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Biomedical subjects

M Moos

Publications and source records attributed to M Moos.

At least 55 records · Page 3Linked to original sources
PubMed1996

Successful collection and transplantation of peripheral blood stem cells in cancer patients using large-volume leukaphereses.

It was the aim of our study to determine the collection efficiency and yield of CD34+ cells in 88 cancer patients (pts, 44 males/44 females) who underwent 154 large-volume leukaphereses (LV-LPs). The diagnoses were as follows: 18 patients had Non-Hodgkin's lymphoma, 9 Hodgkin's disease, 24 multiple myeloma, 6 acute leukemia, 27 breast cancer, and 4 patients had solid tumors of different types. During the course of LV-LPs, 20 liters (1) of blood were processed at a median flow-rate of 85 ml/min (CS 3000 Baxter) and 130 ml/min (COBE Spectra), respectively. Peripheral blood stem cells (PBSC) were collected following granulocyte colony-stimulating factor (G-CSF)-supported cytotoxic chemotherapy. A 31% and 21% mean decrease in the platelet and white blood count was noted at the end of the LV-LPs when compared with the pre-leukapheresis values. The aphereses were well tolerated without adverse effects. The level of circulating CD34+ cells was closely related to the number of CD34+ cells contained in the respective leukapheresis product (R = 0.89, P < 0.001). Compared with 270 patients who underwent 838 regular 10 1 LPs, the yield of CD34+ cells/kg was almost two-fold greater (4.84 +/- 0.63 x 10(6) [Mean +/- SEM] vs. 2.60 +/- 0.16 x 10(6), P < 0.001). The antigenic profile of CD34+ cells was assessed in 54 separate products collected on the occasion of 27 LV-LPs following the processing of 10 1 and 20 1, respectively. The intra-individual comparison included differentiation- as well as lineage-associated markers (CD38, Thy-1, c-kit, CD33, CD45RA). No difference in the subset composition was observed between the first and second product, arguing against a preferential release of particular CD34+ cell subsets during the procedure. As shown by molecular biological or immunocytochemical examination, the likelihood of harvesting malignant cells using large-volume aphereses was not increased in comparison with regular leukaphereses. Single harvests of > or = 2.5 x 10(6) CD34+ cells/kg could be obtained in 74% of the patients, compared with 52% in case of regular LPs. As the majority of patients were autografted with more than 2.5 x 10(6) CD34+ cells/kg following high-dose therapy, hematological recovery in general was rapid and not related to the type of apheresis product used. Considering patient comfort and savings in resource utilization, large-volume leukaphereses have become the standard procedure for PBSC collection in our center.

Adolescent↗
PubMed1996

A role for Rho in receptor- and G protein-stimulated phospholipase C. Reduction in phosphatidylinositol 4,5-bisphosphate by Clostridium difficile toxin B.

Receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins) activate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-hydrolyzing phospholipase C (PLC) enzymes by activated alpha of free beta gamma subunits of the relevant G proteins. To study whether low molecular weight G proteins of the Rho family are involved in receptor signaling to PLC, we examined the effect of Clostridium difficile toxin B, which glucosylates and thereby inactivates Rho proteins, on the regulation of PLC activity in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor (mAChR) subtype. Toxin B treatment of HEK cells did not affect basal PLC activity, but potently and efficiently inhibited mAChR-stimulated inositol phosphate formation. PLC activation by the endogenously expressed thrombin receptor and by the direct G protein activators, A1F-4 and guanosine 5'-[gamma-thio]triphosphate (GTP gamma S), studied in intact and permeabilized cells, respectively, were also inhibited by toxin B treatment. C3 exoenzyme, which ADP-ribosylates Rho proteins, mimicked the inhibitory effect of toxin B on GTP gamma S-stimulated PLC activity. Finally both toxin B and C3 exoenzyme significantly reduced, by 40 to 50%, the total level of PtdIns(4,5)P2 in HEK cells, without affecting the levels of phosphatidylinositol and phosphatidylinositol 4-phosphate. Accordingly, When PLC activity was measured with exogenous PtdIns(4,5)P2 as enzyme substrate, Ca(2+)- as well as GTP gamma S- or A1F-4-stimulated PLC activities were not altered by prior toxin B treatment. In conclusion, evidence is provided that toxin B and C3 exoenzyme, apparently by inactivating Rho proteins, inhibit G protein-coupled receptor signalling to PLC, most likely by reducing the cellular substrate supply.

ADP Ribose Transferases↗
PubMed1996

High incidence of a second BCR-ABL fusion in chronic myeloid leukemia revealed by interphase cytogenetic analysis on blood and bone marrow smears.

The t(9;22)(q34;q11) is the single most common chromosomal abnormality in leukemias. Recently, dual-color fluorescence in situ hybridization (FISH) protocols for the detection of the BCR-ABL fusion, which is the molecular counterpart of this translocation, have been described. In the present study, we analyzed blood or bone marrow smears of 46 patients (34 with chronic myeloid leukemia [CML] and 12 with acute lymphoblastic leukemia [ALL]) for the presence of a BCR-ABL fusion. On these clinical routine samples, hybridization was performed with high efficiency and the BCR-ABL fusion was detected reliably. This series includes one case with a Philadelphia chromosome (Ph) on banding analysis and negative reverse transcriptase polymerase chain reaction (RT-PCR) results. Surprisingly, in 13 of the 34 CML patients (4 of 17 patients with chronic phase and 9 of 17 patients with blast crisis), and in 1 of the 12 ALL patients, an additional BCR-ABL fusion was diagnosed in 4% to 72.5% of interphase cells. In 10 of these 14 patients, banding data are available; only in two cases was the additional Ph detected by metaphase analysis. The data from this interphase cytogenetic analysis indicate that an additional Ph occurs more frequently than would be assumed based on banding analysis.

Adult↗
PubMed1996

Purification of bovine brain adenylyl cyclase with a novel derivative of forskolin: evidence for a high specific activity form of the enzyme.

An improved affinity support for the purification of adenylyl cyclase was prepared from 7-desacetyl-7-aminoethylaminocarbonyl forskolin. This analog allows convenient synthesis of an affinity matrix that is chemically stable, with-standing repeated use for up to two years, and efficient, yielding purifications of adenylyl cyclase from solubilized bovine brain membranes of 2,000-6,000 fold in a single step. Immunoblotting data suggest that the majority of the enzyme purified in this fashion differs from forms described previously. Since the specific activity of this preparation is substantially higher than that described in previous reports, it is possible that the purification described here selects, presumably on the basis of affinity for forskolin, for a form of adenylyl cyclase with higher specific activity than any described previously.

Adenylyl Cyclases↗
PubMed1996

Structural features of a close homologue of L1 (CHL1) in the mouse: a new member of the L1 family of neural recognition molecules.

We have identified a close homologue of L1 (CHL1) in the mouse. CHL1 comprises an N-terminal signal sequence, six immunoglobulin (Ig)-like domains, 4.5 fibronectin type III (FN)-like repeats, a transmembrane domain and a C-terminal, most likely intracellular domain of approximately 100 amino acids. CHL1 is most similar in its extracellular domain to chicken Ng-CAM (approximately 40% amino acid identity), followed by mouse L1, chicken neurofascin, chicken Nr-CAM, Drosophila neuroglian and zebrafish L1.1 (37-28% amino acid identity), and mouse F3, rat TAG-1 and rat BIG-1 (approximately 27% amino acid identity). The similarity with other members of the Ig superfamily [e.g. neural cell adhesion molecule (N-CAM), DCC, HLAR, rse] is 16-11%. The intracellular domain is most similar to mouse and chicken Nr-CAM, mouse and rat neurofascin (approximately 60% amino acid identity) followed by chicken neurofascin and Ng-CAM, Drosophila neuroglian and zebrafish L1.1 and L1.2 (approximately 40% amino acid identity). Besides the high overall homology and conserved modular structure among previously recognized members of the L1 family (mouse/human L1/rat NILE; chicken Ng-CAM; chicken/mouse Nr-CAM; Drosophila neuroglian; zebrafish L1.1 and L1.2; chicken/mouse neurofascin/rat ankyrin-binding glycoprotein), criteria characteristic of L1 were identified with regard to the number of amino acids between positions of conserved amino acid residues defining distances within and between two adjacent Ig-like domains and FN-like repeats. These show a collinearity in the six Ig-like domains and four adjacent FN-like repeats that is remarkably conserved between L1 and molecules containing these modules (designated the L1 family cassette), including the GPI-linked forms of the F3 subgroup (mouse F3/chicken F11/human CNTN1; rat BIG-1/mouse PANG; rat TAG-1/mouse TAX-1/chicken axonin-1). The colorectal cancer molecule (DCC), previously introduced as an N-CAM-like molecule, conforms to the L1 family cassette. Other structural features of CHL 1 shared between members of the L1 family are a high degree of N-glycosidically linked carbohydrates (approximately 20% of its molecular mass), which include the HNK-1 carbohydrate structure, and a pattern of protein fragments comprising a major 185 kDa band and smaller fragments of 165 and 125 kDa. As for the other L1 family members, predominant expression of CHL1 is observed in the nervous system and at later developmental stages. In the central nervous system CHL1 is expressed by neurons, but, in contrast to L1, also by glial cells. Our findings suggest a common ancestral L1-like molecule which evolved via gene duplication to generate a diversity of structurally and functionally distinct yet similar molecules.

Animals↗
PubMed1996

High-dose therapy with peripheral blood progenitor cell transplantation in low-grade non-Hodgkin's lymphoma.

It was the objective of our study to evaluate the efficacy of a sequential high-dose therapy with peripheral blood progenitor cell (PBPC) support in patients with low-grade non-Hodgkin's lymphoma (NHL). Since July 1991, 48 patients (23 male/25 female) with a median age of 43 years (range 26-55) were included in the study. At the time of entry, 28 patients were in first and seven in second or higher remission. Twelve patients had relapse of disease and one patient had tumor progression. PBPC were collected during granulocyte colony-stimulating factor (G-CSF)-enhanced leukocyte recovery following treatment with high-dose cytarabine and mitoxantrone (HAM). A median of two leukaphereses (range 2-7) resulted in 6.9 x 10(6) CD34+ cells/kg (median, range 2.1 x 10(6)-38.8 x 10(6)). A comparison was made between the harvests obtained from patients in first remission and those from patients in second remission, in relapse or progressive disease. Patients mobilized in first remission tended to have a greater collection efficiency for CD34+ cells comprising a significantly greater proportion of more primitive CD34+/Thy-1+ progenitor cells. Conversely, leukapheresis (LP) products collected during first remission contained a significantly smaller proportion of CD34+/CD45RA+ cells and CD34+/c-kit+ cells, subsets which reflect a more differentiated progenitor cell stage. Following high-dose therapy and PBPC autografting, the median time to reach platelets > or = 20 x 10(9)/l and neutrophils > or = 0.5 x 10(9)/l and 12 and 13 days, respectively. Two patients died of treatment-related toxic organ failure. Thirty-nine patients are alive in remission after a median follow-up time of 15 months (range 1-31), while seven patients relapsed between 5 and 29 months post-transplantation. Except for one patient autografted in first remission, the patients with relapse had a history of previous relapse or progressive disease. Since the probability of disease-free survival appears to be related to the disease status at the time of autografting, PBPC-supported high-dose therapy including total body irradiation should be investigated further for patients with low-grade NHL while they are in first remission.

Adult↗
PubMed1995

High-dose therapy with peripheral blood progenitor cell support in patients with non-Hodgkin's lymphoma.

Between September 1991 and April 1995, high-dose therapy with peripheral blood progenitor cell (PBPC) support was administered to 105 patients with non-Hodgkin's lymphoma (NHL). Thirty-three patients had high-grade NHL, while 72 patients had different forms of low- or intermediate-grade NHL. Except for three patients who received G-CSF during steady-state hematopoiesis, PBPCs were collected following cytokine-supported cytotoxic chemotherapy. This included G-CSF or the sequential administration of interleukin 3 (IL-3) and GM-CSF. Assessing bone marrow (BM) samples before the start of chemotherapy and leukapheresis (LP) products collected during cytokine-enhanced marrow recovery, a 2.3-fold greater mean concentration of CD34- cells was found in peripheral blood (p < 0.005). The blood-derived progenitor cells were enriched with a particular subset of more primitive progenitors, as the mean proportion of CD34+/Thy-1+ cells in LP products was three-fold greater in comparison to premobilization BM samples, respectively (p < 0.001). In contrast, the mean proportion of CD34+/CD19+ and CD19+ cells in LP products was 8.8- and 80-fold smaller compared to BM samples, respectively (p < 0.001). Following high-dose conditioning therapy including TBI in 74 patients, reinfusion of PBPC resulted in rapid and sustained engraftment in the majority of patients, while in seven patients an unsubstituted platelet count of greater than 20 x 10(9)/l was reached between 31 and 51 days. Five patients died of treatment-related complications between 13 and 188 days following transplantation. The probability of long-term disease-free survival at 30 months in patients autografted while they were in first remission was 70% in high-grade and 83% in low-grade NHL, respectively. The data may provide the rationale for the use of PBPC-supported high-dose regimens as first-line treatment for patients at high risk of treatment failure.

Adult↗
PubMed1995

Sequential high-dose treatment with peripheral blood progenitor cell transplantation in patients with multiple myeloma.

In June 1992, we started a dose-escalated cytotoxic therapy with peripheral blood progenitor cell (PBPC) transplantation in patients with chemosensitive multiple myeloma (MM). At the time of best response to conventional treatment, 70 patients received high-dose cyclophosphamide (HD-CY) or, in case of pre-existing heart disease, dose-escalated ifosfamide/mitoxantrone followed by filgrastim (R-metHuG-CSF, 300 micrograms/day). PBPC collection was commenced when CD34+ cells were detectable using direct immunofluorescence analysis. Fifty-four out of 70 patients were successfully harvested (> or = 2.5 x 10(6) CD34+ cells/kg body weight [BW]) after the first cycle of HD chemotherapy. Conditioning therapy consisted of 140 mg/m2 melphalan plus TBI (14.4 Gy hyper-fractionated) or 200 mg/m2 melphalan in patients not eligible for TBI because of previous radiotherapy. To date, 56 patients have been transplanted. Autografts contained a median of 3.4 x 10(6) CD34+ cells/kg BW. Following reinfusion of PBPC, rapid engraftment was achieved in 54 out of 56 patients with a median of 14 days (range 9-23) to reach 0.5 x 10(9)/l neutrophils and 10 days (range 5-22) for an unsubstituted platelet count of > 20 x 10(9)/l. One patient died of transplantation-related complications. Sequential HD treatment improved the remission status (European Bone Marrow Transplantation criteria) in 19 out of 46 patients (9 patients too early). Of note, in 11 patients the immunofixation became negative and a polyclonal immunoglobulin reconstitution was achieved. Our protocol provides an effective treatment strategy for patients with advanced MM combined with low treatment-related toxicity.

Adult↗
PubMed1995

Detection of minimal residual disease by polymerase chain reaction in B cell malignancies.

It was the aim of this study to examine the prognostic value of the detection of minimal residual disease (MRD), with the help of the polymerase chain reaction (PCR), in patients with non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM) who underwent sequential high-dose therapy with peripheral blood progenitor cell (PBPC) support, and in patients with acute myeloid leukemia (AML) of the subclass M4Eo who underwent high-dose consolidation therapy. Basis for the application of a PCR assay in these disease entities are the following specific gene rearrangements: the t(14;18) translocation in a high percentage of NHL, the clonal rearrangement of the Ig heavy chain locus resulting in a unique complementary determining region 3 (CDR3) for MM region and the inversion 16 characteristic for the M4Eo subclass of AML. Before the G-CSF-supported cytotoxic chemotherapy was given, 65% of the 52 patients with low- and intermediate-grade NHL enrolled into the study had PCR+ bone marrow (BM) and/or peripheral blood (PB) samples. The majority of patients (29 of 52) were autografted with a PCR+ transplant. The proportion of harvests containing t(14;18)+ cells was two-fold less in patients mobilized in first remission than in those with a history of previous treatment failure. This was also reflected when examining the B cell contents of the harvests measured as CD19+ cells with a 3.3-fold smaller proportion of CD19+ cells in leukapheresis (LP) products of patients mobilized in first remission. Patients who received a PCR- transplant are in remission and remained PCR- in BM and PB samples post-transplantation. Conversion to PCR-negativity in BM and PB samples post-transplantation was observed in 11 of 19 patients who were also in remission. In contrast, 6 of 29 patients who were autografted with PCR+ products relapsed, while 4 of them presented with PCR- samples on several occasions post-transplantation. In patients with MM, the assessment of MRD in PBPC harvests was based on the CDR3 regions of the Ig heavy chain locus as a marker for clonality. The great majority of LP products (17 out of 19) contained tumor cells. To prove positive enrichment procedures for the elimination of tumor cells, CD34+ and CD19+ cell fractions obtained from LP samples in an experimental setting via preparative flow cytometry were analyzed for MRD resulting in PCR-negativity for all CD34+ fractions. The results of the four patients with AML M4Eo and inversion 16 are preliminary, with a tendency of persistence of PCR-positivity after finishing the high-dose consolidation therapy. In one case, recurrence of disease was accompanied by an increase of the signal strength in the PCR assay. Longer follow-up periods are necessary to determine the prognostic value of these PCR findings in the different disease entities.

Acute Disease↗
PubMed1995

Characterization of apple 18 and 31 kd allergens by microsequencing and evaluation of their content during storage and ripening.

Patients with tree pollinosis frequently report allergic reactions after ingestion of apples. The severity of apple allergy has been related to the variety of apples and their degree of maturity. To generate a serum pool that is representative of various IgE-binding patterns of apple-allergic sera, serum samples from 34 patients allergic to tree pollens were screened. Only 24 serum samples reacted to the apple extract. Pooled serum was used to identify allergens in apples. An efficient and consistent extraction method for apple fruits was used to compare the immunoreactivities of extracts of different varieties (McIntosh, Red Delicious, Granny Smith, and Golden Delicious) of freshly picked and store-purchased apples. We found that Golden Delicious apples had the greatest amount of the 18 kd allergen, which has been reported to be a potent IgE-binding apple allergen. Store-purchased apples contained higher concentrations of the 18 kd allergen than freshly picked apples. In our study only 37.5% of sera reacted to the 18 kd protein, whereas 75% of the sera reacted to a 31 kd allergen. Other immunoreactive bands in apple extracts included proteins of 50, 38, 16, 14, and 13 kd. The amino-terminal amino acid sequences of the two major allergens, 18 kd and 31 kd, were determined. These sequences shared approximately 50% identity with disease resistance proteins of various plants or Bet v 1 in birch tree pollens. The appearance of various allergens was also investigated in mature apples during storage. The amount of 18 kd allergen increased significantly when apples were stored at 4 degrees C. However, under controlled atmospheric conditions in which oxygen- and carbon dioxide-induced ripening were regulated, the amount of 18 kd allergen remained unaffected. Because ripening and maturation were not associated with increases in 18 kd allergen content, the observed changes might be induced by factors related to disease resistance.

Adolescent↗
PubMed1995

Anti-dorsalizing morphogenetic protein is a novel TGF-beta homolog expressed in the Spemann organizer.

We have identified a novel growth factor in Xenopus, which is most closely related to human Bone Morphogenetic Protein-3. Its expression peaks during gastrulation, most prominently in the Spemann organizer, and persists in the posterior neural floor plate and prechordal plate during neurulation. Injection of the corresponding mRNA into dorsal blastomeres results in dose-dependent suppression of dorsal and anterior structures, even in the presence of lithium chloride. Overexpression of the gene downregulates the dorsalizing factors noggin, goosecoid and follistatin, as well as the dorsal markers NCAM, muscle actin and MyoD; conversely, ventral markers are induced. We therefore designate this gene product Anti-Dorsalizing Morphogenetic Protein (ADMP). Though development of dorsoanterior structures is suppressed when exogenous ADMP is injected, the gene is induced by lithium chloride treatment or activin, both of which are known to produce the opposite effect. Thus, the expression of ADMP resembles that of several dorsalizing signals, but its product exerts dorsal-suppressing activity. This suggests that ADMP may moderate organizer-associated dorsalizing influences. These findings are also consistent with the recently advanced proposal of dorsally expressed inhibitory activin-like signals.

Amino Acid Sequence↗
PubMed1995

Models of risk assessments for biologicals or related products in the European Union.

In the context of veterinary biologicals, environmental risk assessment means the evaluation of the risk to human health and the environment (which includes plants and animals) connected with the release of such products. The following categories or types of veterinary biologicals can be distinguished: non-genetically modified organisms (non-GMOs) (inactivated/live) GMOs (inactivated/live) carrier products related products (e.g. non-specific "inducers'). Suitable models used in risk assessment for these products should aim to identify all possible adverse effects. A good working model should lead, at least, to a qualitative judgement on the environmental risk of the biological product (e.g. negligible, low, medium, severe, unacceptable). Quantifiable outcomes are rare; therefore, the producer of a biological product and the European control authorities should accept only models which are based on testable points and which are relevant to the type of product and its instructions for use. In view of animal welfare aspects, models working without animals should be preferred. In recent years, some of these methods have been integrated into safety tests described in European Union Directives and in monographs of the European Pharmacopoeia. By reviewing vaccine/registration problems (e.g. Aujeszky's disease live vaccine for pigs, and vaccinia-vectored rabies vaccine), several models used in risk assessment are demonstrated and discussed.

Animals↗
PubMed1995

DNA sequencing from single phage plaques using solid-phase magnetic capture.

Many operations encountered in molecular cloning are labor-intensive and time-consuming. One case that is often troublesome is the subcloning of cDNA clones from lambda gt11 phage into plasmid vectors. In situations where several clones have been isolated, time could be saved by a means of assessing insert size and sequence unambiguously without subcloning, particularly where degenerate PCR or low-stringency hybridization approaches are taken to identify multiple members of a gene family. We describe a simple and reliable strategy for efficient sequencing of small amounts of lambda phage DNA, lysates or individual phage plaques. The strategy combines the advantages of universal lambda phage primers, rapid air thermal cycling, streptavidin magnetic bead capture of highly purified single-stranded templates and the unparalleled clarity of T7 DNA polymerase sequence. We routinely obtain 350-500 bases of unambiguous sequence from each reaction. It takes only hours from lifting phage plaques to finishing the sequencing reactions. The method provides an alternative to thermal cycle sequencing that has comparable sensitivity and affords sequence data of much higher clarity.

Bacteriophage lambda↗
PubMed1994

Cartilage-derived morphogenetic proteins. New members of the transforming growth factor-beta superfamily predominantly expressed in long bones during human embryonic development.

Partially purified extracts from newborn calf articular cartilage were found to induce cartilage and bone when subcutaneously implanted in rats. This activity showed characteristics of bone morphogenetic proteins (BMPs). Degenerate oligonucleotide primer sets derived from the highly conserved carboxyl-terminal region of the BMP family were designed and used in reverse transcription-polymerase chain reactions with poly(A)+ RNA from articular cartilage as template to determine which BMPs are produced by chondrocytes. Two novel members of the transforming growth factor-beta (TGF-beta) superfamily were identified and designated cartilage-derived morphogenetic protein-1 (CDMP-1) and -2 (CDMP-2). Their carboxyl-terminal TGF-beta domains are 82% identical, thus defining a novel subfamily most closely related to BMP-5, BMP-6, and osteogenic protein-1. Northern analyses showed that both genes are predominantly expressed in cartilaginous tissues. In situ hybridization and immunostaining of sections from human embryos showed that CDMP-1 was predominantly found at the stage of precartilaginous mesenchymal condensation and throughout the cartilaginous cores of the developing long bones, whereas CDMP-2 expression was restricted to the hypertrophic chondrocytes of ossifying long bone centers. Neither gene was detectable in the axial skeleton during human embryonic development. The cartilage-specific localization pattern of these novel TGF-beta superfamily members, which contrasts with the more ubiquitous presence of other BMP family members, suggests a potential role for these proteins in chondrocyte differentiation and growth of long bones.

Amino Acid Sequence↗
PubMed1994

Single-step affinity purification, partial structure and properties of human platelet cGMP inhibited cAMP phosphodiesterase.

The human platelet cilostamide- and cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) was rapidly purified approximately 19,000-fold to apparent homogeneity using single step affinity chromatography on the isothiocyanate derivative of cilostamide coupled to aminoethyl agarose. Within 24 h, 30 micrograms of enzyme protein was obtained from 20 ml of packed platelets. Vmax for cAMP and cGMP was 6.1 and 0.9 mumol/min per mg protein, respectively. Several polypeptides (110/105, 79, 62, 55/53 kDa) were identified after SDS-PAGE, all of which were immunologically related to cGI-PDE and represented approx. 5, 20, 50 and 20% of the total protein, respectively. Limited proteolysis of the cGI-PDE with chymotrypsin produced a major fragment of approximately 47 kDa (and at least two smaller peptides) with catalytic activity and sensitivity to cGMP and OPC 3911 similar to controls. Phosphorylation of the cGI-PDE by cAMP-dependent protein kinase (A-kinase) resulted in maximal incorporation of 0.6-1.8 mol of 32P/mol 110/105 and 79 kDa polypeptides; much lower and variable amounts of phosphate were incorporated into the 62 and 55/53 kDa polypeptides. After digestion of cGI-PDE with several proteinases a number of peptides were isolated and sequenced. Most of the peptide sequences obtained could be aligned within the carboxy terminal domain of the deduced sequence of the human cardiac cGI-PDE. These and other results suggest that the subunit size of the intact platelet cGI-PDE is 110 kDa and that proteolytic fragments of 79, 62 and 55/53 kDa are produced during purification. The smaller fragments (62 and 55/53 kDa) contain the catalytic domain; the larger fragments (110 and 79 kDa) also contain the regulatory domain with phosphorylation sites for A-kinase.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1994

Detection of chimeric BCR-ABL genes on bone marrow samples and blood smears in chronic myeloid and acute lymphoblastic leukemia by in situ hybridization.

The presence of BCR-ABL fusion genes has important diagnostic and prognostic implications in chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). The CML-specific chimeric BCR-ABL gene with a break involving the major breakpoint cluster region (M-bcr) of the BCR-gene has been detected by means of fluorescence in situ hybridization (FISH). In this study, we present a FISH protocol that allows the detection of breaks in both the major and the minor breakpoint cluster region (m-bcr). Three hybridization signals of D107F9, a yeast artificial chromosome (YAC)-derived probe spanning the breakpoint regions of the BCR gene, were indicative of the translocation events. To increase the specificity further, this probe was combined with cos-abl 8, a cosmid probe flanking the breakpoint within the ABL gene for dual-color hybridization. Samples of 21 patients with CML, the ALL-derived cell line SUP-B15, and of seven patients with Philadelphia chromosome (Ph1)-positive ALL (three of them with breakpoints within m-bcr) were examined. BCR-ABL fusion was detected in all cases with high specificity (false-positive nuclei: mean, 0.1%). On cytogenetic preparations, the percentages of BCR-ABL-positive interphase cells ranged from 53% to 91%. Comparable efficiencies were achieved on blood smears. In conclusion, hybridization with D107F9 and cos-abl 8 allows unambiguous diagnosis of BCR-ABL genes and is likely to become an important tool for the monitoring of therapies in patients with CML and ALL.

Adult↗
PubMed1994

The role of cysteine 78 in fluorosulfonylbenzoyladenosine inactivation of rat liver S-adenosylhomocysteine hydrolase.

Inactivation of rat liver S-adenosylhomocysteine hydrolase by the site-directed reagent 5'-p-fluorosulfonylbenzoyladenosine (FSBA) is associated with the formation of a disulfide bond between Cys-78 and Cys-112 (Takata, Y., and Fujioka, M. (1984) Biochemistry 23, 4357-4362; Gomi, T., Ogawa, H., and Fujioka, M. (1986) J. Biol. Chem. 261, 13422-13425). To characterize the inactivation mechanism more precisely, the properties of four hydrolase proteins mutated at Cys-78 or Cys-112 were compared to those of the wild-type enzyme. When Cys-78 was mutated to either a serine or an alanine, proteins with greatly reduced enzymatic activity were obtained, large effects on kinetic constants were observed, and enzymatic activity was not affected by incubation with FSBA. When Cys-112 was mutated to either a serine or an alanine, the activity was similar to the wild-type protein, only small changes in the kinetic constants were observed, and the enzyme was inactivated more rapidly upon incubation with FSBA. FSBA inactivation of the C112A mutant protein was accompanied by the formation of a disulfide between Cys-78 and Cys-52. The data indicate that FSBA initially reacts with Cys-78 and that Cys-78 has an important role in the structure of the enzyme.

Adenosine↗