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PubMed2026

DNM1L depletion leads to accelerated heteroplasmy shifting of m.10191C allele through ATG7-dependent pathways.

Nucleotide composition bias in mitochondrial DNA (mtDNA) makes the heavy strand prone to form a DNA secondary structure called a guanine quadruplex (G4). This secondary structure has been shown to inhibit polymerase processivity in vitro. We previously identified pathogenic mtDNA variants that lead to increased G4-forming propensity, including a T to C mutation at m.10191 (m.10191 T > C) that causes Leigh syndrome. Cells treated with G4 binding agent (G4BA) berberine show a reduction in m.10191C pathogenic heteroplasmy levels. To help better understand the underlying mechanism behind berberine-induced heteroplasmy shift, we examined the relationship between mitochondrial fission and berberine-mediated shift. Here we show that knockdown of the fission factor DNM1L leads to an accelerated heteroplasmy shift towards the healthy mtDNA allele, lowering m.10191C by 10% in 3 weeks, compared to the 5 weeks required for berberine alone. The specific mechanism involves ATG7, as knockdown of ATG7 is able to partially delay this accelerated heteroplasmy shift. Taken together, we show that DNM1L knockdown is able to accelerate berberine-induced m.10191C heteroplasmy shifting through an autophagy-related mechanism.

Humans↗
PubMed2000

Lipocalins as a scaffold.

The concept of scaffolds that can be equipped with artificial biochemically active sites has gained recent interest in the field of protein design. Members of the lipocalin protein family represent promising model systems in this respect. Especially prototypic lipocalins, such as the retinol-binding protein or the bilin-binding protein (BBP), exhibit a structurally simple one-domain fold with a conformationally well conserved beta-barrel as their central motif. This type of supersecondary structure is made of a cylindrically closed beta-sheet of eight antiparallel strands. At the open end of the barrel the beta-strands are connected by four loops in a pairwise manner so that a pocket for the ligand is formed. In a rational protein design study a metal-binding site was functionally grafted on the solvent-exposed surface of the beta-barrel, whereby the rigid backbone conformation permitted the spatially defined arrangement of three His side chains. In a combinatorial protein design approach, the natural ligand pocket of a lipocalin was reshaped. In this manner variants of the BBP were engineered which exhibit high affinity and remarkable specificity for haptens like fluorescein and digoxigenin. The so-called 'anticalins', i.e. artificial lipocalins recognizing prescribed ligands, could provide an interesting alternative to recombinant antibody fragments. Consequently, the use of lipocalins as a scaffold opens new applications for members of this functionally diverse protein family in biotechnology and medicine.

Animals↗
PubMed2002

Microsatellite instability and its relevance to cutaneous tumorigenesis.

Increasing evidence suggests that human tumors sequentially accumulate multiple mutations that cannot be explained by the low rates of spontaneous mutations in normal cells (2-3 mutations/cell). The mathematical models estimate that for the solid tumors to develop, as many as 6-12 mutations are required in each tumor cell. Therefore, to account for such high mutation rates, it is proposed that tumor cells are genetically unstable, i.e. they have genome-wide mutations at short repetitive DNA sequences called microsatellites. Microsatellite repeats are scattered throughout the human genome, primarily in the non-coding regions, and can give rise to variants with increased or reduced lengths, i.e. microsatellite instability (MSI). This instability has been reported in an increasing number of cutaneous tumors including: melanocytic tumors, basal cell carcinomas and primary cutaneous T-cell lymphomas. Moreover, MSI has been observed in skin tumors arising in the context of some hereditary disorders such as Muir-Torre syndrome, Von Recklinghausen's disease and disseminated superficial porokeratosis. While MSI in some of these disorders reflects underlying DNA replication errors, the mechanism of instability in others is still unknown. Thus far, MSI is considered to be a distinct tumorigenic pathway that reveals surprising versatility. The ramifications for cutaneous neoplasms warrant further investigation.

Animals↗
PubMed2000

A simple method for the rapid generation of recombinant adenovirus vectors.

Recombinant adenoviruses are useful vectors for basic research. When the vectors are used for delineating protein function, several viruses, each containing a mutated version of the transgene are compared at the same time. However, methods to generate multiple vectors simultaneously within a short time period are cumbersome. In this report, we show that a novel backbone plasmid, when cotransfected with routinely used shuttle vectors into HEK293 cells allowed for production of recombinant viruses in an average of 14 days. The recombinant viruses had no detectable wild-type virus contamination by A549 plaque assay and only three to 300 E1a copies per 109 adenovirus genomes by a sensitive PCR-based assay. Further culturing or serial amplification did not result in wild-type revertants nor did cultures show increased levels of E1a copy number by quantitative PCR. Thus, recombinant adenovirus vectors can be produced very simply, rapidly and with little to no contaminating wild-type particles. This system should facilitate the generation of multiple genetic variants by eliminating the need for time-consuming plaque purification and the need to manipulate and screen very large plasmids. We call this the RAPAd.I system.

Adenoviridae↗
PubMed2002

Molecular and morphological studies on the Anopheles minimus group of mosquitoes in southern China: taxonomic review, distribution and malaria vector status.

Mosquitoes of the Anopheles minimus group (Diptera: Culicidae) from nine Provinces of southern China were identified morphologically and by molecular characterization, using single-strand conformation polymorphisms (SSCPs) and sequence data for the D3 region of the 28S ribosomal DNA and the mitochondrial COII locus. Species A and C (sensu Green et al., 1990) of the An. minimus complex were found to be sympatric in Yunnan Province. Species A occurs eastward from Yunnan through southern Guangxi, Hainan, Guangdong and Taiwan Provinces, whereas species C occurs northward to northern Guangxi, Guizhou and Sichuan Provinces. Morphological and molecular evidence (based on specimens from the field and four isofemale lines) shows that An. minimus forms A and B (sensu Yu & Li, 1984) are morphological variants of species A, which is accepted as An. minimus Theobald sensu stricto (type-locality: Pokfulam, Hong Kong). The so-called subspecies x of An. minimus (sensu Baba, 1950) is reinterpreted as An. aconitus Dönitz. The distribution and vector status of members of the An. minimus group are discussed in relation to the historical and current transmission of malaria and filariasis in China. Both An. minimus A and C have been implicated as widespread vectors of malaria, whereas only species A has been found in Hainan, where An. minimus s.l. was a vector of Bancroftian filariasis. The presence of An. aconitus in Hainan and Yunnan Provinces is confirmed, but the occurrence of An. varuna Iyengar and An. fluviatilis James, which were previously recorded in China, could not be verified.

Animals↗
PubMed2000

A tuftelin-interacting protein (TIP39) localizes to the apical secretory pole of mouse ameloblasts.

Enamel biomineralization is a complex process that involves interactions between extracellular matrix proteins. To identify proteins interacting with tuftelin, a potential nucleator of enamel crystallites, the yeast two-hybrid system was applied to a mouse tooth expression library and a tuftelin-interacting protein (TIP) was isolated for further characterization. Polyclonal antibodies were prepared against two recombinant variants of this protein. Both antibodies identified a major protein product in tooth organs at 39 kDa, and this protein has been called TIP39. Northern analysis showed TIP39 messenger RNA in multiple organs, a pattern similar to that of tuftelin messenger RNA. In situ hybridization of mandibles of 1-day-old mice detected TIP39 RNA in secretory ameloblasts and odontoblasts. Immunolocalization of TIP39 and tuftelin in cultured ameloblast-like cells showed that these two proteins colocalize. Within the developing tooth organ, TIP39 and tuftelin immunolocalized to the apical pole of secretory ameloblasts (Tomes' processes) and to the newly secreted extracellular enamel matrix. TIP39 amino acid sequence appears to be highly conserved with similarities to proteins in species as diverse as yeast and primates. Available sequence data and the findings reported here suggest a role for TIP39 in the secretory pathway of extracellular proteins.

Ameloblasts↗
PubMed1988

Identification of viridans streptococci by three commercial systems.

The API 20S (Analytab Products, Plainview, NY), the GPI card (Vitek Systems, St. Louis, MO) and the RapSTR system (Innovative Diagnostics, Atlanta, GA) were compared with conventional biochemicals for the identification of viridans streptococci. One hundred nine clinical isolates were tested that included the following species: intermedius (38) sanguis II (20), bovis (variant) (14), mitis (14), salivarius (11), sanguis I (6), constellatus (3), mutans (2), and uberis (1). With initial testing, a correct species call was made with 72% of the isolates with the GPI card, 62% with the RapSTR, and 50% with the API 20S. Identifications of viridans streptococci group or those that needed additional biochemicals for species identification occurred with 28% of isolates with the API 20S, 8% with the RapSTR, and 9% with the GPI card. Incorrect identifications occurred with 6% of the isolates tested by the GPI card, 20% with the API 20S, and 30% with the RapSTR. Most discrepancies with the RapSTR were with 66% of the intermedius isolates, whereas most, 55%, of misidentifications with the API 20S were with sanguis II isolates. No identifications were made with 2% and 13% of isolates with the API 20S and GPI, respectively.

Humans↗
PubMed1988

Paraimmunoblastic variant of small lymphocytic lymphoma/leukemia.

We report 16 cases of a distinctive, biologically aggressive variant of small lymphocytic lymphoma/leukemia (SLL/L) that is characterized by the diffuse proliferation of cells normally comprising the pseudoproliferation centers (so-called paraimmunoblasts). Demographically, the patients differed in no significant regard from patients with SLL/L of usual type. Rapidly progressive, generalized lymphadenopathy was the dominant clinical finding in 15 of the 16 patients; one patient presented with symptoms related to lymphomatous involvement of the stomach and regional lymph nodes. Splenomegaly was observed in five patients. Seven patients, two of whom had a history of indolent-phase chronic lymphocytic leukemia, had an absolute lymphocytosis at diagnosis. In most patients, bone marrow involvement was noted at diagnosis. It consisted predominantly of small lymphocytic infiltrates indistinguishable from those observed in SLL/L of usual type; significant paraimmunoblastic infiltration was infrequent and generally occurred late in the disease course. Immunohistochemical and cytogenetic study further substantiated the hypothesized relationship of these cases to SLL/L. Findings included (a) coexpression of sIg and Leu-1 antigen in the majority of cases and (b) the presence of a t(11;14) (q13;q32) chromosome translocation in two of three cases with analyzable metaphases. Although treatment protocols were not uniform, follow-up data indicated an accelerated clinical course. Eleven patients have died of their disease between 3 and 39 months after diagnosis; the median survival was 28 months.

Adult↗
PubMed2005

Segmental facial anhidrosis and tonic pupils with preserved deep tendon reflexes: a novel autonomic neuropathy.

A 31-year-old woman had exertional right-sided hemifacial flushing and sweating. Examination demonstrated slightly dilated pupils with absent constriction to light and a tonic near response and redilatation, features consistent with Adie syndrome. Neurological examination was otherwise normal, including preservation of deep tendon reflexes. Magnetic resonance imaging of brain and spine were normal. The combination of unilateral loss of sudomotor and vasomotor activity without loss of ocular sympathetic innervation fulfills the diagnosis of Harlequin syndrome. The combination of Harlequin and Adie syndromes has been called Ross syndrome, but the preservation of deep tendon reflexes precludes a diagnosis of Ross syndrome in our patient. This previously undescribed variant adds further complexity to the spectrum of autonomic neuropathies.

Adie Syndrome↗
PubMed1998

The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system.

Interruption of translation in Escherichia coli can lead to the addition of an 11-residue carboxy-terminal peptide tail to the nascent chain. This modification is mediated by SsrA RNA (also called 10Sa RNA and tmRNA) and marks the tagged polypeptide for proteolysis. Degradation in vivo of lambda repressor amino-terminal domain variants bearing this carboxy-terminal SsrA peptide tag is shown here to depend on the cytoplasmic proteases ClpXP and ClpAP. Degradation in vitro of SsrA-tagged substrates was reproduced with purified components and required a substrate with a wild-type SsrA tail, the presence of both ClpP and either ClpA or ClpX, and ATP. Clp-dependent proteolysis accounts for most degradation of SsrA-tagged amino-domain substrates at 32 degrees C, but additional proteases contribute to the degradation of some of these SsrA-tagged substrates at 39 degrees C. The existence of multiple cytoplasmic proteases that function in SsrA quality-control surveillance suggests that the SsrA tag is designed to serve as a relatively promiscuous signal for proteolysis. Having diverse degradation systems able to recognize this tag may increase degradation capacity, permit degradation of a wide variety of different tagged proteins, or allow SsrA-tagged proteins to be degraded under different growth conditions.

Adenosine Triphosphatases↗
PubMed1982

'Carcinoid' tumours of the breast: the morphological spectrum of argyrophil carcinomas.

Fourteen 'carcinoid' tumours of the breast are described. They are separable into five with and nine without intracellular mucin. All the tumours are argyrophil, but none is argentaffin. Four tumours studied ultrastructurally contain dense-core granules. Argyrophil carcinomas represent the endocrine analogues of ductal carcinoma in situ, of invasive ductal carcinoma and probably of lobular carcinoma also. Current views vary between the one that the so-called carcinoid is a rare and totally distinct entity to the view, at the other extreme, that it is a very common variant of conventional breast cancer. On the basis of our findings, an intermediate view is justified: argyrophil carcinomas constitute about 5% of breast carcinomas and some varieties at least have non-argyrophil analogues. Factors influencing the prognosis in individual cases are discussed. Argyrophil carcinomas of the breast form a tumour spectrum with a wide range of morphological and histochemical appearances and a variable prognosis.

Aged↗
PubMed1986

Genetic polymorphism of esterase B3 in human leukocytes.

Human tissues contain an esterase activity called ESB3, detectable by starch gel electrophoresis followed by staining with alpha-naphthyl butyrate. Using mononuclear leukocytes, we demonstrated an electrophoretic variant of ESB3. Family studies suggest that the variant is inherited as a simple Mendelian trait; individuals with the ESB3 2-1 phenotype are heterozygotes, designated ESB3(1)ESB3(2), to distinguish them from the more common homozygotes, ESB3(1)ESB3(1). The frequency of the ESB3(2) allele is estimated to be 0.035 in U.S. Whites. No homozygotes for this allele have yet been found. Our studies suggest that the enzyme from ESB3 1 individuals exists primarily as a trimer of three identical subunits with a molecular weight of approximately 58,000 daltons. The genetic variant (ESB3(2) allele) appears to be the result of a mutation that does not affect the charge of the subunit, but rather reduces its ability to form and maintain the trimeric structure.

Chromatography, Gel↗
PubMed1997

Searching through subsets: a test of the visual indexing hypothesis.

This paper presents three experiments investigating the claim that the visual system utilizes a primitive indexing mechanism (sometimes called FINSTS; Pylyshyn, 1989) to make non-contiguous features directly accessible for further visual processing. This claim is investigated using a variant of the conjunction search task in which subjects search among a subset of the items in a conjunction search display for targets defined by a conjunction of colour and orientation. The members of the subset were identified by virtue of the late onset of the objects' place-holders. The cued subset was manipulated to include either homogeneous distractors or mixed distractors. Observers were able to select a subset of three items from among fifteen for further processing (Experiment 1); furthermore, a reaction time advantage for homogeneous subsets over mixed subsets was observed, indicating that more than one of the subset is selected for further specialized processing. The homogeneous subset advantage held for subsets of two to five items (Experiment 2), and the time required to process the cued subset did not increase with increased dispersion of the items (Experiment 3). These results support the basic claim of the indexing theory: the claim that multiple visual indexes are used in selecting objects for visual processing.

Adult↗
PubMed1979

[The 4 c/sec rhythm (case report and differential diagnosis) (author's transl)].

Report on a female patient with a 3 c/sec rhythm. The 3 c/sec rhythm is a special case of the 4 +/- 1 c/sec rhythm. The characteristics of this variant are reviewed in tabulated form. The differential diagnosis has to take into consideration other slow posterior rhythms like the so-called delta-parenrhythmia and delta-parenrhythmia, abortive forms of spike and wave complexes and the abnormal delta-rhythms and sinusoidal delta-rhythms in the infantile EEG.

Adult↗
PubMed1992

Hirudisins. Hirudin-derived thrombin inhibitors with disintegrin activity.

Recombinant hirudin variants have been designed which inhibit alpha-thrombin by the hirudin mechanism and which in addition exhibit disintegrin activity. These proteins, called "hirudisins," have been engineered by replacing the Ser-Asp-Gly-Glu sequence at the tip of hirudin's finger-like structure (residues 32-35) by Arg-Gly-Asp-Ser (RGDS) to yield hirudisin and Lys-Gly-Asp-Ser (KGDS) to obtain hirudisin-1. Comparison of thrombin inhibition activities showed that hirudisin is 2-fold more potent (K(i) = 160 +/- 70 fM) than hirudisin-1 (K(i) = 370 +/- 44 fM) and recombinant (r)-hirudin (K(i) = 270 +/- 50 fM). alpha-Thrombin-stimulated platelet aggregation was effectively inhibited by r-hirudin, hirudisin, and hirudisin-1 with IC50 of 5.7 to 6.8 nM. Unlike r-hirudin, hirudisin inhibits ADP-induced platelet aggregation (IC50 = 65 microM) 3- to 5-fold stronger than the linear GRGDS- and RGDS-peptide. Direct interaction of hirudisin with purified glycoprotein IIb-IIIa demonstrated that antiplatelet aggregation activity is due to the integrin-directed RGD motif. Disintegrin activity of hirudisin relative to that of reduced and carboxymethylated hirudisin suggests that the conformational strain favors binding to integrins. On the basis of these results, hirudisins appear to be interesting molecules for the design of potential antithrombotic agents with antithrombin as well as antiplatelet aggregation activities.

Adenosine Diphosphate↗
PubMed2004

Hepatic and extrahepatic expression of the new iron regulatory protein hemojuvelin.

BACKGROUND AND OBJECTIVES: Hereditary hemochromatosis (HH) is a common disorder of iron overload. A rare variant of the disease, juvenile hemochromatosis, is an early-onset form which is caused by mutations in a recently identified gene, called HJV or HFE2. A previous report based on Northern blotting showed human HJV mRNA expression only in the skeletal muscle, liver and heart. DESIGN AND METHODS: In this study we analyzed the expression of HJV mRNA in a number of human and mouse tissues by a sensitive reverse transcription-polymerase chain reaction method. We also studied the expression of HJV protein in mouse tissues using Western blotting. A polyclonal rabbit antibody was raised against a synthetic peptide which was designed based on the predicted sequence of human and mouse HJV protein. RESULTS: Human HJV mRNA expression was detected in the liver, heart, esophagus, pancreas, descending colon, ileocecum and skeletal muscle. Mouse tissues that were positive for expression included brain, liver, heart, lung, stomach, spleen, kidney, duodenum, jejunum, ileum, colon, skeletal muscle, testis and blood. By Western blotting, HJV protein expression was detected in the mouse liver, heart, kidney, brain and muscle. INTERPRETATION AND CONCLUSIONS: The facts that HJV protein is expressed in the liver and mutations in the HJV gene induce hepatic iron accumulation point to a possibility that HJV protein may modulate iron transport in hepatocytes. The wide expression of HJV as shown in the present study suggests that its role in regulating iron allocation could be extended to other tissues beyond the liver.

Adult↗
PubMed2006

[Male hormonal contraception].

Human reproduction is a complex phenomenon remaining at the center of interest of many medical and extra-medical disciplines and the search for new contraceptive methods is an important part of research effort. The development of effective forms of male hormonal contraception (MHC) is one of the priorities of the World Health Organization (WHO Task Force on Methods of Regulation of Male Fertility). The principle of MHC consists in the suppression of spermatogenesis while preserving all other functional aspects of the male glands (especially the sexual functions, bone metabolism and lean muscle mass). It is possible to stop spermatogenesis by blocking gonadotrophin (FSH and LH) secretion by testosterone administration (isolated or in combination with other hormones). The currently existing variants of MHC are based on testosterone application (which has in the same time the role of substitutive treatment--the so-called androgen "add-back") either in monotherapy (oral, intramuscular, transdermal and subcutaneous form) or in combination with various progestins (levonorgestrel, norethisterone, desogestrel, eronogestrel, medroxyprogesterone), antiandrogens or GnRH analogues. The most promising, at present, seem to be the methods which use a combination of depot testosterone preparations with long-acting progestins. To what extent will the role of MHC be important in the future--this can only be judged after establishing their effectiveness, full reversibility, safe application, acceptability, and financial accessibility in clinical practice.

Contraceptive Agents, Male↗
PubMed1978

[Some questions about blood group nomenclature].

Author in introductory part of the paper deals with several basic principles of the scientifical nomenclature, then with the up to date definition of "blood-groups". The so called "blood-groups" in the human and in animals represent biochemical, more exactly structurechemical polymorphisms, i.e. they are genetically regulated inherited variants of some functionally basic molecules. Consequently the expression "blood-groups"--which originally implied Landsteiner's ABO-system and it's groups--nowadays include various types (erythrocyte-membrane, serum, enzym etc.) of structural polymorphisms. Author recommends to sign serum samples ("Serotypes") as anti-A, anti-B and anti-A + B. Definition of "sub-groups" and "variants" is given. Author deals with the nomenclature of Rh-system including Rh-phenotypes and genotypes. At last description of symbols used in blood-group systems is given.

ABO Blood-Group System↗