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Biomedical subjects

M Anton

Publications and source records attributed to M Anton.

At least 37 records · Page 2Linked to original sources
PubMed2000

Bacterial and nonbacterial expression of wild-type and mutant human phospholipid hydroperoxide glutathione peroxidase and purification of the mutant enzyme in the milligram scale.

15-Lipoxygenases and phospholipid hydroperoxide glutathione peroxidases are counterparts in the metabolism of hydroperoxy lipids and a balanced regulation of both enzymes is essential for normal cell function. Glutathione peroxidases contain selenocysteine as catalytically active amino acid and this selenocysteine is encoded by a TGA stop codon. Detailed protein chemical investigations on phospholipid hydroperoxide glutathione peroxidases and crystal trials have been hampered in the past by limited protein supply. There is no efficient natural source for large-scale enzyme preparation and overexpression of the functional protein in recombinant systems has not been reported so far. To avoid problems with recognition of the selenocysteine stop codon we mutated the selenocysteine to a cysteine and expressed the Sec46Cys mutant in milligram amounts in the baculovirus/insect cell system and as His-tag fusion protein in Escherichia coli. The recombinant enzyme species were purified by conventional fast protein liquid chromatography (nonfusion protein) or by affinity chromatography on a nickel matrix (His-tag protein). Surprisingly, we found that both protein variants were functional although their specific activities were reduced when compared with the wild-type enzyme. Basic protein chemical and enzymatic properties of the purified enzyme species were determined and monoclonal antibodies which recognize the native phospholipid hydroperoxide glutathione peroxidases were raised using our enzyme preparations as antigen. The described strategies for overexpression of mutant phospholipid hydroperoxide glutathione peroxidase species and their purification from recombinant sources provide sufficient amounts of enzyme for future protein chemical investigations and detailed crystal trials.

Animals↗
PubMed2000

ARF- and coatomer-mediated peroxisomal vesiculation.

The authors characterized on a molecular level the clofibrate-inducible 26-kDa integral peroxisomal membrane protein (Pmp26p, Pex11-1p) of rat liver. By screening cDNA databases with the obtained Pex11-1p-cDNA, a second homologous cDNA was identified that codes for a polypeptide with slightly larger molecular mass than Pex11-1p. The authors call this polypeptide Pex11-2p. Studies on the topology of Pex11-1p revealed two transmembrane domains with the N- and C-terminus facing the cytoplasm. The C-terminal tail of Pex11-1p ends in a consensus dilysine motif of the type -KXKXX-COOH, which is known to be involved in the ADP-ribosylation factor (ARF)1-coat protein (COP) I coat (ARF)1-dependent membrane recruitment to Golgi membranes. Studies with isolated peroxisomes incubated in the presence of cytosol, adenosine triphosphate and GTP gamma S, indeed, provided evidence for specific binding of ARF and coatomer to peroxisomes. Expression of Pex11-1p in Chinese hamster ovary (CHO) wild-type cells led to a twofold increase in the number of peroxisomes, but expression in a temperature-sensitive CHO mutant, defective in coatomer, induced elongation and tubulation of peroxisomal structures, rather than numerical proliferation. The obtained results for the first time offer a mechanism explaining Pex11-1p-, as well as ARF- and coatomer-mediated peroxisomal vesiculation. Two models are presented that may explain how these observations fit in with peroxisome biogenesis.

ADP-Ribosylation Factor 1↗
PubMed2000

The role of tumour suppressors and viral oncoproteins in cervical carcinogenesis.

This article summarizes current knowledge of the cervical carcinogenesis with a special focus on the molecular mechanisms involving the interaction of cellular tumour suppressors (p53, RB, p73) with viral oncoproteins (E6, E7). The E6-induced degradation of p53 protein results in the inhibition of apoptosis, inability to repair DNA and fixation of mutations. The p53-dependent tumourigenesis is influenced by interaction not only with E6/HPV 16, 18 but also with MDM2, bcl-2 and RB protein. The polymorphism of p53 seems to contribute to malignant transformation of cervix. On contrary, there are experimental data showing that p53 may not be the only factor playing role in malignant transformation in cervical cancer. It has been generally agreed that viral oncoprotein E6 is a critical step in the onset of malignant transformation of cervix. There is a vast number of experimental and clinical studies confirming the validity of E6 induced cervical cancer including alteration of the genotype and phenotypic characteristics of the transforming cells. The modern tools of molecular biology offer an exact diagnosis as well as relevant targets for gene therapy of the cervical cancer.

Female↗
PubMed2000

European Society of Gene Therapy (ESGT) Seventh Meeting. 26-28 November 1999, Munich, Germany.

This meeting gave an excellent overview of the recent developments in gene therapy. Much research effort has focused on the improvement and de novo construction of gene vectors, on characterizing their mechanisms of action and on their interactions with, and in, living organisms. The continual improvements in understanding disease at a molecular level and the progress in cell biology, immunology and related fields have opened the way for novel gene therapy approaches. The gene therapeutic strategy has proven to be feasible and efficient in numerous preclinical (animal) models of a variety of diseases. On the other hand, applying this experience to humans has turned out to be difficult. Currently, the field is rapidly moving into clinical applications. No major limiting side effects have been observed in patients in the phase I and later stage trials presented. Nonetheless, years of preclinical and clinical research will be required before gene therapy can be considered a reliable and efficient therapeutic approach with broad applicability.

Journal Article↗
PubMed1999

Shape and specificity in mammalian 15-lipoxygenase active site. The functional interplay of sequence determinants for the reaction specificity.

Previous mutagenesis studies along with molecular modeling using the x-ray coordinates of the rabbit 15-lipoxygenase have led to the suggestion that the size of the substrate binding pocket may play an essential role in determining the oxygenation specificity of 5-, 12-, and 15-lipoxygenases. Based on the x-ray crystal structure of rabbit 15-lipoxygenase, Ile(593) appeared to be important in defining size and shape of the substrate-binding site in 15-lipoxygenases. We found that substitution of Ile(593) with alanine shifted the positional specificity of this enzyme toward 12-lipoxygenation. To compare the importance of position 593 with previously defined determinants for the oxygenation specificity, we introduced small (alanine-scan) or large amino acids (phenylalanine-scan) at critical positions surrounding the putative fatty acid-binding site, so that the volume of the pocket was either increased or decreased. Enlargement or alteration in packing density within the substrate binding pocket in the rabbit 15-lipoxygenase increased the share of 12-lipoxygenase products, whereas a smaller active site favored 15-lipoxygenation. Simultaneous substitution of both large and small residues in the context of either a 15- or 12-lipoxygenase indicated that there is a functional interplay of the sequence determinants for lipoxygenation specificity. If the 15-lipoxygenase active site is enlarged excessively, however, no lipoxygenation was observed anymore. Together these results indicate the importance of the overall size and shape of the arachidonic acid binding pocket in defining the specificity of lipoxygenase reaction.

Animals↗
PubMed1999

Human papillomavirus genotype spectrum in Czech women: correlation of HPV DNA presence with antibodies against HPV-16, 18, and 33 virus-like particles.

Because the biological spectrum of human papillomavirus (HPV) genotypes present in cervical cancer lesions varies according to the geographical region studied, and because little genotype information is available for Central and Eastern European countries, we studied the endemic HPV-genotype spectrum in cervical samples collected from women visiting gynaecological departments of selected hospitals in the Czech Republic. In a series of 389 samples, 171 (44.0%) were positive for HPV DNA using a consensus-primer polymerase chain reaction (PCR). Genotyping of the HPV PCR products was done using dot-blot hybridisation with type-specific oligonucleotide probes and thermocycle DNA sequencing. Twenty-two different HPV types were detected, HPV-16 being the most prevalent type irrespective of severity of the lesions (55.0%). Multiple HPV types were found in 16.4% of our HPV-DNA-positive samples. The prevalence of HPV infection was 23.0% in women with normal findings and 59.4% in patients with cervical neoplasia, and increased significantly with the severity of the disease: 52.9% in low-grade lesions, 58.0% in high-grade lesions, and 73.5% in cervical carcinomas (P for trend < .00001). In the sera of 191 subjects, 89 with normal findings and 102 with different forms of cervical neoplasia, the prevalence of HPV-specific IgG antibodies was tested by an enzyme-linked immunosorbent assay (ELISA) using virus-like particles (VLPs) of HPV-16, -18, and -33. Antibodies were significantly more prevalent in HPV-DNA-positive than in HPV-DNA-negative women and there was no association with age. In agreement with the results of HPV genotyping, antibodies reactive with HPV-16 VLPs were the most frequent and, moreover, their prevalence increased with the cervical lesion severity. About half of the subjects with smears in which either HPV-16 or HPV-33 DNA had been detected possessed antibodies reactive with homotypic VLPs. With HPV-18-DNA-positive subjects, however, fewer than 25% displayed homotypic antibodies. In general, subjects older than 30 years of age had antibodies reactive to HPV-specific VLPs more often than subjects younger than 30 years of age. In women with benign findings, the seropositivity to HPV-16, -18, and -33 VLPs increased with age, whereas in women with cervical neoplasia the seropositivity decreased with age.

Adult↗
PubMed1998

Probing the substrate alignment at the active site of 15-lipoxygenases by targeted substrate modification and site-directed mutagenesis. Evidence for an inverse substrate orientation.

For oxygenation of polyenoic fatty acids by 12- and 15-lipoxygenases the methyl terminus of the substrate constitutes the signal for the initial hydrogen abstraction. In contrast, for 5-lipoxygenases an inverse head to tail substrate orientation has been proposed. However, recent structure-based sequence alignments suggested a conserved uniform substrate orientation for 5S- and 15S-lipoxygenation. Oxygenation of 15S-HETE derivatives by various wild-type and mutant lipoxygenases was investigated, and the evidence proved an inverse substrate orientation: (i) Substrate affinity and Vmax of 15S-HETE oxygenation by arachidonic acid 15-lipoxygenases are >1 order of magnitude lower than the corresponding data for polyenoic fatty acids. 5S,15S- and 14R, 15S-DiH(P)ETE were identified as major reaction products. (ii) Methylation of the carboxylate group of 15S-HETE augmented the reaction rate and shifted the reaction specificity strongly toward 5S-lipoxygenation. In contrast, methyl arachidonate was less effectively oxygenated than the free acid. Methylation of 15S-HETrE(8,11,14), which lacks the C5-C6 double bond, was without major impact on the oxygenation rate and on the product specificity. (iii) Introduction of a bulky glycerol moiety at the carboxylic group of 15S-HETE reversed the kinetic effects of methylation and led to a 14R-oxygenation of the substrate. (iv) When the product pattern of 15S-HETE oxygenation by the recombinant wild-type rabbit 15-lipoxygenase was compared with that formed by the Arg403Leu mutant, 5S- and 8S-lipoxygenations were augmented and 14R, 15S-DiH(P)ETE formation was impaired. (v) Phe353Leu or Ile418Ala mutation of the same enzyme, which favored 12S-HETE formation from arachidonic acid, strongly augmented 8S-lipoxygenation of 15S-HETE methyl ester. These kinetic data and the alterations in the product specificity are consistent with the concept of an inverse head to tail substrate orientation during the oxygenation of 15S-HETE methyl ester and/or of free 15S-HETE by 15-LOXs. For 5S- and 8S-lipoxygenation, 15-HETE may slide into the substrate binding pocket with its carboxy terminus approaching the doubly allylic methylenes C-7 or C-10 to the non-heme iron.

Animals↗
PubMed1998

Peroxisome biogenesis: involvement of ARF and coatomer.

Peroxisomal membrane protein (Pmp)26p (RnPex11p), a major constituent of induced rat liver peroxisomal membrane, was found to contain a COOH-terminal, cytoplasmically exposed consensus dilysine motif with the potential to bind coatomer. Biochemical as well as immunocytochemical evidence is presented showing that peroxisomes incubated with preparations of bovine brain or rat liver cytosol recruit ADP-ribosylation factor (ARF) and coatomer in a strictly guanosine 5'-O-(3-thiotriphosphate)-dependent manner. Consistent with this observation, ldlF cells expressing a temperature-sensitive mutant version of the epsilon-subunit of coatomer exhibit elongated tubular peroxisomes possibly due to impaired vesiculation at the nonpermissive temperature. Since overexpression of Pex11p in Chinese hamster ovary wild-type cells causes proliferation of peroxisomes, these data suggest that Pex11p plays an important role in peroxisome biogenesis by supporting ARF- and coatomer-dependent vesiculation of the organelles.

ADP-Ribosylation Factors↗
PubMed1998

Intra- and extracellular expression of rabbit reticulocyte 15-lipoxygenase in the Baculovirus/insect cell system.

Rabbit reticulocyte 15-lipoxygenase was expressed as intracellular enzyme and as export protein in High Five cells. While intracellular expression in the baculovirus/insect cell system was already reported for various mammalian lipoxygenases, we have developed a strategy for secretion of this cytosolic enzyme using the signal sequence of human interleukin-2. Expression levels of 10 mg/liter (intracellular strategy) and 18 mg/liter (extracellular strategy) were obtained. The recombinant enzyme expressed as intracellular protein was purified to apparent homogeneity by anion-exchange chromatography on a Mono-Q column with an overall recovery of 80% enzyme activity. For the final enzyme preparation, a specific linoleic acid oxygenase activity of 16.7 micromol 13-hydroperoxyoctadeca-9,11-dieic acid formation mg-1 min-1 was determined, which corresponds to a molecular turnover number of 21 s-1. Similar turnover rates have been reported for the native rabbit 15-lipoxygenase. Extracellularly expressed recombinant 15-lipoxygenase exhibited a heterogeneity in anion-exchange chromatography. Three major peaks of 15-lipoxygenase activity were eluted from a Mono-Q column and the relative amounts of these isoforms varied from batch to batch of enzyme expression. One of the major isoenzymes which cochromatographed with the native 15-lipoxygenase was purified to homogeneity from the cell-free culture supernatant and exhibited a specific activity of 5.1 micromol 13-hydroperoxyoctadeca-9,11-dienoic acid formation mg-1 min-1 (turnover rate of 6.1 s-1). The recombinant enzyme species were characterized with respect to their protein-chemical and enzymatic properties and no differences to the native rabbit 15-lipoxygenase were detected.

Animals↗
PubMed1998

Computer assisted orthopaedic surgery with image based individual templates.

Recent developments in computer assisted surgery offer promising solutions for the translation of the high accuracy of the preoperative imaging and planning into precise intraoperative surgery. Broad clinical application is hindered by high costs, additional time during intervention, problems of intraoperative man and machine interaction, and the spatially constrained arrangement of additional equipment within the operating theater. An alternative technique for computerized tomographic image based preoperative three-dimensional planning and precise surgery on bone structures using individual templates has been developed. For the preoperative customization of these mechanical tool guides, a desktop computer controlled milling device is used as a three-dimensional printer to mold the shape of small reference areas of the bone surface automatically into the body of the template. Thus, the planned position and orientation of the tool guide in spatial relation to bone is stored in a structural way and can be reproduced intraoperatively by adjusting the position of the customized contact faces of the template until the location of exact fit to the bone is found. No additional computerized equipment or time is needed during surgery. The feasibility of this approach has been shown in spine, hip, and knee surgery, and it has been applied clinically for pelvic repositioning osteotomies in acetabular dysplasia therapy.

Acetabulum↗
PubMed1998

Requirement for both Shc and phosphatidylinositol 3' kinase signaling pathways in polyomavirus middle T-mediated mammary tumorigenesis.

Transgenic mice expressing the polyomavirus (PyV) middle T antigen (MT) develop multifocal mammary tumors which frequently metastasize to the lung. The potent transforming activity of PyV MT is correlated with its capacity to activate and associate with a number of signaling molecules, including the Src family tyrosine kinases, the 85-kDa Src homology 2 subunit of the phosphatidylinositol 3' (PI-3') kinase, and the Shc adapter protein. To uncover the role of these signaling proteins in MT-mediated mammary tumorigenesis, we have generated transgenic mice that express mutant PyV MT antigens decoupled from either the Shc or the PI-3' kinase signaling pathway. In contrast to the rapid induction of metastatic mammary tumors observed in the strains expressing wild-type PyV MT, mammary epithelial cell-specific expression of either mutant PyV MT resulted in the induction of extensive mammary epithelial hyperplasias. The mammary epithelial hyperplasias expressing the mutant PyV MT defective in recruiting the PI-3' kinase were highly apoptotic, suggesting that recruitment of PI-3' kinase by MT affects cell survival. Whereas the initial phenotypes observed in both strains were global mammary epithelial hyperplasias, focal mammary tumors eventually arose in all female transgenic mice. Genetic and biochemical analyses of tumorigenesis in the transgenic strains expressing the PyV MT mutant lacking the Shc binding site revealed that a proportion of the metastatic tumors arising in these mice displayed evidence of reversion of the mutant Shc binding site. In contrast, no evidence of reversion of the PI-3' kinase binding site was noted in tumors derived from the strains expressing the PI-3' kinase binding site MT mutant. Tumor progression in both mutant strains was further correlated with upregulation of the epidermal growth factor receptor family members which are known to couple to the PI-3' kinase and Shc signaling pathways. Taken together, these observations suggest that PyV MT-mediated tumorigenesis requires activation of both Shc and PI-3' kinase, which appear to be required for stimulation of cell proliferation and survival signaling pathways, respectively.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Spermatic cord hematoma in a collegiate football player: a case report.

OBJECTIVE: In order to expand the athletic trainer's awareness of acute scrotal injuries, the objective is to present a case of a collegiate football player sustaining a spermatic cord hematoma injury. BACKGROUND: Hematomas tend to resolve with conservative management. However, untreated testicular injuries may have serious ramifications, including ischemic atrophy, necrosis, and secondary infection. Exploratory surgery remains the standard for any acute scrotal lesion that cannot be diagnosed by physical examination or by diagnostic testing. Surgical exploration, however, is unnecessary in cases of scrotal hematoma. DIFFERENTIAL DIAGNOSIS: Epididymitis, incarcerated inguinal hemia, testicular torsion, testicular hematocele, and scrotal trauma with hematoma formation. TREATMENT: Conservative management including bedrest, ice, slight elevation of the lower extremities, and nonsteroidal anti-inflammatory drugs. UNIQUENESS: Noncontact scrotal injuries resulting in moderate swelling, pain, and disability are rare in athletes. This case study presents an athlete who suffered a moderate adductor strain 1 month before the formation of a spermatic cord hematoma. Comparison of the two episodes may be found in the subjective history. The question arises as to whether or not the hematoma formation was a possible complication of the initial adductor muscle injury. CONCLUSIONS: Although scrotal injuries are not life threatening, untreated testicular injures can have immediate and severe consequences. It is essential that athletes with acute scrotal injuries accompanied by swelling and tenderness be evaluated by a physician with training in clinical urology.

Journal Article↗
PubMed1997

Quail egg yolk: a novel cryoprotectant for the freeze preservation of Poitou jackass sperm.

For many years, attempts have been made to establish a sperm bank for the Poitou jackass population which is threatened with extinction. Unfortunately, no cryopreservation technique has ever been described for spermatozoa of this species. In an attempt to find a suitable technique, we studied the relative effectiveness of chicken egg yolk and quail egg yolk in preserving the motility and characteristics of movement of Poitou jackass spermatozoa during the freezing-thawing process. Semen was diluted to 60 x 10(6) sperm/ml in a preservation medium containing 4% (v/v) glycerol with 0, 2, 5, 10, 15, or 20% (v/v) of chicken or quail egg yolk. The chemical composition of these two eggs was compared. Effects were assessed using an automated analyzer which measured curvilinear velocity (VCL), straight line velocity (VSL), and the velocity of the average path. Linearity was defined as VSL/VCL x 100. The amplitude of the lateral head displacement was also measured. It was found that after the freeze-thaw process, quail egg yolk improved the percentages of motile and progressively undulating spermatozoa and the movement characteristics compared with chicken egg yolk. The optimal concentration of quail egg yolk was 10%. The general composition of the two types of egg yolk were similar, but quail egg yolk contained significantly more phosphatidylcholine, less phosphatidylethanolamine, and a smaller ratio of polyunsaturated to saturated fatty acids than chicken egg yolk. The improvement of motility for frozen-thawed Poitou jackass spermatozoa using frozen-thawed quail egg yolk compared to chicken egg yolk may be due to the differences in composition of the two yolks.

Animals↗
PubMed1997

Reprogramming of telomerase by expression of mutant telomerase RNA template in human cells leads to altered telomeres that correlate with reduced cell viability.

Telomerase synthesizes telomeric DNA by copying the template sequence of its own RNA component. In Tetrahymena thermophila and yeast (G. Yu, J. D. Bradley, L. D. Attardi, and E. H. Blackburn, Nature 344:126-131, 1990; M. McEachern and E. H. Blackburn, Nature 376:403-409, 1995), mutations in the template domain of this RNA result in synthesis of mutant telomeres and in impaired cell growth and survival. We have investigated whether mutant telomerase affects the proliferative potential and viability of immortal human cells. Plasmids encoding mutant or wild-type template RNAs (hTRs) of human telomerase and the neomycin resistance gene were transfected into human cells to generate stable transformants. Expression of mutant hTR resulted in the appearance of mutant telomerase activity and in the synthesis of mutant telomeres. Transformed cells were not visibly affected in their growth and viability when grown as mass populations. However, a reduction in plating efficiency and growth rate and an increase in the number of senescent cells were detected in populations with mutant telomeres by colony-forming assays. These results suggest that the presence of mutant telomerase, even if coexpressed with the wild-type enzyme, can be deleterious to cells, likely as a result of the impaired function of hybrid telomeres.

Cell Survival↗
PubMed1997

A heterotrimeric G protein-phospholipase A2 signaling cascade is involved in the regulation of peroxisomal motility in CHO cells.

Peroxisomal motility was studied in vivo in CHO cells following transfection with a green fluorescent protein construct containing the C-terminal peroxisomal targeting signal 1 (GFP-PTS1). Time-lapse imaging and evaluation of difference images revealed that peroxisomes attach to microtubules in a Ca2+ requiring step and are transported in an ATP-dependent manner. Following microinjection of guanosine-5'-O-(3-thiotri-phosphate) (GTP(gamma)S), peroxisomal movements were arrested, indicating regulation by GTP-binding proteins. The effect of GTP(gamma)S was mimicked by AlF4- and mastoparan, two drugs which are known to activate heterotrimeric G proteins. Pertussis toxin which prevents Gi/Go protein activation completely abolished the effect of GTP(gamma)S and mastoparan on peroxisomal motility suggesting that the G protein belongs to the Gi/Go class. At least one effector of the G protein is phospholipase A2 as demonstrated by the observation that the phospholipase A2 activating protein peptide efficiently blocks peroxisomal motility, and that the effect of mastoparan and AlF4- is largely abolished by various phospholipase A2 inhibitors. In summary, these data provide evidence for a new type of regulation of organelle motility mediated by a Gi/Go-phospholipase A2 signaling pathway. This type of regulation has not been observed so far with other cell organelles such as mitochondria, the endoplasmic reticulum or axonal vesicles. Thus, motility is regulated individually for each cell organelle by distinct mechanisms enabling the cell to fulfill its vital functions.

Animals↗
PubMed1997

DNA flow cytometry: a predictor of a high-risk group in cervical cancer.

We studied the relationship between the 5-year disease-free interval or the occurrence of distant metastases, and the flow cytometric nuclear DNA content in a group of 55 patients treated by radiation for squamous cell carcinoma of the uterine cervix, stages Ib-IIIb (FIGO). The diploid DNA content was associated with a better prognosis and a lower incidence of distant metastases, while aneuploid tumors tended to be prognostically unfavorable and had distant metastases more often. We consider the flow cytometric nuclear DNA content a prospective prognostic parameter in squamous cell carcinoma of the uterine cervix treated by radiotherapy.

Aneuploidy↗