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At least 595 records · Page 33Linked to original sources
PubMed1998

Coupled plasmon-waveguide resonance spectroscopy studies of the cytochrome b6f/plastocyanin system in supported lipid bilayer membranes.

The incorporation of cytochrome (cyt) b6f into a solid-supported planar egg phosphatidylcholine (PC) bilayer membrane and complex formation with plastocyanin have been studied by a variant of surface plasmon resonance called coupled plasmon-waveguide resonance (CPWR) spectroscopy, developed in our laboratory. CPWR combines greatly enhanced sensitivity and spectral resolution with direct measurement of anisotropies in refractive index and optical extinction coefficient, and can therefore probe structural properties of lipid-protein and protein-protein interactions. Cyt b6f incorporation into the membrane proceeds in two stages. The first occurs at low protein concentration and is characterized by an increase in total proteolipid mass without significant changes in the molecular order of the system, as demonstrated by shifts of the resonance position to larger incident angles without changing the refractive index anisotropy. The second stage, occurring at higher protein concentrations, results in a decrease in both the mass density and the molecular order of the system, evidenced by shifts of the resonance position to smaller incident angles and a large decrease in the membrane refractive index anisotropy. Plastocyanin can bind to such a proteolipid system in three different ways. First, the addition of plastocyanin before the second stage of b6f incorporation begins results in complex formation between the two proteins with a KD of approximately 10 microM and induces structural changes in the membrane that are similar to those occurring during the second stage of complex incorporation. The addition of larger amounts of plastocyanin under these conditions leads to nonspecific binding to the lipid phase with a KD of approximately 180 microM. Finally, the addition of plastocyanin after the completion of the second phase of b6f incorporation results in tighter binding between the two proteins (KD approximately 1 microM). Quantitation of the binding stoichiometry indicates that two plastocyanin molecules bind tightly to the dimeric form of the cyt b6f complex, assuming random insertion of the cytochrome into the bilayer. The structural basis for these results and formation of the proteolipid membrane are discussed.

Cyanobacteria↗
PubMed2000

The splice variant D3nf reduces ligand binding to the D3 dopamine receptor: evidence for heterooligomerization.

The D3 dopamine receptor belongs to the D2-like family of dopamine receptors. As with other members of this group, the D3 dopamine receptor gene contains introns which allow for alternative splicing of gene products. The best characterized of the human D3 dopamine receptor mRNA splice variants encodes a truncated protein called D3nf. The D3 dopamine receptor and D3nf were epitope-tagged and expressed in Sf9 insect cells by recombinant baculovirus infection. The D3 dopamine receptor showed saturable, high affinity binding of agonists and antagonists, consistent with reported D3 dopamine receptor pharmacology. When the D3 dopamine receptor and D3nf were co-expressed, the apparent density of D3 dopamine receptor expression, as determined by radioligand binding, was significantly lowered compared to D3 dopamine receptor expressed alone. This effect of D3nf was specific for the D3 dopamine receptor, since co-expression with the D2 dopamine receptor or beta2-adrenoceptor had no effect on binding. Confocal immunofluorescence studies were used to confirm that both D3 dopamine receptor and D3nf were well expressed on the cell surface and densitometric analysis of cell surface membrane protein confirmed that D3nf did not significantly alter the amount of D3 dopamine receptor expressed. Photoaffinity labelling with [125I]azidonemonapride showed that the amount of ligand bound by membranes co-expressing D3 dopamine receptor and D3nf was significantly less than that bound by membranes expressing D3 dopamine receptor alone. The greatest decrease in binding was observed in the D3 dopamine receptor oligomeric forms. Ligand binding to dimers and tetramers was reduced by 69 and 46%, respectively, indicating effects of a protein-protein interaction. Co-immunoprecipitation confirmed that the D3DR and D3nf interact with each other. These data indicate that D3nf heterodimerizes with the D3 dopamine receptor and decreases the capacity of D3 dopamine receptor to bind ligand.

Alternative Splicing↗
PubMed2004

Structures of the yeast ribonucleotide reductase Rnr2 and Rnr4 homodimers.

Class I ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides. Eukaryotic RNRs comprise two subunits, the R1 subunit, which contains substrate and allosteric effector binding sites, and the R2 subunit, which houses a catalytically essential diiron-tyrosyl radical cofactor. In Saccharomyces cerevisiae, there are two variants of the R2 subunit, called Rnr2 and Rnr4. Rnr4 is unique in that it lacks three iron-binding residues conserved in all other R2s. Nevertheless, Rnr4 is required to activate Rnr2, and the functional species in vivo is believed to be a heterodimeric complex between the two proteins. The crystal structures of the Rnr2 and Rnr4 homodimers have been determined and are compared to that of the heterodimer. The homodimers are very similar to the heterodimer and to mouse R2 in overall fold, but there are several key differences. In the Rnr2 homodimer, one of the iron-binding helices, helix alphaB, is not well-ordered. In the heterodimer, interactions with a loop region connecting Rnr4 helices alphaA and alpha3 stabilize this Rnr2 helix, which donates iron ligand Asp 145. Sequence differences between Rnr2 and Rnr4 prevent the same interactions from occurring in the Rnr2 homodimer. These findings provide a structural rationale for why the heterodimer is the preferred complex in vivo. The active-site region in the Rnr4 homodimer reveals interactions not apparent in the heterodimer, supporting previous conclusions that this subunit does not bind iron. When taken together, these results support a model in which Rnr4 stabilizes Rnr2 for cofactor assembly and activity.

Amino Acid Sequence↗
PubMedDate not supplied

Retro-regression--another important multivariate regression improvement.

We review the serious problem associated with instabilities of the coefficients of regression equations, referred to as the MRA (multivariate regression analysis) "nightmare of the first kind". This is manifested when in a stepwise regression a descriptor is included or excluded from a regression. The consequence is an unpredictable change of the coefficients of the descriptors that remain in the regression equation. We follow with consideration of an even more serious problem, referred to as the MRA "nightmare of the second kind", arising when optimal descriptors are selected from a large pool of descriptors. This process typically causes at different steps of the stepwise regression a replacement of several previously used descriptors by new ones. We describe a procedure that resolves these difficulties. The approach is illustrated on boiling points of nonanes which are considered (1) by using an ordered connectivity basis; (2) by using an ordering resulting from application of greedy algorithm; and (3) by using an ordering derived from an exhaustive search for optimal descriptors. A novel variant of multiple regression analysis, called retro-regression (RR), is outlined showing how it resolves the ambiguities associated with both "nightmares" of the first and the second kind of MRA.

Journal Article↗
PubMed2003

MIM-B, a putative metastasis suppressor protein, binds to actin and to protein tyrosine phosphatase delta.

We have found that MIM-B, a putative metastasis suppressor protein, is implicated in actin cytoskeletal control and interaction with a protein tyrosine phosphatase (PTP). MIM was originally described as a protein whose mRNA was Missing in Metastasis, as it was found not to be present in metastatic bladder carcinoma cell lines [Lee, Y. G., Macoska, J. A., Korenchuk, S. and Pienta, K. J. (2002) Neoplasia 4, 291-294]. We further characterized a variant of MIM, which we call MIM-B, and which we believe may be a link between tyrosine kinase signalling and the actin cytoskeleton. We have shown, using purified proteins and cell extracts, that MIM-B is an actin-binding protein, probably via a WASP (Wiskott-Aldrich syndrome protein)-homology 2 domain at its C-terminus. We have also found that MIM-B binds to the cytoplasmic domain of receptor PTPdelta. Expression of full-length MIM-B induces actin-rich protrusions resembling microspikes and lamellipodia at the plasma membrane and promotes disassembly of actin stress fibres. The C-terminal portion of MIM-B is localized in the cytoplasm and does not affect the actin cytoskeleton when expressed, while the N-terminal portion localizes to internal vesicles and probably targets the protein to membranes. We postulate that MIM-B may be a regulator of actin assembly downstream of tyrosine kinase signalling and that this activity may explain the involvement of MIM in the metastasis of cancer cells.

Actins↗
PubMed1998

Pigmented atypical fibroxanthoma.

AIM: The purpose of this report is to call attention to a pigmented variant of atypical fibroxanthoma that resembles malignant melanoma, both clinically and histopathologically. METHODS AND RESULTS: Thirty-eight cases of atypical fibroxanthoma were examined for the presence of pigmented areas. Four such cases were found. Neoplastic cells showed erythrophagocytosis and accumulation of haemosiderin pigment in the cytoplasm. In three cases, immunohistochemical studies using a battery of antibodies were performed. Neoplastic cells were strongly positive for vimentin and weakly positive for CD68, whereas they were negative for melanocytic markers, including S100 protein, HMB45, and the monoclonal antibody NK1-C3 to melanoma-associated antigen. CONCLUSIONS: Pigmented atypical fibroxanthoma represents a poorly recognized variant of the neoplasm that may be easily misinterpreted as malignant melanoma. To the best of our knowledge, this is the first report of erythrophagocytosis in atypical fibroxanthoma.

Aged↗
PubMed2003

Degeneracy and function of the ubiquitous RVXF motif that mediates binding to protein phosphatase-1.

Most interactors of protein phosphatase-1 (PP1) contain a variant of a so-called "RVXF" sequence that binds to a hydrophobic groove of the catalytic subunit. A combination of sequence alignments and site-directed mutagenesis has enabled us to further define the consensus sequence for this degenerate motif as [RK]-X(0-1)-[VI]-[P]-[FW], where X denotes any residue and [P] any residue except Pro. Naturally occurring RVXF sequences differ in their affinity for PP1, and we show by swapping experiments that this binding affinity is an important determinant of the inhibitory potency of the regulators NIPP1 and inhibitor-1. Also, inhibition by NIPP1-(143-224) was retained when the RVXF motif (plus the preceding Ser) was swapped for either of two unrelated PP1-binding sequences from human inhibitor-2, i.e. KGILK or RKLHY. Conversely, the KGILK motif of inhibitor-2 could be functionally replaced by the RVXF motif of NIPP1. Our data provide additional evidence for the view that the RVXF and KGILK motifs function as anchors for PP1 and thereby promote the interaction of secondary binding sites that determine the activity and substrate specificity of the enzyme.

Amino Acid Sequence↗
PubMed2003

Human population structure and its effects on sampling Y chromosome sequence variation.

The excess of rare variants in global sequencing studies of the nonrecombining portion of the Y chromosome (NRY) has been interpreted as evidence for the effects of human demographic expansion. However, many NRY polymorphisms are geographically localized and the effect of different geographical sampling on patterns of NRY variation is unknown. We use two sampling designs to detect population structure and its effects on patterns of human NRY polymorphism. First, we sequence 26.5 kb of noncoding Y chromosome DNA from 92 globally distributed males representing 35 populations. We find that the number of polymorphisms with singleton variants is positively correlated with the number of populations sampled and that there is a significant negative correlation of Tajima's D (TD) and Fu and Li's D (FD) statistics with the number of pooled populations. We then sequence the same region in a total of 73 males sampled from 3 distinct populations and find that TD and FD values for the 3 pooled and individual population samples were much less negative than those in the aforementioned global sample. Coalescent simulations show that a simple splitting model of population structure, with no changes in population size, is sufficient to produce the negative values of TD seen in our pooled samples. These empirical and simulation results suggest that observed levels of NRY population structure may lead to an upward bias in the number of singleton variants in global surveys and call into question inferences of population expansion based on global sampling strategies.

Asian People↗
PubMed2005

Interchromosomal segmental duplications explain the unusual structure of PRSS3, the gene for an inhibitor-resistant trypsinogen.

Homo sapiens possess several trypsinogen or trypsinogen-like genes of which three (PRSS1, PRSS2, and PRSS3) produce functional trypsins in the digestive tract. PRSS1 and PRSS2 are located on chromosome 7q35, while PRSS3 is found on chromosome 9p13. Here, we report a variation of the theme of new gene creation by duplication: the PRSS3 gene was formed by segmental duplications originating from chromosomes 7q35 and 11q24. As a result, PRSS3 transcripts display two variants of exon 1. The PRSS3 transcript whose gene organization most resembles PRSS1 and PRSS2 encodes a functional protein originally named mesotrypsinogen. The other variant is a fusion transcript, called trypsinogen IV. We show that the first exon of trypsinogen IV is derived from the noncoding first exon of LOC120224, a chromosome 11 gene. LOC120224 codes for a widely conserved transmembrane protein of unknown function. Comparative analyses suggest that these interchromosomal duplications occurred after the divergence of Old World monkeys and hominids. PRSS3 transcripts consist of a mixed population of mRNAs, some expressed in the pancreas and encoding an apparently functional trypsinogen and others of unknown function expressed in brain and a variety of other tissues. Analysis of the selection pressures acting on the trypsinogen gene family shows that, while the apparently functional genes are under mild to strong purifying selection overall, a few residues appear under positive selection. These residues could be involved in interactions with inhibitors.

Chromosomes, Human↗
PubMed1996

Structure, genomic organization, replication and variability of hepatitis C virus.

Hepatitis C virus (HCV) is an enveloped, single-stranded RNA virus that has been classified in the Flaviviridae family. The genome of 9400 nucleotides comprises two non-coding regions in 5' and 3' flanking a large reading frame which codes for a polyprotein of 3000 amino acids; this polyprotein is further cleaved into structural (C, E1, E2) and non-structural (NS1, NS2, NS3, NS4, NS5) proteins. The positive RNA acts as a cap-independent messenger; the transcription is mediated by the NS5 RNA polymerase. After the maturation step, the virion is liberated by budding through the cytoplasmic membrane. As for many other RNA viruses, the HCV genome exhibits a high degree of variability, especially in the E2/NS1, E1, NS3 and NS5b regions. Conversely the 5' non-coding region is highly conserved, at least in part, and can be used for diagnostic purposes by PCR technique. Six genotypes of HCV have already been reported, numbered from 1 to 6 in Simmonds' classification. The same genotype can be divided into subtypes (for instance, genotype 1 comprises three subtypes: 1a, 1b and 1c). Various minor variants of the same strain, called quasispecies, are commonly present in the blood of the same patient. Strains of genotype 1b--which is the most widespread worldwide--are correlated with more severe clinical manifestations, greater viral loads and lower response to interferon treatment. The high variability of the HCV genome contributes greatly to the difficulty of designing potent vaccines.

DNA, Viral↗
PubMed1986

The virology of variola minor. Correlation of laboratory tests with the geographic distribution and human virulence of variola isolates.

Several groups of variola isolates were compared in DNA structure, and by four independent biologic markers. Isolates of variola minor from Europe and South America (alastrim virus) could be distinguished from African isolates of variola minor by DNA structure and by two of the four biologic markers. Taken as a group, the properties of African isolates, in general, differed from those of variola major, but this difference was confined to properties which depended (in the laboratory) on the recent history of the virus concerned. The suggestion made previously that there was an "intermediate" or "African" variety of variola virus is discounted. Laboratory tests did not distinguish any individual African isolate from variola major virus. It is concluded that a virus which may be called "alastrim" represents a "fixed" variant of variola virus, whose distribution is consistent with the dramatic spread of variola minor through the Americas and Europe in the early part of this century, and that variola minor in Africa in recent years was due to variola virus which was not alastrim and which laboratory evidence fails to identify as an entity distinguishable from variola major virus.

Africa, Western↗
PubMed2005

Kinetic folding mechanism of PDZ2 from PTP-BL.

PDZ domains represent a large family of protein-interaction modules associated with a variety of unrelated proteins with different functions. We report a complete characterization of the kinetic folding mechanism of a fluorescent variant of PDZ2 from PTP-BL, investigated under a variety of different experimental conditions. For this purpose, we engineered a fluorescent variant of this protein Y43W (called pseudo-wild-type, pWT43). The results suggest the presence of a high-energy intermediate in the folding of PDZ2, as revealed by a pronounced non-linear dependence of the unfolding rate constant on denaturant concentration. Such an intermediate may or may not be detectable depending on the experimental conditions, giving rise to apparent two-state folding under stabilizing conditions (e.g. in the presence of sodium sulfate). Interestingly, even under these conditions, three-state folding can be restored by selectively destabilizing the native-like rate-limiting barrier by one specific mutation (V44A). Finally, we show that data taken on pWT43 under different experimental conditions (e.g. different pH values from 2.1 to 8.0 or in the presence of a stabilizing salt) and also data on a site-directed conservative mutant can be rationalized in terms of a simple reaction scheme involving a single set of intermediates and transition states.

Amino Acid Substitution↗
PubMed1987

Restricted replication of herpes simplex virus type 1 in murine embryonal carcinoma cells.

Herpes simplex virus type 1 (HSV-1) has a broad host range but the KOS strain of HSV-1 did not replicate efficiently in murine embryonal carcinoma (EC) cells. The yield of infectious HSV-1 from EC cells was 100- to 1000-fold lower than that from fibroblast cell lines of mouse, monkey or human origin. The thymidine kinase (TK) gene of HSV-1 is expressed early during the infectious cycle. The levels of TK mRNA and of TK activity in infected EC cells were only two- to threefold lower than levels from infected fibroblast cells. Infected EC cells supported replication of about half as much HSV-1 DNA as did fibroblast cells. The reduced yield of infectious virus was consistent with a paucity of virions in infected EC cells examined by electron microscopy, suggesting a major block late during the HSV-1 infectious cycle. We isolated a variant strain of HSV-1, called KOSEC, which replicated as efficiently in EC cells as in mouse fibroblasts. KOSEC infected EC and fibroblast cells, synthesized more TK mRNA, more TK enzyme, and more HSV-1 DNA than did the same cells infected with the KOS stain. Both HSV-1 strains induced similar levels of synthesis of gD, an early viral glycoprotein. By co-infection of EC cells with the KOS and KOSEC virus, both the elevated virus yield and the elevated TK synthesis seen in KOSEC-infected cells appeared to be recessive. Apparently a viral mutation that affects expression of some early viral functions can also overcome the EC cell restriction to HSV-1 replication.

Animals↗
PubMed2005

High-level, inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum using versatile expression vectors.

Vectors have been developed for inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum in which expression of the gene of interest is driven by strong, regulated promoters from bacteriocin operons found in L. sakei strains. The activity of these promoters is controlled via a two-component signal transduction system, which responds to an externally added peptide pheromone. The vectors have a modular design, permitting easy exchange of all essential elements: the inducible promoter, the cognate regulatory system, the gene of interest, the antibiotic resistance marker and the replicon. Various variants of these so-called 'pSIP' vectors were constructed and tested, differing in terms of the bacteriocin regulon from which the regulatory elements were derived (sakacin A or sakacin P), the regulated promoter selected from these regulons, and the replicon (derived from p256 or pSH71). Using beta-glucuronidase (GusA) and aminopeptidase N (PepN) as reporters, it was shown that the best vectors permitted inducible, pheromone-dose-dependent gene expression at very high levels, while displaying moderate basal activities when not induced. The most effective set-up was obtained using a vector containing the pSH71 replicon, the orfX promoter from the sakacin P regulon, and the cognate regulatory genes, in a L. sakei host. GusA levels obtained with this set-up were approximately ten times higher than the levels obtained with prototype pSIP versions, whereas PepN levels amounted to almost 50% of total cellular protein.

Bacteriocins↗
PubMed2003

Comment on "Ehrenfest times for classically chaotic systems".

In a recent Rapid Communication [P. G. Silvestrov and C. W. J. Beenakker, Phys. Rev. E 65, 035208(R) (2002)], the authors, Silvestrov and Beenakker, introduce a way to lengthen the Ehrenfest time tau for fully chaotic systems. We disagree with several statements made in their paper, and address the following points essential to their conclusions: (1) it is not true that all semiclassical approximations for chaotic systems fail at a so-called "log time" tau proportional, variant -ln( variant Planck's over 2pi ), differing only by a numerical coefficient; and (2) the limitation of the semiclassical approximation as expressed in the authors' Eq. (8) is not limited by their argument leading to Eq. (12).

Comment↗
PubMed2005

Exact asymptotic results for the Bernoulli matching model of sequence alignment.

Finding analytically the statistics of the longest common subsequence (LCS) of a pair of random sequences drawn from c alphabets is a challenging problem in computational evolutionary biology. We present exact asymptotic results for the distribution of the LCS in a simpler, yet nontrivial, variant of the original model called the Bernoulli matching (BM) model. We show that in the BM model, for all c , the distribution of the asymptotic length of the LCS, suitably scaled, is identical to the Tracy-Widom distribution of the largest eigenvalue of a random matrix whose entries are drawn from a Gaussian unitary ensemble.

Binomial Distribution↗
PubMed1977

Haematological changes associated with the McLeod phenotype of the Kell blood group system.

The McLeod phenotype is inherited as an X-linked characteristic. The red cells have weak antigenicity in the Kell blood group and lack Kx, a precursor-like substance that appears to be necessary for proper biosynthesis of Kell antigens. Kx antigen is also required for establishment of normal cell morphology. Absence of Kx antigen causes a membrane abnormality, in which the most prominent feature is acanthocytosis, and a compensated haemolytic state. The X-linked gene that determines normal Kx production is called X1k. Inheritance of a variant allele at the Xk locus is responsible for lack of Kx synthesis and the McLeod phenotype. The Xk locus is inactivated by the Lyon effect, and female carriers of the variant gene exhibit blood group mosaicism in the Kell system and have a dual red cell population of acanthocytes and discocytes.

Acanthocytes↗
PubMed1987

HLA and non-HLA phenotyping and genotyping in Austral and Gambier Polynesian Archipelagos.

Forty-one individuals from Rurutu Island (Austral Archipelagos) and 41 individuals from the Gambier Archipelagos have been typed for HLA; for blood groups ABO, Rh, MNSs, P, Kell, Kp, Lewis, Lutheran, Kidd; for the electrophoretic systems G6PD, 6-PGD, PGM1, PGM2, AcP, ADA, GPT, Est-D, GLO I, and for the immunoglobulin allotypes Gm and Km. There is a high degree of homogeneity among these Polynesian populations and among other Polynesian populations previously typed. However, small differences exist between the populations of the two archipelagos, possibly due to endogamy or to the smallness of the samples studied. A variant of HLA-Bw22 (called Bw22x) is described.

Blood Group Antigens↗