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PubMed2002

Characterization of visuoconstructional disabilities in patients with probable dementia of Alzheimer's type.

The goal of this study was to systematically examine alterations underlying the graphical copying performance of Alzheimer's patients at different stages of cognitive decline, in light of a theoretical framework of visuoconstructional processing (Guérin, Ska, & Belleville, 1999). Eight patients with a diagnosis of probable Alzheimer's disease were studied. A group of 24 elderly subjects, of similar age to the patient group and slightly higher education, served as controls. The severity of the cognitive decline of each patient was measured with a standard neuropsychological battery. All subjects were administered a copying task and tasks measuring visual exploration, judgment of spatial relations and graphical planning. Group and individual results were analyzed. The group results suggested that visuoconstructional apraxia in patients with Alzheimer's type dementia is characterized by impaired visual exploration and judgment of spatial relations. Individual results revealed between-patient variability related to the severity of cognitive decline. Given the small number of patients, these observations need to be replicated with a larger-scaled study.

Aged↗
PubMed1992

Membrane fusion of Semliki Forest virus involves homotrimers of the fusion protein.

Infection of cells with enveloped viruses is accomplished through membrane fusion. The binding and fusion processes are mediated by the spike proteins in the envelope of the virus particle and usually involve a series of conformational changes in these proteins. We have studied the low-pH-mediated fusion process of the alphavirus Semliki Forest virus (SFV). The spike protein of SFV is composed of three copies of the protein heterodimer E2E1. This structure is resistant to solubilization in mild detergents such as Nonidet P-40 (NP40). We have recently shown that the spike structure is reorganized during virus entry into acidic endosomes (J. M. Wahlberg and H. Garoff, J. Cell Biol. 116:339-348, 1992). The original NP40-resistant heterodimer is dissociated, and the E1 subunits form new NP40-resistant protein oligomers. Here, we show that the new oligomer is represented by an E1 trimer. From studies that use an in vitro assay for fusion of SFV with liposomes, we show that the E1 trimer is efficiently expressed during virus-mediated membrane fusion. Time course studies show that both E1 trimer formation and fusion are fast processes, occurring in seconds. It was also possible to inhibit virus binding and fusion with a monoclonal antibody directed toward the trimeric E1. These results give support for a model in which the E1 trimeric structure is involved in the SFV-mediated fusion reaction.

Animals↗
PubMed2004

Structural and energetic characterization of nucleic acid-binding to the fingers domain of Moloney murine leukemia virus reverse transcriptase.

Reverse transcriptase is an essential retroviral enzyme that replicates the single-stranded RNA genome of the retrovirus producing a double-stranded DNA copy, which is subsequently integrated into the host's genome. We have previously reported that processive DNA synthesis of Moloney murine leukemia virus reverse transcriptase (MMLV RT) is severely compromised by substitution of an Ala for the fingers domain residue Arg 116. In order to further investigate the role of Arg 116 in interactions of MMLV RT with nucleic acids, we have determined the crystal structure of the R116A N-terminal fragment and characterized the binding of two self-complementary DNA duplexes [d(CATGCATG)2 and d(CGCGCGCG)2] to both the wild-type and R116A fragments by isothermal titration calorimetry. The resultant thermodynamic profiles extrapolated to 25 degrees C reveal that binding of the wild-type N-terminal fragment to both DNA duplexes is enthalpy-driven and characterized by an unfavorable entropy. Although the temperature dependence of the respective protein-DNA binding enthalpies is markedly different reflecting distinct heat capacity changes, the binding free energies are nearly identical and relatively invariant to temperature (DeltaG approximately -6.0 kcal x mol(-1)). In contrast to the wild-type fragment, the R116A fragment exhibits no measurable affinity for either DNA duplex, yet its crystal structure reveals no significant changes when compared to the wild-type structures. We suggest that hydrogen-bonding interactions involving the fingers domain residue Arg 116 are critical for DNA binding as well as processive DNA synthesis by MMLV RT.

Amino Acid Substitution↗
PubMed1996

Overexpression of the herpes simplex virus type 1 tegument protein VP22 increases its incorporation into virus particles.

The tegument of herpes simplex virus type 1 (HSV-1) virus particles is a complex assemblage of virus proteins whose relative proportions within virions are essentially constant for a particular strain of virus. To examine the processes controlling incorporation into the tegument, we constructed a HSV-1 recombinant that expresses two copies of gene UL49, which encodes the major tegument protein VP22. One copy specifies the unmodified form of VP22 under the control of the native promoter while the second expresses an epitope-tagged version of the protein via the human cytomegalovirus immediate early promoter. In cells infected with the recombinant virus, the overall levels of VP22 synthesized were about fivefold higher than those for wild-type virus, due to the high levels of expression of tagged protein. Analysis of virus particles revealed that the amount of VP22 in the tegument was approximately two- to threefold higher in recombinant virions and L-particles than in particles produced by wild-type virus. These results provide the first evidence that, for certain proteins, the level of polypeptide synthesis can act as a controlling factor for the amount of protein incorporated into tegument.

Amino Acid Sequence↗
PubMed1998

The first strand transfer during HIV-1 reverse transcription can occur either intramolecularly or intermolecularly.

Reverse transcription is a complicated process that involves at least two cDNA transfer reactions to produce a full-length copy DNA of the retroviral RNA genome. Because one retrovirus particle contains two identical genomic RNA molecules, the transfers can occur in an intramolecular or intermolecular manner. The mechanism of the first transfer step (minus-strand strong-stop cDNA transfer) has been studied previously in detail in transduction experiments with spleen necrosis virus vectors containing genetic markers. Different results have been reported with respect to the type of strand transfer mechanism. In this study, we analyzed the first strand transfer for human immunodeficiency virus type 1 (HIV-1). Two genetically marked genomes were copackaged into virions and reverse transcription was initiated within these particles upon permeabilization by NP-40 and addition of dNTPs. To test whether intrastrand or interstrand transfer had occurred, the cDNA products of this endogenous reverse transcription reaction were extracted from the virions and analyzed for the presence of restriction enzyme recognition sites provided by the genetic markers. The results of this analysis demonstrated that the first DNA transfer reaction occurs in a random manner, with approximately the same contribution of intrastrand and interstrand transfers. The ability to perform intermolecular strand transfer was lost upon extraction of the dimeric RNA template from the virion particle.

Base Sequence↗
PubMed1990

Specific truncations of an acetolactate synthase gene from Brassica napus efficiently complement ilvB/ilvG mutants of Salmonella typhimurium.

The expression of an acetolactate synthase (ALS) gene isolated from the cruciferous plant Brassica napus was investigated in Salmonella typhimurium. Using an expression plasmid containing the highly active trc (trp-lac) promoter, several plant ALS constructs were made containing successive in-frame truncations from the 5' end of the coding region. Functional complementation by these plant ALS constructs of a S. typhimurium mutant devoid of ALS enzymic activity was assayed on minimal medium. Truncations which eliminated a large portion of the transit peptide coding sequence proved to act as efficient ALS genes in the bacterial host. Truncations close to the putative processing site of the plant protein were inactive in the complementation test. A full length copy of the gene, including the entire transit peptide coding region, was also inactive. The efficiency of the complementation, estimated by comparison to the growth rate of wild-type S. typhimurium, was found to correlate with levels of ALS activity in the transformed bacteria. Specific mutations, known to produce herbicide resistance in plants, were introduced into the truncated ALS coding sequence by site-directed mutagenesis. When expressed in bacteria these constructs conferred a herbicide resistance phenotype on the host. The potential of this system for mutagenesis and enzymological studies of plant proteins is discussed.

Acetolactate Synthase↗
PubMed1998

PsbY, a novel manganese-binding, low-molecular-mass protein associated with photosystem II.

We describe two related manganese-binding polypeptides with L-arginine metabolizing enzyme activity that can be detected as distinct components (designated PsbY-A1 and PsbY-A2, previously called L-AME) in membranes containing Photosystem II (PS II) from spinach. The polypeptides are bitopic and appear to exist in a heterodimeric form, but only in the chlorophyll a/b lineage of plants. Both proteins are encoded in the nucleus. In spinach and in Arabidopsis thaliana they are both derived from a single-copy gene (psbY) that is translated into a precursor polyprotein of approximately 20 kDa. The processing of the polyprotein is complex and includes at least four cleavage steps. Both polypeptides are exposed N-terminally to the lumenal and C-terminally to the stromal face of the thylakoid membrane.

Amino Acid Sequence↗
PubMed2005

Copper chaperone cycling and degradation in the regulation of the cop operon of Enterococcus hirae.

Extensive insight into copper homeostasis has recently emerged. The Gram-positive bacterium Enterococcus hirae has been a paradigm for many aspects of the process. The cop operon of E. hirae consists of four genes that encode a repressor, CopY, a copper chaperone, CopZ, and two CPx-type copper ATPases, CopA and CopB. CopA and CopB accomplish copper uptake and export, respectively, and the expression of the cop operon is regulated by copper via the CopY repressor and the CopZ chaperone. The functions of the four Cop proteins have been extensively studied in vivo as well as in vitro and a detailed understanding of the regulation of the cop operon by copper has emerged.

Adenosine Triphosphatases↗
PubMed1994

Sequence of the 3'-noncoding region of the luteinizing hormone receptor gene and identification of two polyadenylation domains that generate the major mRNA forms.

We present 6.2 kb of the 3'-noncoding region sequence of the rat luteinizing hormone receptor (LHR) gene and identification of two functional polyadenylation (pA) domains, H1 (nt 2368-2491) and H2 (nt 5579-5768) responsible for 3'-end processing of the 2.6/2.3 kb and the 5.8 kb LHR mRNA, respectively. Two identical copies of pA elements AAUAUA in H1 and of AAUAAA in H2 account for micro-heterogeneous poly(A) addition at each of the two pA regions. Both LH holoreceptor and major splice variant form B (lacking the first 266 bp of exon 11) are identified in H1-terminated (2.6 kb and 2.3 kb) and H2-terminated (5.8 kb) mRNA transcripts. A rodent repetitive DNA LINE R domain 3' of H1 within the major 5.8 kb species and a B2 element downstream of H2 were identified. Alignment of the 3'-noncoding region of LHR with TSH, FSH and beta 2-adrenergic receptors indicate that H1 pA signal is unique to the LHR and may represent an insertion domain.

Alternative Splicing↗
PubMedDate not supplied

Antigenic variation in Trypanosoma equiperdum.

Trypanosoma equiperdum is an African trypanosome that causes dourine in horses. Like the other African trypanosomes, T. equiperdum escapes elimination by the immune system of its host by using an elaborate system of antigenic variant. The trypanosomes are covered by a coat consisting of a single protein called the variable surface glycoprotein (VSG) that acts as the major trypanosome immunogen. As the host responds to one VSG, trypanosomes covered with another VSG become dominant. There is a loose order of appearance of these VSG during the infection. The factors that affect the timing of VSG expression and the effective size of the VSG repertoire in T. equiperdum are reviewed. The VSG genes are generally activated by a process of duplicative transposition involving the duplication of a silent VSG gene and inserting a copy of the gene into an expression site. The order of VSG expression is related to the amount of homology between the silent gene and the expression site. The genes expressed late in infection lack extensive homology with the expression site and depend on homology with the gene in the expression site. The genes coding for VSG expressed late in infection are hybrid genes because of this mode of transfer. This transfer mechanism allows the trypanosome to create complex VSG genes from parts of several different silent genes that are each pseudogenes. Additionally, data are presented showing that only a limited portion of the VSG is actually seen by the host immune system. These factors indicate that the effective VSG repertoire is greater than the number of VSG genes in the trypanosome genome.

Animals↗
PubMed2006

A novel method for improvement of visualization of power spectra for sorting cryo-electron micrographs and their local areas.

In a context of automation of cryo-electron microscopy, we developed a novel method for improving visibility of diffraction rings in the power spectra of cryo-electron micrographs of vitreous ice (without carbon film or high concentration of diffracting material). We used these enhanced spectra to semi-automatically detect and remove micrographs and/or local areas introducing errors in the global 3D map (drifted and charged areas) or those unable to increase global signal-to-noise ratio (non-diffracting areas). Our strategy also allows a detection of micrographs/areas with a strong astigmatism. These images should be removed when using algorithms that do not correct astigmatism. Our sorting method is simple and fast since it uses the normalized cross-correlation between enhanced spectra and their copies rotated by 90 degrees. It owes its success mainly to the novel pre-processing of power spectra. The improved visibility also allows an easier visual check of accuracy of sorting. We show that our algorithm can even improve the visibility of diffraction rings of cryo-electron micrographs of pure water. Moreover, we show that this visibility depends strongly on ice thickness. This algorithm is implemented in the Xmipp (open-source image processing package) and is freely available for implementation in any other software package.

Algorithms↗
PubMed2002

Conventional and nonconventional roles of the nucleolus.

As the most prominent of subnuclear structures, the nucleolus has a well-established role in ribosomal subunit assembly. Additional nucleolar functions, not related to ribosome biogenesis, have been discovered within the last decade. Built around multiple copies of the genes for preribosomal RNA (rDNA), nucleolar structure is largely dependent on the process of ribosome assembly. The nucleolus is disassembled during mitosis at which time preribosomal RNA transcription and processing are suppressed; it is reassembled at the end of mitosis in part from components preserved from the previous cell cycle. Expression of preribosomal RNA (pre-rRNA) is regulated by the silencing of individual rDNA genes via alterations in chromatin structure or by controlling RNA polymerase I initiation complex formation. Preribosomal RNA processing and posttranscriptional modifications are guided by a multitude of small nucleolar RNAs. Nearly completed ribosomal subunits are exported to the cytoplasm by an established nuclear export system with the aid of specialized adapter molecules. Some preribosomal and nucleolar components are transiently localized in Cajal bodies, presumably for modification or assembly. The nonconventional functions of nucleolus include roles in viral infections, nuclear export, sequestration of regulatory molecules, modification of small RNAs, RNP assembly, and control of aging, although some of these functions are not well established. Additional progress in defining the mechanisms of each step in ribosome biogenesis as well as clarification of the precise role of the nucleolus in nonconventional activities is expected in the next decade.

Active Transport, Cell Nucleus↗
PubMed2003

Gene conversion drives GC content evolution in mammalian histones.

To examine the evolutionary influence of gene conversion on DNA base composition, I analysed an exhaustive dataset of histone paralogous genes from human and mouse. I show that those gene copies that belong to subfamilies of very similar sequences (presumably undergoing gene conversion) have a higher GC content than unique gene copies (presumably not undergoing gene conversion). Thus, it seems that gene conversion is a biased process that tends to increase the DNA GC content, a conclusion that has implications for the evolution of isochores in vertebrates.

Animals↗
PubMed2001

Psychomotor slowing and planning deficits in schizophrenia.

The relative contribution of cognitive and motor processing to psychomotor slowing in schizophrenia was investigated using three tasks: a simple line-copying task and a more complex figure-copying task, both following a reaction paradigm, and a standard psychomotor test, the Digit Symbol Test (DST). Various movement variables of the task performances were derived from recordings made with the aid of a digitizing tablet. The patients with schizophrenia appeared to be about one-third slower in their total performance time on all three tasks when compared with healthy controls, which suggests a general psychomotor slowing in this group. When itemized over the various movement variables, this slowing was found in both initiation time and movement time in the copying tasks and in the DST in the time to match the symbol and the digit, but not in writing the digit. Furthermore, in the figure-copying task it was found that increased figure complexity or decreased familiarity prolonged the initiation time. These latency increases were not significantly larger for the schizophrenia group as a whole, but only for a subgroup of patients with higher scores on negative symptoms. Regarding reinspection time, the effects of familiarity were larger in the schizophrenia group as a whole. These group findings suggest that patients tend to plan their actions less in advance, which, in the case of the more complex or unfamiliar task conditions, is a less sophisticated planning strategy. Given the longer latencies in patients with more severe negative symptoms, it seems that these patients have problems with turning a plan into action. The present study provides evidence of psychomotor slowing and planning deficits in schizophrenia.

Adult↗
PubMed2003

Functional characterization of transgene integration patterns by halo fluorescence in situ hybridization: electroporation versus retroviral infection.

Gene transfer techniques are essential for bioengineering and gene therapy. Retroviral vectors constitute an important tool in this context as integrase-catalyzed genomic anchoring is one of the best defined results of nonhomologous recombination, occurring at loci with favorable expression properties. On the basis of a retroviral expression cassette, this study focuses on differences regarding the genomic targets after its transfer by either retroviral infection or electroporation. Fluorescence in situ hybridization (FISH) of clones generated by infection revealed for the majority an association of the transgene with the nuclear matrix while this was true only for a minority of single copy clones if the same construct was transferred by electroporation. Our results demonstrate that the process of integration is distinct for both gene transfer routes and confirm that retroviral survival strategies favor integration next to scaffold/matrix attachment regions. These results may be one key to explain recent consequences of gene therapy trials that have led to the deregulation of endogenous genes.

Animals↗
PubMedDate not supplied

Enhanced production of human mini-proinsulin in fed-batch cultures at high cell density of Escherichia coli BL21(DE3)[pET-3aT2M2].

Synthesis of recombinant protein (human mini-proinsulin) is investigated in fed-batch cultures at high cell concentration of recombinant Escherichia coli BL21(DE3)[pET-3aT2M2]. Transcription of the recombinant gene is controlled by a T7 promoter system. The human mini-proinsulin is characterized by a C-chain peptide consisting of only nine amino acids, whereas the C-chain peptide of natural human proinsulin is made up of 35 amino acids. It is expressed in a fusion protein with a small fusion partner (a peptide with 18 amino acids) and finally aggregated into insoluble inclusion bodies in cytoplasm of recombinant E. coli. The fermentative production of this small fusion mini-proinsulin may be of great advantage in enhancing the yield of human insulin. To find an optimum induction strategy, effects of various key cultivation variables on the mini-proinsulin production are examined in high cell density fed-batch cultures. No general correlation is found between preinduction specific growth rate and recombinant protein synthesis, which confers a flexibility in choosing the feeding strategy of preinduction media for achieving the high cell density cultures. A culture temperature below 37 degrees C is unfavorable for recombinant gene expression, and the T7-based expression system is almost completely repressed at 30 degrees C. The nutrient glucose and yeast extract concentration in postinduction feed media is optimized by applying a statistical method for medium optimization, i.e. response surface methodology, and an effective amount of inducer molecule (IPTG) is determined to maximize the specific recombinant protein formation. The mini-proinsulin production in E. coli culture is significantly influenced by the volumetric feed rate of postinduction media, which is shown to be closely related to the plasmid copy number in the recombinant cell. Consequently, in a single-stage fed-batch process, the mini-proinsulin concentration is increased up to 7 g/L, approximately 62 wt % of which corresponds to mature human insulin. A two-stage fed-batch fermentation process, with recombinant cell growth occurring at a constant growth rate and constant cell concentration in a growth fermenter and mini-proinsulin production in an induction fermenter, is designed, and its efficacy in increasing volumetric productivity of mini-proinsulin is demonstrated.

Colony Count, Microbial↗
PubMed1998

Three different prohormones yield a variety of Hydra-RFamide (Arg-Phe-NH2) neuropeptides in Hydra magnipapillata.

The freshwater polyp Hydra is the most frequently used model for the study of development in cnidarians. Recently we isolated four novel Arg-Phe-NH2 (RFamide) neuropeptides, the Hydra-RFamides I-IV, from Hydra magnipapillata. Here we describe the molecular cloning of three different preprohormones from H. magnipapillata, each of which gives rise to a variety of RFamide neuropeptides. Preprohormone A contains one copy of unprocessed Hydra-RFamide I (QWLGGRFG), II (QWFNGRFG), III/IV [(KP)HLRGRFG] and two putative neuropeptide sequences (QLMSGRFG and QLMRGRFG). Preprohormone B has the same general organization as preprohormone A, but instead of unprocessed Hydra-RFamide III/IV it contains a slightly different neuropeptide sequence [(KP)HYRGRFG]. Preprohormone C contains one copy of unprocessed Hydra-RFamide I and seven additional putative neuropeptide sequences (with the common N-terminal sequence QWF/LSGRFGL). The two Hydra-RFamide II copies (in preprohormones A and B) are preceded by Thr residues, and the single Hydra-RFamide III/IV copy (in preprohormone A) is preceded by an Asn residue, confirming that cnidarians use unconventional processing signals to generate neuropeptides from their precursor proteins. Southern blot analyses suggest that preprohormones A and B are each coded for by a single gene, whereas one or possibly two closely related genes code for preprohormone C. Northern blot analyses and in situ hybridizations show that the gene coding for preprohormone A is expressed in neurons of both the head and foot regions of Hydra, whereas the genes coding for preprohormones B and C are specifically expressed in neurons of different regions of the head. All of this shows that neuropeptide biosynthesis in the primitive metazoan Hydra is already rather complex.

Amino Acid Sequence↗
PubMed1980

Preferential inclusion of extrachromosomal genetic elements in yeast meiotic spores.

During meiosis and sporulation in the yeast Saccharomyces cerevisiae, extrachromosomal traits are efficiently transmitted to haploid spores. Although the pattern of inheritance of chromosomal traits reflects the mechanism of regular chromosomal segregation in meiosis, it is not known what processes are reflected by the efficient inheritance of extrachromosomal traits. Because extrachromosomal genetic elements in yeast are present in multiple copies, perpetuation of an extrachromosomal trait could occur by the passive envelopment of a subset of copies or by an active sequestering of all or a subset of copies within the four spores. We show that only subsets of the four extrachromosomal nucleic acids commonly found in yeast are transmitted through meiosis--55% of mitochondrial DNA copies, 82% of the 2-micron DNA plasmids, and about 70% of the L and M double-stranded RNAs. However, electron micrographs of serial sections through yeast asci indicate that the four spore enclose only 30% of the total ascus material. Thus these extrachromosomal elements are preferentially included within the spores, indicating that their inheritance is not a random process. Transmission of mitochondrial DNA can be accounted for by the observed enclosure of 52% of the mitochondrial volume within the spores. The high transmission frequencies of the double-stranded RNAs (which exist as virus-like particles in the cytoplasm) and 2-micron DNA must indicate that either these nucleic acids are actively recruited from the cytoplasm by some mechanism or they are associated in some way with the nucleus during meiosis.

DNA, Mitochondrial↗