palatal measuring appliance presented here together with the purpose-developed storage and evaluation equipment permits for the first time continuous 24-hour measurement of tongue contact with the palate. The clinical observation is confirmed by the presented results. Nasal obstruction is associated with lower tongue-palate contact times. In our probands, these times fell by an average of 72% after forced mouth breathing. Since complex movements within the mouth cavity cannot be directly observed, functional analysis relating to the tongue position was previously impossible, at least over a longer period. The measuring device presented here is suitable for analyzing in more detail the diagnostically difficult complex of tongue movements and breathing habits. It might therefore conceivably be used to assess myofunctional disturbances and therapeutic methods.

Child↗

PubMed2001

Thermodynamic linked-function analysis of Mg(2+)-activated yeast pyruvate kinase.

Yeast pyruvate kinase (YPK) is regulated by intermediates of the glycolytic pathway [e.g., phosphoenolpyruvate (PEP), fructose 1,6-bisphosphate (FBP), and citrate] and by the ATP charge of the cell. Recent kinetic and thermodynamic data with Mn(2+)-activated YPK show that Mn(2+) mediates the allosteric communication between the substrate, PEP, and the allosteric effector, FBP [Mesecar, A., and Nowak, T. (1997) Biochemistry 36, 6792, 6803]. These results indicate that divalent cations modulate multiligand interactions, and hence cooperativity with YPK. The nature of multiligand interactions on YPK was investigated in the presence of the physiological divalent activator Mg(2+). The binding interactions of PEP, Mg(2+), and FBP were monitored by fluorescence spectroscopy. The binding data were subject to thermodynamic linked-function analysis to determine the magnitudes of the multiligand interactions governing the allosteric activation of YPK. The two ligand coupling free energies between PEP and Mg(2+), PEP and FBP, and FBP and Mg(2+) are 0.88, -0.38, and -0.75 kcal/mol, respectively. The two-ligand coupling free energies between PEP and Mn(2+) and FBP and Mn(2+) are more negative than those with Mg(2+) as the cation. This indicates that the interactions between the divalent cation and PEP with YPK are different for Mg(2+) and Mn(2+) and that the interaction is not simply electrostatic in nature, as originally hypothesized. The magnitude of the heterotropic interaction between the metal and FBP is similar with Mg(2+) and Mn(2+). The simultaneous binding of Mg(2+), PEP, and FBP to YPK is favored by 3.21 kcal/mol compared to independent binding. This complex is destabilized by 3.30 kcal/mol relative to the analogous YPK-Mn(2+)-PEP-FDP complex. Interpretation of K(d) values when cooperative binding occurs must be done with care as these are not simple thermodynamic constants. These data demonstrate that the divalent metal, which activates phosphoryl transfer in YPK, plays a key role in modulating the various multiligand interactions that define the overall allosteric properties of the enzyme.

Adenosine Triphosphate↗

PubMed2004

Functional analysis of novel sonic hedgehog gene mutations identified in basal cell carcinomas from xeroderma pigmentosum patients.

Altered sonic hedgehog (SHH) signaling is crucial in the development of basal cell carcinomas (BCC), the most common human cancer. Mutations in SHH signal transducers, PATCHED and SMOOTHENED, have already been identified, but SHH mutations are extremely rare; only 1 was detected in 74 sporadic BCCs. We present data showing unique SHH mutations in BCCs from repair-deficient, skin cancer-prone xeroderma pigmentosum (XP) patients, which are characterized by high levels of UV-specific mutations in key genes involved in skin carcinogenesis, including PATCHED and SMOOTHENED. Thus, 6 UV-specific SHH mutations were detected in 5 of 33 XP BCCs. These missense SHH alterations are not activating mutations for its postulated proto-oncogene function, as the mutant SHH proteins do not show transforming activity and induce differentiation or stimulate proliferation to the same level as the wild-type protein. Structural modeling studies of the 4 proteins altered at the surface residues, G57S, G64K, D147N, and R155C, show that they do not effect the protein conformation. Interestingly, they are all located on one face of the compact SHH protein suggesting that they may have altered affinity for different partners, which may be important in altering other functions. Additional functional analysis of the SHH mutations found in vivo in XP BCCs will help shed light on the role of SHH in skin carcinogenesis. In conclusion, we report for the first time, significant levels of SHH mutations found only in XP BCCs and none in squamous cell carcinomas, indicating their importance in the specific development of BCCs.

Animals↗

PubMed1977

[Functional analysis in orthodontics: diagnosis of mandibular positions and movements].

Conventional gnathological concepts concerning border movements, intermaxillary relations and terminal hinge movement of the mandible have been complemented by definitions obtained from observations in children, particularly orthodontic patients. While gnathologists tend to prefer the hand guided technique for diagnostic centric relation to the unguided method, the orthodontists on the other hand do not attribute the necessary importance to the guidance of the mandible into centric relation. By prevention or elimination of occlusal interferences, RCP/IP discrepancies, of balancing and particularly hyperbalance contacts, by possibly creating a type of occlusion as conceived by gnathology, the orthodontist may--within certain limits--be able to prevent TMJ disturbances and/or peridontal disease. Hopefully it may be at the same time possible to obtain more stable results of successful orthodontic treatment. The described functional analysis is designed to invite a more dynamic concept of the masticatory system.

Child↗

PubMed2003

Identification and functional analysis of ZIC3 mutations in heterotaxy and related congenital heart defects.

Mutations in the zinc finger transcription factor ZIC3 cause X-linked heterotaxy and have also been identified in patients with isolated congenital heart disease (CHD). To determine the relative contribution of ZIC3 mutations to both heterotaxy and isolated CHD, we screened the coding region of ZIC3 in 194 unrelated patients, including 61 patients with classic heterotaxy, 93 patients with heart defects characteristic of heterotaxy, and 11 patients with situs inversus totalis. Five novel ZIC3 mutations in three classic heterotaxy kindreds and two sporadic CHD cases were identified. None of these alleles was found in 97 ethnically matched control samples. On the basis of these analyses, we conclude that the phenotypic spectrum of ZIC3 mutations should be expanded to include affected females and CHD not typical for heterotaxy. This screening of a cohort of patients with sporadic heterotaxy indicates that ZIC3 mutations account for approximately 1% of affected individuals. Missense and nonsense mutations were found in the highly conserved zinc finger-binding domain and in the N-terminal protein domain. Functional analysis of all currently known ZIC3 point mutations indicates that mutations in the putative zinc finger DNA binding domain and in the N-terminal domain result in loss of reporter gene transactivation. It is surprising that transfection studies demonstrate aberrant cytoplasmic localization resulting from mutations between amino acids 253-323 of the ZIC3 protein, indicating that the pathogenesis of a subset of ZIC3 mutations results at least in part from failure of appropriate nuclear localization. These results further expand the phenotypic and genotypic spectrum of ZIC3 mutations and provide initial mechanistic insight into their functional consequences.

Amino Acid Sequence↗

PubMed2004

Functional analysis of virion host shutoff protein of pseudorabies virus.

During lytic infection, the virion host shutoff (vhs) protein of alphaherpesviruses causes the degradation of mRNAs nonspecifically. In this work, we cloned the vhs gene (UL41 open reading frame) of pseudorabies virus (PRV; TNL strain) by PCR, and its nucleotide sequences were determined. The PCR product of vhs gene was subcloned into the prokaryotic pET32b expression vector, and production of the recombinant vhs protein was examined by SDS-PAGE. Result of Western blotting demonstrated that our recombinant vhs protein reacted with antiserum against a synthetic peptide of 17 amino acids of the vhs protein. After purification with nickel-chelate affinity chromatography, the purified recombinant vhs protein exhibited in vitro ribonuclease activity as expected. We further cloned the vhs gene into eukaryotic expression vectors and investigated the intracellular function of vhs protein by DNA transfection. By transient transfection and CAT assay, we found the CAT activity was reduced in the presence of vhs, indicating that degradation of mRNA of the CAT gene was caused by the vhs. Furthermore, our results showed that the plaque formation of pseudorabies virus was blocked by exogenous vhs. Taken together, we have cloned the vhs gene of pseudorabies virus (TNL strain) and conducted functional analysis of the recombinant vhs protein in vitro as well as in vivo.

Amino Acid Sequence↗

PubMed1997

Functional analysis of the mouse myelin/oligodendrocyte glycoprotein gene promoter in the oligodendroglial CG4 cell line.

Myelin/oligodendrocyte glycoprotein (MOG) is a late phylogenetic acquisition among vertebrates that is found only in mammals. MOG is a minor component of myelin protein, representing approximately 0.01-0.05% of the total. Regulatory elements in the MOG gene were identified by transfecting the oligodendroglial CG4 cell line with chimeric MOG-luciferase genes. Only a few hundred base pairs upstream of the coding sequence were necessary for high-level activity of the mouse MOG promoter. More distal recognition sites may exist, because silencing activity, indicative of negative regulatory elements, was detected upstream of base pair 657. Transcriptional activity of chimeric MOG- and myelin basic protein-luciferase genes was greater in CG4 cells than in 3T3 fibroblasts or C6 glioblastoma, demonstrating their superiority for functional analysis of myelin gene regulatory elements.

Animals↗

PubMed1996

Nucleotide sequence and functional analysis of the luxE gene encoding acyl-protein synthetase of the lux operon from Photobacterium leiognathi.

Nucleotide sequence of the luxE gene GenBank Accession No. U66407 from Photobacterium leiognathi PL741 has been determined, and the amino acid sequence of acyl-protein synthetase encoded by the luxE gene is deduced. Nucleotide sequence reveals that the luxE gene encodes acyl-protein synthetase, which is a component of the fatty acid reductase complex that is responsible for converting fatty acid to aldehyde as substrate in the luciferase-catalyzed bioluminescence reaction. The acyl-protein synthetase encoded by the luxE gene has a calculated M, 43,128 and comprises 373 amino acid residues. Alignment and comparison of acyl-protein synthetases from P. leiognathi, P. phosphoreum, Vibrio fischeri, V. harveyi and Xenorhabdus luminescens shows that they are homologous; there is 75.5% homologous (44.2% identity and 31.3% similarity) among these species. Functional analysis illustrates that the specific segment sequence lying before or in the luxE gene might from potential loops omega o omega e1, omega e2 as mRNA stability loop and/or for sub-regulation by alternative modulation in the lux operon. The gene order of the luxE gene in the lux and the lum operons is<--ter-lumQ-lumP-R&R-luxC-luxD-luxA-luxB -luxN-luxE-->(R&R: regulatory region; ter; transcriptional terminator), whereas the R&R is the regulatory region for the lum and the lux operons, and ter is the transcriptional terminator for the lum operon.

Acyltransferases↗

PubMed1995

Functional analysis of the human calcyclin gene promoter in a panel of human melanoma cell lines.

By comparing two subsequent human tumor stages we previously described calcyclin as a new potential melanoma associated neoplastic progression marker positively linked with metastasis. In this study the calcyclin expression levels in a representative panel of human melanoma cell lines were correlated with the occurrence of DNase I hypersensitive (DH) regions and potential enhancer elements in a 6 kb genomic fragment spanning the human calcyclin gene. Examination of the chromatin structure of the transcription unit revealed no qualitative differences in DH sites within the panel of tested human melanoma cells, but especially the sequences around the transcription start site and a 1.5 kb upstream region appeared more accessible to the nuclease in frequently (BLM, MV3) as compared to poorly (530, 1F6) metastasizing cells. The genomic fragments that harbor one or more DH sites were subjected to functional analysis by luciferase reporter gene assays. Thus, an enhancer element was detected between 361 and 167 bp upstream of the transcription start site. This enhancer displayed equal activating potential (2-3 fold) both in weakly and in frequently metastasizing cells and was apparently recognized by transcription factors present in both types of human melanoma cells lines. We conclude that, in addition to a slight amplification of the encoding gene, the elevated calcyclin mRNA levels are only reflected in a selectively increased accessibility of the chromatin structure to DNaseI in metastasizing melanoma cells.

Calcium-Binding Proteins↗

PubMed1993

Functional analysis of tumor-associated lymphocytes from gynecological tumors.

Tumor-Associated Lymphocytes (TAL) were isolated from peritoneal fluids of six ovarian cancer patients and pleural effusion from eight breast cancer patients, respectively. In one case we obtained ascitic fluid as well as pleural effusion because of intraabdominal metastatic breast carcinoma. The collected cells were cultured in a complete medium and supplemented with human interleukin-2 (nIL-2) in a concentration of 1000 Units/ml. Phenotyping was not always possible due to rapid decay of the cells. Cytotoxicity was determined with a fluorescence-based assay, in some cases at different stages of cell growth. In two cases TAL from ascitic fluids showed increased cytotoxic activity after a longer cultivation period. TAL from pleural effusions showed cytotoxic activity against the target cell lines in two cases only. Some of these TAL did not proliferate any more but died within 24 h. With the functional analysis we wanted to investigate the cytotoxic potential against natural killer (NK)-sensitive and NK-resistant (Raji) cell lines. The results demonstrate the ability of some of the TAL populations to destroy tumor cells.

Adult↗

PubMed1990

Functional analysis of cryopreserved veins. Preliminary report.

Functional comparisons of cryopreserved and fresh canine vein endothelium, smooth muscle, and connective tissue were performed. Morphometric analysis of saphenous vein endothelium revealed no significant loss of endothelial integrity as a result of cryopreservation. Endothelial cell culture revealed similar numbers of clonogenic intimal cells from cryopreserved and fresh saphenous, cephalic, and jugular veins. Smooth muscle function was assessed by measurement of the isometric force generated by vein rings in response to norepinephrine, serotonin, and potassium chloride. There was no significant difference in the dose responses of cryopreserved and fresh saphenous veins to the reagents tested. Similar results were obtained for the cephalic and jugular vein experiments with norepinephrine. The maximum tensions generated in response to norepinephrine were 52% of fresh control segments. Connective tissue function was assessed by quantitation of 3H-proline incorporation. The results indicate that cryopreserved veins retained approximately 43.5% of values of fresh vein collagen synthesis. Finally, eight cryopreserved cephalic vein autografts were placed as femoral artery grafts and were removed electively after 1 to 8 weeks. All grafts were patent. Both light and electron microscopy demonstrated that the cryopreserved veins remained intact in vivo and that arteriolization occurred as described for fresh autografts in the literature. In conclusion, cryopreserved veins retain much of their cellular and tissue functions on thawing. Transplantation of cryopreserved veins suggests that cryopreservation does not change the sequence of histologic events associated with the use of autologous fresh vein as an arterial substitute.

Animals↗

PubMed2001

Functional analysis of mutations in SLC7A9, and genotype-phenotype correlation in non-Type I cystinuria.

Cystinuria (OMIM 220100) is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids that results in nephrolithiasis of cystine. Mutations in SLC3A1, which encodes rBAT, cause Type I cystinuria, and mutations in SLC7A9, which encodes a putative subunit of rBAT (b(o,+)AT), cause non-Type I cystinuria. Here we describe the genomic structure of SLC7A9 (13 exons) and 28 new mutations in this gene that, together with the seven previously reported, explain 79% of the alleles in 61 non-Type I cystinuria patients. These data demonstrate that SLC7A9 is the main non-Type I cystinuria gene. Mutations G105R, V170M, A182T and R333W are the most frequent SLC7A9 missense mutations found. Among heterozygotes carrying these mutations, A182T heterozygotes showed the lowest urinary excretion values of cystine and dibasic amino acids. Functional analysis of mutation A182T after co-expression with rBAT in HeLa cells revealed significant residual transport activity. In contrast, mutations G105R, V170M and R333W are associated to a complete or almost complete loss of transport activity, leading to a more severe urinary phenotype in heterozygotes. SLC7A9 mutations located in the putative transmembrane domains of b(o,+)AT and affecting conserved amino acid residues with a small side chain generate a severe phenotype, while mutations in non-conserved residues give rise to a mild phenotype. These data provide the first genotype-phenotype correlation in non-Type I cystinuria, and show that a mild urinary phenotype in heterozygotes may associate with mutations with significant residual transport activity.

Amino Acid Sequence↗

PubMed1996

Comparative and functional analysis of the AP2 promoter indicates that conserved octamer and initiator elements are critical for activity.

AP-2 is a developmentally-regulated transcription factor expressed in ectodermal cell lineages. The AP-2 protein is essential for neural tube, craniofacial and body wall morphogenesis and has been implicated in oncogenesis. Here we report the isolation of the AP-2 promoter from human, mouse and chicken. The initiation sites for the human gene have been mapped in a variety of cell lines, including several derived from breast tumours. Initiation occurs just upstream of an IR3-like repetitive element, present in the human and mouse genes, but absent in chicken. The cis-acting elements responsible for promoter activity in human HeLa cells have been mapped both in vivo and in vitro. The proximal promoter contains binding sites for transcription factors AP-2, NF-1 and octamer proteins, but lacks a TATA box motif. Functional analysis demonstrates that the octamer binding site is the critical component of basal promoter activity. In addition, the promoter relies on an initiator element for efficient start site utilization. There is an excellent correlation between the requirement for the initiator and octamer elements in transcription assays and the conservation of these cis-acting sequences between chicken, mouse and human.

Animals↗

PubMed1988

Acquisition and functional analysis of manding with autistic students.

Three autistic students were trained to request a specific object from an adult "supplier" with the sentence, "Give me--" and to deliver that object to another adult, the "director." Subsequently, the degree to which the object offered by the supplier controlled the "Give me--" verbal response was assessed by delivering to the student an object other than the one requested. Despite knowing the names of all objects used in the experiment, students accepted and delivered to the director any object offered by the supplier regardless of its match with the requested object. After training to say "That's not it. Give me--" when nonrequested objects were offered, students responded differentially to requested and nonrequested objects, suggesting control of the "Give me--" response by the requested object, a characteristic of a mand. These results generalized across settings and objects. Results are discussed in terms of the training technique to establish manding and the functional analysis of the resulting verbal behavior.

Autistic Disorder↗

PubMedDate not supplied

Functional analysis of DNA sequences required for conidium-specific expression of the SpoC1-C1C gene of Aspergillus nidulans.

The SpoC1-C1C gene is centrally located within the A. nidulans conidium-specific SpoC1 gene cluster. With one exception, the 14 genes within the cluster are coordinately regulated. C1C transcript is first detected late in conidiation, coincidental with the appearance of mature conidia, and accumulates approximately 1000-fold in conidia. We show that C1C expression is restricted to conidia, with mRNA abundance decreasing immediately after induction of germination. C1C transcription and translation are not temporally separated and, similar to C1C RNA abundance, a C1C::beta-galactosidase fusion protein is first detected with the appearance of mature conidia and decreases after induction of germination. Cell-specific C1C expression requires both a position-dependent mechanism of regulation, responsible for repression in hyphae, and a position-independent mechanism of regulation, responsible for developmental expression. We show by functional analysis of upstream DNA sequences that a 10-bp sequence and two adjacent 6-bp direct repeats are necessary for position-independent, condium-specific expression of both the intact C1C gene and the reporter gene. At least one repeat (CAACAT) is required for normal levels of expression. We find that the C1C gene is not a direct target of the BrlAp and AbaAp developmental regulators, but of a yet unidentified conidium-specific transcriptional activator.

Amino Acid Sequence↗