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P M Larsen

Publications and source records attributed to P M Larsen.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1992

Changes in the metaphase transit times and the pattern of sister chromatid separation in stamen hair cells of Tradescantia after treatment with protein phosphatase inhibitors.

Stamen hair cells from the spiderwort plant, Tradescantia virginiana, exhibit remarkably predictable metaphase transit times, making them uniquely suitable for temporal studies on mitotic regulation. In this study, we describe two kinds of experiments that test whether protein phosphatase activity is a necessary prerequisite for entry into anaphase in living, mitotic cells. We treated cells at specific points during prophase, prometaphase and metaphase with the broad-spectrum protein phosphatase inhibitor, alpha-naphthyl phosphate (administered by microinjection), or with the naturally occurring, potent phosphatase inhibitors okadaic acid, microcystin-LR or microcystin-RR (administered by perfusion), and we have observed changes in the metaphase transit time that are primarily dependent on the time of initial exposure to the inhibitor. Maximal extensions of the metaphase transit time result from alpha-naphthyl phosphate microinjections initiated in mid-metaphase, 10-20 min after nuclear envelope breakdown. Perfusions with okadaic acid started during a specific interval in mid-metaphase, 15-20 min after nuclear envelope breakdown, resulted in a statistically significant extension of the metaphase transit time. Perfusions with either microcystin-LR or microcystin-RR initiated 15-26 min after nuclear envelope breakdown extended the metaphase transit times significantly. Treatments of cells with okadaic acid or with either of the microcystins initiated outside this mid-metaphase interval either were without effect or, alternatively, resulted in a significant shortening of the metaphase transit time. In addition to their effects on the timing of anaphase onset, treatments with these protein phosphatase inhibitors also resulted in a remarkable change in the way in which these cells enter anaphase. Sister chromatid separation in stamen hair cells typically requires only 5 seconds, but after treatment with any of these inhibitors some, but not all, of the chromatids split apart at anaphase onset. Those that split begin to migrate toward the spindle pole regions, while those that fail to split remain at the metaphase plate. Later, more of the paired chromatids split apart and begin moving toward the spindle pole regions. Those that fail to separate remain at the metaphase plate. This process can be repeated several times before all of the chromatids have separated. Thus, entry into anaphase becomes extremely asynchronous, and as much as 30 min can transpire between the centromeric separation of the first and last chromosomes. Some of the chromosomes complete their anaphase movements before others have even split apart at the metaphase plate. Asynchronous separation did not result in a permanent segregation anomaly.(ABSTRACT TRUNCATED AT 400 WORDS)

Anaphase↗
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

1,2-Dioctanoylglycerol accelerates or retards mitotic progression in Tradescantia stamen hair cells as a function of the time of its addition.

We have treated living, intact stamen hair cells from the spiderwort plant, Tradescantia virginiana, with 0.5 microgram/ml or 60 micrograms/ml 1,2-dioctanoylglycerol, a potent and permeant activator of protein kinase C, and have observed the rates of progression of mitosis from prophase through anaphase. We have found that in addition to the concentration used, the time of initial treatment with 1,2-dioctanoylglycerol defines the response by the cells. The cells rapidly undergo nuclear envelope breakdown when this diglyceride is added in very late prophase, 0 to approximately 8 min prior to the time of normal nuclear envelope breakdown. Anaphase onset occurs 28 min after nuclear envelope breakdown, rather than after the 33 min interval observed in untreated cells. Rapid progression through metaphase is also observed if cells are treated with 0.5 microgram/ml 1,2-dioctanoylglycerol during prometaphase, up to 15 min after nuclear envelope breakdown. The addition of 0.5 microgram/ml 1,2-dioctanoylglycerol in late metaphase, approximately 26 min after nuclear envelope breakdown, results in sister chromatid separation slightly ahead of its normal time, 33 min after nuclear envelope breakdown, and in precocious cell plate vesicle aggregation, 3-5 min earlier than that observed in untreated cells. Treatment of cells with 60 micrograms/ml of 1,2-dioctanoylglycerol at any point during the interval from 0 to approximately 5 min prior to nuclear envelope breakdown results in precocious entry into anaphase. If cells are treated with either 0.5 microgram/ml or 60 micrograms/ml 1,2-dioctanoylglycerol earlier than 20 min before nuclear envelope breakdown, they do not enter mitosis, but instead revert to interphase without dividing. When 1,2-dioctanoylglycerol is added at other times during mitosis, the rate of subsequent mitotic progression is dramatically slowed; the cells require greater than 55 min to progress from nuclear envelope breakdown to anaphase onset, though once in anaphase, the cells progress onward to cytokinesis at normal rates. Treatments o of cells with 1,3-dioctanoylglycerol at any point during prophase, prometaphase, or metaphase are without effect on the rate of subsequent mitotic progression. The shifts in response by cells treated at specific times with 1,2-dioctanoylglycerol during mid- and late metaphase may be indicative of the existence of one or more regulatory switch points (i.e., checkpoints) just prior to anaphase onset.

Anaphase↗
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

No effect of intralesional injection of interferon on moderate cervical intraepithelial neoplasia.

Women with histologically confirmed cervical intraepithelial neoplasia grade 2 (CIN 2) were treated in a double blind investigation of treatment with intralesioneal interferon alpha-2b (Intron a). Before treatment commenced, the existence and the types of human papilloma-virus (HPV) were assessed in [35S] methionine-labelled cervical biopsies by the determination of specific protein markers. Pronounced side effects occurred in all the women treated with interferon and the trial was stopped when it became apparent that there were no obvious beneficial effects. No positive benefits of interferon treatment were detected on either the CIN 2 or on the persistence of HPV types. It is concluded that intralesioneal injection of interferon has no place in the treatment of CIN.

Adult↗
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

[Is hepatitis B antigenemia a risk in adjuvant cytostatic treatment of breast cancer?].

One-hundred and fifteen consecutive patients with breast cancer were examined for hepatitis B. All the patients received adjuvant chemotherapy. The median age was 46 years (range 26-54 years). None of the patients were found to be HBsAg-positive. The prevalence of HbsAg was within the limits of 0-0.026 (95% confidence interval). Vaccination of patients receiving chemotherapy is not indicated. Furthermore, regular HBsAg screening among patients under 55 years cannot be recommended.

Adult↗
PubMed1989

Creatine kinase-BB in the cerebrospinal fluid as a marker of CNS metastases and leptomeningeal carcinomatosis in patients with breast cancer.

To determine whether creatine kinase-BB isoenzyme would be useful in detecting central nervous system metastases secondary to breast cancer, we measured the cerebrospinal fluid (CSF) activity of creatine kinase (CK) and its BB isoenzyme (CK-BB) in 65 consecutive patients suspected of having CNS involvement. All patients underwent neurological evaluation, computer tomography (CT) scan and/or radionuclide scintigraphy and lumbar puncture with CSF examination. Thirty patients had CNS metastases, of whom 18 had parenchymal brain metastases (MET). Twelve had leptomeningeal carcinomatosis (MC), of whom four also had parenchymal brain metastases. Thirty-four patients were concluded not to have CNS involvement, whereas one was considered equivocal. CK-BB activity was significantly higher in patients with CNS metastases than in those without (P less than 0.05). This difference was primarily related to the fact that patients with MC had a significantly higher CK-BB activity than patients without CNS metastases or patients with parenchymal brain metastases only (P less than 0.01 and P less than 0.05, respectively). Taking 0.20 U/l as a tentative cut-off value (the upper limit range of patients without CNS metastases being 0.19 U/l), 10 out of 12 patients with MC had activities above this level. The sensitivity and specificity for having MC were 83% and 87%, and the positive and negative predictive values 60% and 96%, respectively. The sensitivity and negative predictive value for having any CNS metastases were 57% and 72%. Specificity and positive predictive value: 100%. The CSF activity of CK-BB appears to be a contribution in the diagnosis of MC secondary to breast cancer and seems superior to protein and LDH.

Biomarkers, Tumor↗
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↗
PubMed1989

Revelation of specificity of 64K autoantibodies in IDDM serums by high-resolution 2-D gel electrophoresis. Unambiguous identification of 64K target antigen.

Antibodies in serums from newly diagnosed insulin-dependent (type I) diabetes mellitus (IDDM) patients and individuals experiencing early phases of beta-cell destruction specifically immunoprecipitate a minor pancreatic islet cell membrane protein of 64,000 Mr (64K). In this report, we demonstrate the use of two-dimensional (2-D) gel electrophoresis to unambiguously identify the 64K antigen. By nonequilibrium pH-gradient gel electrophoresis in the first dimension and sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second dimension, the 64K protein separates into two components, designated alpha and beta, that differ in size but display identical charge heterogeneity. The high resolution of the 2-D method efficiently separates the 64K components from background proteins in immunoprecipitates from crude detergent lysates of islets. The background proteins were identified as major cellular proteins carried nonspecifically through the immunoprecipitation procedure. The high affinity and specificity of the 64K autoantibodies were demonstrated by the exclusive and greater than 1000-fold purification of this minor protein by immunoprecipitation with IDDM serums. The 2-D analyses did not reveal additional proteins specifically immunoprecipitated by IDDM serums, suggesting that the 64K protein is the only protein antigen specifically and consistently recognized by IDDM autoantibodies in the relatively stringent conditions of immunoprecipitation. Moreover, the 2-D analyses demonstrate that purification of membrane protein fractions from both human and rat islets before the immunoprecipitation efficiently removes background proteins and substantially increases the specificity of 64K autoantibody measurements by traditional methods.

Animals↗
PubMed1989

Quin2-induced metaphase arrest in stamen hair cells can be reversed by 1,2-dioctanoylglycerol but not by 1,3-dioctanoylglycerol.

Elicitor molecules of the polyphosphoinositide cycle, inositol 1,4,5-trisphosphate (InsP3) and 1,2-diacylglycerol (1,2-DAG) play roles in the entry of calcium into the cytosol and in the elevation of protein kinase C activity, respectively. We have treated stamen hair cells of the spiderwort plant, Tradescantia virginiana, with a solution of quin2 (50 microM) or its acetoxymethyl ester, quin2-AM (50 microM) and have retarded the normally predictable rate of progression through metaphase. Metaphase arrest persists for longer than 80 min after treatment with this Ca2+-chelator, and, shortly thereafter, the cells revert to interphase without dividing. Reversal of metaphase arrest results from treatments with calcium chloride (100 microM) after 5 to 8 min or with 1,2-DAG (i.e., 60 micrograms/ml 1,2-dioctanoylglycerol) after 7 to 11 min. In control experiments, metaphase arrest could not be reversed by treatment with either magnesium sulfate or 1,3-dioctanoylglycerol. Anaphase onset was observed in these control cells after post-treatment with calcium chloride (after 4-9 min) or with 1,2-dioctanoylglycerol (after 7-13 min). The treatment of stamen hair cells in very early prophase with H-7, (i.e., 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride), a potent protein kinase C inhibitor, extends the duration of metaphase significantly. Neither H-8 nor HA-1004, less active protein kinase C inhibitors in this class of molecules, alter the duration of metaphase to a significant extent. These results suggest that in cells arrested in metaphyase by quin2, calcium translocation plays a role in the sequence of events which culminate in anaphase.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminoquinolines↗
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

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↗