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S J Fey

Publications and source records attributed to S J Fey.

At least 37 records · Page 2Linked to original sources
PubMed1995

Two-dimensional gel analysis of human endometrial proteins: cyclic changes in the expression of specific proteins during the normal menstrual cycle.

High resolution two-dimensional (2-D) gel electrophoresis was used to compare the patterns of [35S]methionine-labelled cellular proteins in endometrial tissue from healthy, normally menstruating women. Samples of endometrial tissue were incubated with [35S]methionine for 20 h, and total cell lysates were processed for 2-D gel electrophoresis. Using this technique it was possible to study proteins with iso-electric points (pI) ranging from 3.5 to 11 and relative molecular weights (M(r)) ranging from 10,000 to 300,000 Da. The fluorograms were compared by computer-aided analysis whereby a total of 1095 [35S]-labelled proteins were resolved on the iso-electric focusing gels (IEF, pI 3.5-7) and 488 on the non-equilibrium pH gradient electrophoresis (NEPHGE) gels (pI 6.5-11). Of the proteins on the IEF gels, 125 showed differential expression during the menstrual cycle. Of these, 36 were maximally expressed in proliferative phase endometrium, 26 in the interval phase and 63 in secretory and/or late secretory phase endometrium. Correspondingly, on the NEPHGE gels a total of 61 proteins exhibited cyclical variation, of which 30 were more prominent in proliferative phase, 13 in interval phase and 18 in secretory phase endometrium. This study shows that 2-D gel electrophoresis is eminently suited to the identification of proteins whose expression varies in a cyclical manner during the menstrual cycle. Further investigations should be carried out to isolate and characterize these proteins with the aim of establishing useful markers for specific endometrial phases of the menstrual cycle.

Adult↗
PubMed1995

Human endometrial proteins with cyclic changes in the expression during the normal menstrual cycle: characterization by protein sequence analysis.

Endometrial proteins showing cyclic expression during the normal menstrual cycle were localized on two-dimensional (2-D) electrophoresis gels separating proteins with isoelectric points (pl) ranging from 3.5 to 7 and relative molecular weights ranging from 10 to 300 kDa. Menstrual cycle-related proteins were excised from several 2-D gels, concentrated by one-dimensional (1-D) sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis, and cleaved in situ by trypsin. The tryptic fragments were extracted and separated by reverse phase high performance liquid chromatography (RP-HPLC). Finally, the partial amino-terminal amino acid sequence of selected tryptic fragments were determined for each protein. We aimed at characterizing the 21 menstrual cycle-related proteins that were visible on silver-stained 2-D electrophoresis gels. Of the proteins being maximally synthesized in the proliferative phase endometrium, we identified proteins associated mainly with the cytoskeleton: vimentins, keratin, tropomyosin and tubulin, but also proteins such as proliferating cell nuclear antigen and beta-galactoside binding lectin. The partial amino acid sequences for another two proteins did not match any protein sequence in the Protein Identification Resource (PIR) and Swissprot databases. In the group of proteins having maximal synthesis in the secretory phase endometrium, we identified creatine kinase chain B and an isocitrate dehydrogenase-homologous protein, both of which are involved in energy metabolism. However, we also identified the annexin IV precursor, the 14-3-3 protein homologue also called stratifin or the epithelial cell marker protein 1 and the 21K tumour protein. Finally, four of the proteins were present in too low amounts to allow characterization. Interestingly, most of the identified proteins have not previously been described as having a menstrual cycle-related synthesis in the human endometrium. It may be considered that the concentration of some of the cycle-related proteins may be used in clinical situations to reflect specific endometrial phases.

Amino Acid Sequence↗
PubMed1995

Two-dimensional gel electrophoresis of rat islet proteins. Interleukin 1 beta-induced changes in protein expression are reduced by L-arginine depletion and nicotinamide.

Interleukin (IL)-1 beta-mediated damage to beta-cells in isolated islets of Langerhans depends upon de novo synthesis of proteins that have not been fully identified. Further, IL-1 beta-induced and tumor necrosis factor alpha-induced islet damage partly depends on the intracellular production of the nitric oxide (NO) radical. IL-1 beta has also been reported to induce the synthesis of cellular defense proteins, e.g., heme-oxygenase and heat shock proteins 70 and 90. Nicotinamide, while in itself inactive, inhibited IL-1 beta-induced NO production in a time- and dose-dependent manner. To enable the identification of IL-1 beta-induced proteins with possible protective and deleterious effects, we characterized the effects of IL-1 beta, nicotinamide, and NO synthesis inhibition by L-arginine depletion on rat islet protein expression detected by high-resolution two-dimensional gel electrophoresis. More than 1,600 proteins were reproducibly detected in control rat islets. Incubation with IL-1 beta-, nicotinamide-, or L-arginine-depleted control medium upregulated 29, 3, and 1 protein, respectively, and downregulated 4, 0, and 1 protein, respectively. Addition of nicotinamide and L-arginine depletion reduced the upregulation of 16 and 20 IL-1 beta-induced proteins, respectively. The identity of these proteins is under study. The demonstrated changes in protein expression caused by IL-1 beta +/- nicotinamide and L-arginine depletion may form the basis for identification of proteins with possible protective and deleterious roles in the initial beta-cell destruction in insulin-dependent diabetes mellitus.

Animals↗
PubMed1995

Cloning and expression of cytokine-inducible nitric oxide synthase cDNA from rat islets of Langerhans.

An inducible nitric oxide (NO) synthase isoform (iNOS) is specifically induced in the beta-cells of interleukin (IL)-1 beta-exposed rat islets, suggesting a role for NO in the pathogenesis of type I diabetes. The aim of this study was to clone and characterize iNOS cDNA from cytokine-exposed islets. Neither NO production nor iNOS transcription could be detected in rat islets or in rat insulinoma RIN-5AH beta-cells cultured in the absence of cytokines. Addition of IL-1 beta alone or in combination with tumor necrosis factor-alpha induced a concentration- and time-dependent expression of the iNOS gene and associated NO production (measured as nitrite) from both islets and RIN cells. iNOS transcripts were cloned by reverse transcriptase-polymerase chain reaction from the cytokine-exposed rat islets and RIN cells, and DNA sequence analysis revealed a near 100% identity to the recently published iNOS cDNA cloned from cytokine-exposed rat hepatocytes and smooth muscle cells. Recombinant rat islet iNOS was transiently and stably expressed in human kidney 293 fibroblasts, and the high enzymatic activity was inhibited by addition of the L-arginine analogs, N omega-nitro-L-arginine methyl ester and aminoguanidine. Two-dimensional gel electrophoresis revealed the recombinant iNOS as a series of spots with the expected molecular mass of 131 kDa and pI values in the range of 6.8 to 7.0. In conclusion, the IL-1 beta-induced iNOS cloned and expressed from rat islets and RIN cells is encoded by the same transcript as the iNOS induced in other cell types.

Amino Acid Oxidoreductases↗
PubMed1991

Chlamydia trachomatis contains a protein similar to the Legionella pneumophila mip gene product.

A 27kDa Chlamydia trachomatis L2 protein was characterized by the use of monoclonal antibodies and by two-dimensional gel electrophoresis. The protein was shown to be located in the membrane of reticulate bodies as well as elementary bodies. Its synthesis could be detected from 10 hours post-infection. Cloning and sequence analysis of the distal part of the gene revealed an open reading frame of 175 amino acids. Comparison of the deduced amino acid sequence with the NBRF data base revealed significant homology between the 27 kDa chlamydial membrane protein and the product of the macrophage infectivity potentiator (mip) gene of Legionella pneumophila.

Amino Acid Sequence↗
PubMed1991

Morphological differentiation and changes in polypeptide synthesis pattern during regeneration of human epidermal tissue developed in vitro.

By incubating multilayered primary cultures of human keratinocytes in low-calcium medium the suprabasal cell layers can be stripped off leaving a basal cell monolayer. When this monolayer is re-fed normal calcium medium a reproducible series of cell kinetic, morphological, and biochemical changes takes place resulting in the reestablishment of a multilayered tissue. Analysis of cell-cycle-specific proteins indicated that, during regeneration, a large cohort of cells became synchronized undergoing DNA replication after 3 days. Examination of culture morphology at the ultrastructural level confirmed the capacity of the basal cell monolayer to gradually reestablish a multilayered, differentiated epithelium. The ultrastructural appearance at 7 days poststripping was similar to that of unstripped cultures and was indicative of a tissue in steady state. Quantitation of cornified envelope formation at different times during regeneration showed that an increasing proportion of the cells were able to undergo terminal differentiation. In general, the pattern of keratin synthesis in the original epidermal explant labelled in vitro was similar to the pattern observed in human epidermis in vivo; however, in contrast to epidermis in vivo the explant also synthesized the hyperproliferative keratins 6 and 16. The in vitro differentiated keratinocytes showed underexpression of several proteins identified as differentiation markers, whereas several basal cell markers were overexpressed compared to the original explant. In addition, the in vitro differentiated keratinocytes synthesized some new proteins, notably keratins 7, 15 and 19. The basal layer remaining after stripping mainly expressed basal cell markers; however, during recovery, some of the differentiation-specific markers (e.g. keratin 10 and 15) were again expressed together with keratin no. 19, which is also expressed during wound healing in vivo. It is suggested that the present system of regenerating epidermal tissue cultures may serve as an experimental model to investigate certain aspects of the regulation of epidermal tissue homeostasis.

Cell Differentiation↗
PubMed1991

Human papillomavirus type 18 E6*, E6, and E7 protein synthesis in cell-free translation systems and comparison of E6 and E7 in vitro translation products to proteins immunoprecipitated from human epithelial cells.

Expression of the E6 and E7 transforming genes of human papillomavirus type 18 (HPV18) occurs via structurally bicistronic mRNAs in which the downstream open reading frame (ORF) E7 is preceded either by the full-length ORF E6 or by a spliced ORF, E6*. We have used in vitro transcription and translation of HPV18 cDNAs in order to analyze the synthesis of E6*, E6, and E7 proteins and to compare the E6 and E7 in vitro translation products with the authentic proteins immunoprecipitated from cervical cancer cells. In wheat germ extract, in vitro translation resulted in the production of all three proteins, E6*, E6, and E7. In rabbit reticulocyte lysate, however, only the E6 and E7 proteins were produced. The lack of E6* protein was due neither to template RNA degradation nor to an inhibitory influence of the RNA 5' leader sequences, thus indicating the possibility of either inhibition of synthesis or degradation of E6* protein in reticulocyte lysate. The E7 protein was synthesized from both E6*-E7 and E6-E7 RNAs. In vitro-synthesized and authentic HPV18 E7 proteins revealed identical electrophoretic mobilities in two-dimensional gel electrophoresis, thus indicating similar modifications. By using a monoclonal antibody against the N terminus of HPV18 E6* and E6, an 18-kDa protein was detected not only in HPV18-positive but also in HPV18-negative epithelial cells. The 18-kDa proteins and the in vitro-synthesized HPV18 E6 protein exhibited comparable electrophoretic characteristics in two-dimensional gels. These results suggest the possible existence of a cellular protein related to HPV18 E6.

Cell-Free System↗
PubMed1990

Markers for human placental trophoblasts in two-dimensional gel electrophoresis.

Primary cultures of trophoblasts established from human term placentae showed high viability and reproducibility. Two-dimensional gel patterns obtained by metabolically labeling the trophoblasts with [35S]-methionine demonstrated that their pattern of gene expression was stable during the 6-d period investigated. Gel analysis demonstrated the keratins 7, 8, 14, 17, 18, and 19. Analysis of the gel pattern confirmed the presence of a small proportion of contaminating fibroblasts and lymphocytes. The gel patterns were compared with that of skin fibroblasts, peripheral lymphocytes, and epithelial cells to identify a group of proteins that are enriched in the trophoblasts and thus may be used as marker for these cells.

Cells, Cultured↗
PubMed1990

Characterization and identification of early proteins in Chlamydia trachomatis serovar L2 by two-dimensional gel electrophoresis.

The synthesis of early proteins from Chlamydia trachomatis serovar L2 was analyzed by two-dimensional gel electrophoresis. By pulse-label experiments, the synthesis of seven proteins was observed at 2 to 8 h postinfection before the major outer membrane protein was detected at 8 to 10 h after infection. The early proteins were synthesized throughout the 30-h period investigated, but the synthesis of three proteins of 75, 62, and 45 kilodaltons decreased from 26 to 30 h postinfection. Pulse-chase analysis showed that the signals from the same three proteins declined 26 to 30 h after infection. Three of the early proteins were identified as the S1 ribosomal protein, the GroEL-like protein, and DnaK-like protein, respectively.

Animals↗
PubMed1989

Demonstration of in vitro synthesis of human papilloma viral proteins from hand and foot warts.

HPV particles purified from [35S]-methionine labeled and unlabeled halves of single hand and foot warts have been fractionated into empty, light full, and heavy full particles by buoyant density gradient centrifugation, and their proteins analyzed by two-dimensional gel electrophoresis (IEF and NEPHGE) and visualized by either fluorography or silver staining. The L1 coat protein (54 Kd) was found in trace amounts in unmodified and slightly modified forms in the labeled empty and light full particles but could not be detected in the labeled heavy particles. L1 appeared to exist in the three unlabeled particle types in differentially modified forms. A putative L2 protein was also found to be modified (74-80 Kd) and was found preferentially in the unlabeled heavy full particles. The commercial cross-reactive BPV antibody recognized a labeled 58-Kd protein found predominantly in the empty and light full particles and a pair of proteins (41-42 Kd) found unlabeled in the heavy full particles. Besides L1, there were several other proteins (IEF 40 Kd; NEPHGE 42, 38, and 36 Kd) which were detected labeled in the empty particles and in increasing unlabeled amounts in the light full and heavy full particles. Four proteins (IEF 66, 13 and 11 Kd, and NEPHGE 9 Kd) were found exclusively in the full particles and may be involved in packing the viral genome. These observations suggest that a virus particle assembly pathway exists from the empty particles, via the light full, to the mature heavy full particles.

Bovine papillomavirus 1↗
PubMed1988

Future trends in cervical cancer.

The identification of the close association of certain types of human papillomavirus with the development of cervical cancer should lead to an extensive revision of appropriate health policies. Having taken into account the drawbacks inherent in the existing data (stemming from the use of varying nomenclature, diagnostic methods and reliability, registration and screening practices) it is possible to conclude that the incidence of HPV infections, all premalignant and malignant stages of cervical cancer are, or will soon be, increasing in several countries. This rate of increase is fastest for the younger age groups and is despite the introduction of various forms of screening. These trends therefore indicate an urgent need to adopt policies to avert an unnecessary increase in fatalities due to cervical cancer. It is therefore recommended to: (1) establish a routine diagnostic method which can identify either the type of HPV present or the lesions which are progressing; (2) determine the incidence of HPV infections in the general population; (3) disseminate to medical personnel, teachers, and other members of society existing knowledge concerning the dangers associated with this virus and relevant to preventing its further spread; (4) introduce an effective population screening campaign for all sexually active women, preferably involving a yearly examination at a colposcopy clinic; (5) intensify basic and applied HPV research, especially that which could lead to a deeper understanding of viral transmission and infection, identification of cofactors which promote cervical lesion progression, or to the production of a vaccine.

Female↗
PubMed1988

DNA viruses and human cancer.

This review examines some of the evidence which aetiologically implicates various DNA viruses (primarily papillomavirus, hepatitis B virus and Epstein-Barr virus) in certain human cancers (cervical carcinoma, primary liver cell carcinoma, Burkitt's lymphoma and nasopharyngeal carcinoma, respectively). The evidence includes: presence of viral DNA, RNA and proteins in tumours (and cell lines derived from them); occurrence of viruses with apparently different oncogenic potential; their ability to transform cell lines in vitro or cause tumours in animals; epidemiological and serological data. Factors which affect the progression to cancer are briefly considered as they illustrate that there are several stages in tumorigenesis. These factors include the immune system, irradiation, presence of other viruses or carcinogens and treatment. The lack of a single unique characteristic which defines a transformed cell would be expected from the multistep hypothesis and is related to the possible virus-cell interactions that can occur. These form a continuous spectrum ranging from productive infection of a permissive cell, through infection of a non-permissive cell, to the inability of a virus to infect a cell. This spectrum may reflect the absence of increasing numbers of cellular functions necessary for productive virus infection, with cell transformation occurring as a rare type of abortive infection. The evidence, especially for human papillomavirus, indicates that it is quite probable that particular DNA viruses are the causative agents for certain human cancers. Even so other factors can play decisive roles in tumorigenesis. Final aetiological proof will only be obtained when an anti-virus vaccine eradicates one form of human cancer.

Cell Transformation, Viral↗
PubMed1988

Human papilloma virus (HPV) and carcinomas of the head and neck.

Increasing evidence for a causal link between human papilloma virus and carcinomas of the cervix has emerged in recent research. This group of species-specific, epitheliotropic viruses has also been associated with tumours of the head and neck, but the individual reports deal only with relatively small sample numbers. In the present review these reports are considered in relation to the methods employed, and it is concluded that HPV is associated with more than 50% of oral and nasal carcinomas, as well as with carcinomas of the larynx and oesophagus. The clinical relevance and strategies for future work are outlined.

DNA Probes, HPV↗
PubMed1988

MS and the group-specific component.

Sera from 95 MS patients and 227 control persons were examined by IEF to determine the Gc genotype. There was no correlation between MS and the distribution of Gc genotype (0.70 less than P less than 0.80) nor Gc alleles (0.80 less than P less than 0.90) although such a correlation is generally accepted on the basis of a smaller material. Furthermore, Gc correlated with neither disease course nor with the age of disease onset.

Alleles↗
PubMed1987

Proteins present in bovine papillomavirus particles.

Analysis by two-dimensional gel electrophoresis and silver staining of heavy full, light full, and empty bovine papillomavirus particles has shown that the major capsid protein L1 is highly modified. Besides exhibiting at least 13 isoelectric point variants of approximately the same molecular mass (54 kilodaltons), it is suggested that an additional heavier protein chain (69 kilodaltons) is also derived from L1 by glycosylation. These modifications may stabilize the particle structure. Treatment with neuraminidase reduces the number of modification products detectable, with a concomitant increase in the more basic forms of L1. Although it was not possible to detect histones in any of the preparations, proteins of similar molecular mass were detected. Therefore, it is suggested that the basic tails of L1 bind to the DNA in a manner similar to that of histone. Calculation of the theoretical mobilities of the papillomavirus proteins shows good agreement with the actual position of L1 and its isoelectric point variants and suggests that two of the proteins with molecular masses similar to those of the histones may actually be coded by the bovine papillomavirus E7 and E5 open reading frames.

Animals↗
PubMed1986

The type of human papillomavirus present in cervical infections can be determined by the occurrence of specific marker proteins.

Four different proteins have been identified on high resolution two dimensional gels of [35S] methionine labelled human cervical biopsies whose expression correlates with the presence of papillomavirus. They are all basic proteins having molecular weights in the region of 48 to 50 kd and are normally expressed individually in different lesions unless the lesion results from a co-infection of two virus types. Comparison of the occurrence of these marker proteins with the actual HPV type present, determined by in situ filter hybridisation, has shown that two are found exclusively with HPV types 6/11 while the other two are found with types 16/18.

Antigens, Viral↗