Arabidopsis cytosolic alpha-glycan phosphorylase, PHS2, is important during carbohydrate imbalanced conditions.

Arabidopsis thaliana has two isoforms of alpha-glycan phosphorylase (EC 2.4.1.1), one residing in the plastid and the other in the cytosol. The cytosolic phosphorylase, PHS2, acts on soluble heteroglycans that constitute a part of the carbohydrate pool in a plant. This study aimed to define a physiological role for PHS2. Under standard growth conditions phs2 knock-out mutants do not show any clear growth phenotype, and we hypothesised that during low-light conditions where carbohydrate imbalance is perturbed, this enzyme is important. Soil-grown phs2 mutant plants developed leaf lesions when placed in very low light. Analysis of soluble heteroglycan (SHG) levels showed that the amount of glucose residues in SHG was higher in the phs2 mutant compared to wild-type plants. Furthermore, a standard senescence assay from soil-grown phs2 mutant plants showed that leaves senesced significantly faster in darkness than the wild-type leaves. We also found decreased hypocotyl extension in in vitro-grown phs2 mutant seedlings when grown for long time in darkness at 6 °C. We conclude that PHS2 activity is important in the adult stage during low-light conditions and senescence, as well as during prolonged seedling development when carbohydrate levels are unbalanced.

Evolution of UV-B regulation and protection in plants.

Plants have evolved under the influence of UV-B radiation and have acquired systems for monitoring it and investing appropriate resources for protection against it, i.e., filters, quenchers of radicals and reactive oxygen species, and repair systems. An hypothesis for how plants monitor radiation has been presented.

DNA content of granulocytes, monocytes, and lymphocytes in the bone marrow smears of patients with myelodysplastic syndromes.

Recently, we have reported a high incidence of DNA hypodiploidy defined as DNA index (DI) in blasts/promyelocytes from 39 patients with myelodysplastic syndromes (MDS) found to be without a relationship to cytogenetics. In the present study the DNA content (DI) in granulocytes, monocytes, and lymphocytes measured in the same bone marrow smears from the above patients are reported. DNA hypodiploidy was found in mature cells, not only in myeloid cells (granulocytes and monocytes) but also in lymphocytes. A lower mean DI in each cell type of patients compared to controls was found. Pairwise comparison of the mean DI (+/-SE) in 32 patients with normal (n = 22) and abnormal (n = 10) cytogenetics and controls (n = 8) showed a significantly (P < 0.01) lower value for each group of patients, respectively, in all cell types. No difference was found between the two groups of patients. Presence of weak-Feulgen stained nuclei (DI < 0.40) in granulocytes and monocytes was more pronounced in patients expressing DNA hypodiploid immature cell populations, but only occasionally in lymphocytes, suggesting a link to an apoptotic event and intramedullary cell death. DNA hypodiploidy is shown to be a common feature even in mature cell populations in MDS bone marrows. Clonality, by means of DNA content, appears reasonable as regards the granulocytes and monocytes. DNA hypodiploid lymphocytes, on the other hand, might be small blasts (stem cells) or dying cell populations of unknown origin.

Interaction between phosphofructokinase and aldolase from Saccharomyces cerevisiae studied by aqueous two-phase partitioning.

Phosphofructokinase (EC 2.7.1.11) and aldolase (EC 4.1.2.13) have been highly purified from Saccharomyces cerevisiae by improved protocols. Partitioning of the enzymes in aqueous polymer two-phase systems was used to detect complex formation. The partition of each enzyme was found to be affected by the presence of the other enzyme. AMP affected the partition of the individual enzymes as well as the mixture of the two. The activities of the respective enzymes were stimulated in the putative complex in an AMP-dependent manner. Two strictly conserved residues belonging to an acidic surface loop of class II aldolases, are a potential site for electrostatic interaction with the positively charged regions close to the active site in phosphofructokinase.

Aberrant expression of active leukotriene C(4) synthase in CD16(+) neutrophils from patients with chronic myeloid leukemia.

Elevated leukotriene (LT)C(4) synthase activity was observed in peripheral blood granulocyte suspensions from patients with chronic myeloid leukemia (CML). Magnetic cell sorting (MACS) with CD16 monoclonal antibodies (mAbs), which were used to fractionate granulocytes from CML patients and healthy individuals, yielded highly purified suspensions of CD16(+) neutrophils. The purity of these cell fractions was verified by extensive morphologic examination. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses, demonstrating the absence of interleukin-4 messenger RNA (IL-4 mRNA), further confirmed the negligible contamination of eosinophils in these fractions. Notably, purified CML CD16(+) neutrophils from all tested patients transformed exogenous LTA(4) to LTC(4). These cells also produced LTC(4 )after activation with ionophore A23187 or the chemotactic peptide fMet-LeuPhe (N-formylmethionyl-leucyl-phenylalanine). Subcellular fractionation revealed that the enzyme activity was exclusively distributed to the microsomal fraction. Expression of LTC(4) synthase mRNA in CML CD16(+) neutrophils was confirmed by RT-PCR. Furthermore, Western blot analyses consistently demonstrated expression of LTC(4) synthase at the protein level in CML CD16(+) neutrophils, whereas expression of microsomal glutathione S-transferase 2 occurred occasionally. Expectedly, LTC(4) synthase activity or expression of the protein could not be demonstrated in CD16(+) neutrophil suspensions from any of the healthy individuals. Instead, these cells, as well as CML CD16(+) neutrophils, transformed LTA(4) to LTB(4). The results indicate that aberrant expression of LTC(4) synthase is a regular feature of morphologically mature CML CD16(+) neutrophils. This abnormality, possibly associated with malignant transformation, can lead to increased LTC(4) synthesis in vivo. Such overproduction may be of pathophysiological relevance because LTC(4 )has been demonstrated to stimulate proliferation of human bone marrow-derived myeloid progenitor cells. (Blood. 2000;95:1456-1464)

Inverse correlation between Ink4-locus deletions and ICM-DNA hyperdiploidy in childhood acute lymphoblastic leukaemia, relation to clinical characteristics and outcome.

Malignant cells from 72 children with ALL were analysed to investigate the relationship between DNA-ploidy and deletion of the Ink4 locus containing the cell-cycle regulating genes p16ink4a, p15ink4b and p14ARF. Image-DNA cytometry (ICM) was used to assess DNA index (DI), and Southern hybridisation was carried out to detect deletions of the Ink4-locus in the leukaemic cells. A DNA content equal to or exceeding 1.16, indicating hyperdiploidy, was detected in 21/72 patients (29%), 1/72 (1.3%) showed DNA-hypodiploidy, and the remaining 50 patients (69%) had a DI within normal limits. Bi-allelic deletion of at least two of the coding sequences from the Ink4 locus was observed in 23/70 (33%) patients. Mono-allelic deletions within the locus were observed in 10/70 patients (14%), and 37/70 patients (53%) had normal signals for both sequences. Out of the 70 patients that could be analysed by both techniques only two had the combination of DNA hyperdiploidy and Ink4-locus bi-allelic deletion (p = 0.004). DNA hyperdiploidy was not associated with any specific clinical characteristics, but there was a trend for a better prognosis for patients with DNA hyperdiploidy (p = 0.09). Ink4-locus deletion was associated with T-cell phenotype and higher white blood cell counts at diagnosis and poor prognosis (p = 0.0015). Multivariate analysis confirmed that Ink4-locus deletion is an independent prognostic marker and a stronger determinant of outcome than DNA ploidy. When DNA ploidy and Ink4-locus deletions were combined, novel subgroups with significantly different outcome could be observed. A group with DNA index > or = 1.16 and no Ink4-locus bi-allelic deletions had an excellent prognosis (p-EFS 0.93 at 60 months), patients with Ink4-locus bi-allelic deletion and a DNA index < 1.16 fared worst (p-EFS 0.57) and patients with no Ink4 deletions and without hyperdiploidy had an intermediate outcome (p-EFS 0.79). The reason for the inverse correlation between DNA ploidy and Ink4 deletion and their combined impact on prognosis remains unclear, and possible reasons are discussed.

DNA image cytometry in MDS bone marrow smears.

The nuclear DNA content, defined as DNA index (DI) in blasts/promyelocytes (bla/pro), were determined on Feulgen-stained bone marrow smears from 39 patients with myelodysplastic syndromes (MDS) and eight control subjects by the use of image cytometry (ICM). The DI in patients was compared to that of corresponding normal cell types, and to cytogenetic data available in 32/39 patients. The mean DI in bla/pro of patients with MDS was significantly (P < 0.01) lower compared to corresponding cell types in control subjects. By ICM, a DNA aneuploidy in bla/pro was found in 67% of the MDS patients, and 59% expressed DNA hypodiploidy. By cytogenetics, an abnormal karyotype was found in 31%, and 6/9 MDS patients with a 'hypodiploid' abnormal karyotype showed DNA hypodiploidy. Of patients with a normal karyotype (69%), seven (32%) showed a normal, 12 (55%) a lower, and three (14%) a higher DI compared to controls. No difference between groups of MDS patients was found. DNA hypodiploidy is suggested to be a common feature in MDS without a relationship to cytogenetics.

Neutrophil dysplasia is not a specific feature of the abnormal chromosomal clone in myelodysplastic syndromes.

In order to study if dysplastic cells are part of the abnormal chromosomal clone in myelodysplastic syndromes (MDS) we used fluorescence in situ hybridization in combination with standard May-Grünwald-Giemsa morphology (MGG/FISH) to investigate the penetration of chromosomal abnormalities into dysplastic neutrophil granulocytes in five MDS patients with documented monosomy 7 or trisomy 8 in the bone marrow at diagnosis. Neutrophil dysplasia was defined as neutrophil granulocytes with extreme hypogranulation or with nuclear abnormalities of the pelgeroid type. In one patient all dysplastic cells were derived from the abnormal chromosomal clone, while in the other four cases only 6-43% of the hypogranulated neutrophils and 33-40% of the pelgeroid granulocytes exhibited the chromosomal marker. The results suggest that neutrophil dysplasia is not a specific feature of the abnormal chromosomal clone in MDS. It is not clear, however, if the disomic dysplastic cells were derived from a parallel MDS clone with a normal karyotype, or represent residual non-clonal, normal hematopoietic cells.

Actin gene sequence from Euglena gracilis.

The full length coding sequence of the Euglena gracilis actin gene was determined by RT-PCR of Euglena gracilis mRNA. Conserved regions in the actin amino acid sequence were used as guides for the synthesis of degenerate primers. Sequence was obtained for 1,238 nucleotides, of which 1,131 were coding for 377 amino acids. Sequence comparisons showed a similarity with other actins of 56% to 80%. Even though most of the actin amino acid sequence was conserved, some regions showed high divergence, i.e. the DNase I-binding loop at the N-terminal region. The construction of a phylogenetic tree based on actin sequences from different organisms placed Euglena gracilis in a cluster with Trypanosoma brucei and Leishmania major.

Addition of etoposide to CHOP chemotherapy in untreated patients with high-grade non-Hodgkin's lymphoma.

BACKGROUND: Second- and third-generation chemotherapy protocols for the treatment of aggressive non-Hodgkin's lymphomas (NHL) have considerable, and age-related, toxic effects. In addition, they do not seem to prolong overall survival in comparison to standard CHOP chemotherapy. In this phase II study we investigated the feasibility and efficacy of the addition of etoposide to the conventional CHOP regimen. PATIENTS AND METHODS: Toxicity and clinical efficacy were determined in 132 patients with previously untreated high-grade NHL. There were 51 patients in clinical stage I and II and 81 patients in stage III and IV, with a median age of 54 years (range 17-85). Patients received standard-dose CHOP plus etoposide 100 mg/m2 i.v. on day 1 and 200 mg/m2 p.o. on days 2-3. RESULTS: The overall response rate was 84%, with 70% complete and 14% partial responses. The predicted three- and five-year survivals for the group as a whole were 60% and 53%, respectively, and the corresponding disease-free survivals for patients achieving complete remissions were 65% and 56%, respectively. Outcome was not different from that of CHOP-treated patients in a recently completed Nordic study performed during the same time period. Myelosuppression (WHO grade 3-4), observed in 87% of patients and infectious complications (WHO grade 3-4) in 33%, dominated the toxicity profile of this regimen. Fifty-seven of 92 complete responders (62%) received 6-8 CHOP-E cycles with no reductions in planned dose intensity. LDH level higher than normal, extranodal sites = 2, stage III-IV at diagnosis were all indicators of a poor survival. CONCLUSIONS: We conclude that CHOP-E treatment is effective in high-grade NHL. However, mainly due to severe myelosuppression frequent schedule modifications were required and the results are not obviously superior to those of conventional CHOP.

The characterization of plasma membrane-bound tubulin of cauliflower using Triton X-114 fractionation.

The cortical microtubules determine how cellulose microfibrils are deposited in the plant cell wall and are thus important for the control of cell expansion. To understand how microtubules can control microfibril deposition, the components that link the microtubules to the plasma membrane (PM) of plant cells must be isolated. To obtain information on the properties of the tubulin-membrane associations, cauliflower (Brassica oleracea) PM was subjected to Triton X-114 fractionation, and the distribution of alpha- and beta-tubulin was analyzed using immunoblotting. Approximately one-half of the PM-associated tubulin was solubilized by Triton X-114 and 10 to 15% of both alpha- and beta-tubulin was recovered in the detergent phase (indicative of hydrophobic properties) and 30 to 40% was recovered in the aqueous phase. The hydrophobic tubulin could be released from the membrane by high pH extraction with preserved hydrophobicity. A large part of the PM-associated tubulin was found in the Triton-insoluble fraction. When this insoluble material was extracted a second time, a substantial amount of hydrophobic tubulin was released if the salt concentration was increased, suggesting that the hydrophobic tubulin was linked to a high-salt-sensitive protein aggregate that probably includes other components of the cytoskeleton. The hydrophobicity of a fraction of PM-associated tubulin could reflect a direct or indirect interaction of this tubulin with the lipid bilayer or with an integral membrane protein and may represent the anchoring of the cortical microtubules to the PM, a key element in the regulation of cell expansion.

Peripheral blood neutrophil morphology reflects bone marrow dysplasia in myelodysplastic syndromes.

Dysplastic features of cells in peripheral blood and bone marrow were studied in 51 patients with myelodysplastic syndromes (MDS) to evaluate the significance of the degree of neutrophil granulation (G-score) and the percentage of pelgeroid polymorphs (ppp) in the peripheral blood, as indices of dysplastic changes in the bone marrow. There was a good correlation between peripheral blood and bone marrow findings, both for G-score figures (r = 0.92, P < 0.01) and ppp (r = 0.82, P < 0.01). Significantly lower G-score figures were found among patients with an increased percentage of bone marrow blasts (P < 0.05), while high ppp correlated with the presence of ring sideroblasts, the degree of bone marrow fibrosis, and findings of complex chromosomal abnormalities. Patients with a high degree of bone marrow dysplasia had significantly lower G-score (P < 0.01) and significantly higher ppp (P < 0.05) figures, than those with less pronounced myelodysplasia. In addition, extreme hypogranulation (G-score < 150) or very high ppp (> or = 20%) was generally a sign of bi- and tri-lineage dysplasia in the bone marrow. The results thus show that quantitative estimation of peripheral blood polymorph dysplasia by G-score figures and ppp seems to reflect the total degree of bone marrow dysplasia in MDS and may serve as a complement to bone marrow evaluation when the diagnosis of MDS is difficult.

Comparison of the Stereospecificity and Immunoreactivity of NADH-Ferricyanide Reductases in Plant Membranes.

The substrate stereospecificity of NADH-ferricyanide reductase activities in the inner mitochondrial membrane and peroxisomal membrane of potato (Solanum tuberosum L.) tubers, spinach (Spinacea oleracea L.) leaf plasma membrane, and red beetroot (Beta vulgaris L.) tonoplast were all specific for the [beta]-hydrogen of NADH, whereas the reductases in wheat root (Triticum aestivum L.) endoplasmic reticulum and potato tuber outer mitochondrial membrane were both [alpha]-hydrogen specific. In all isolated membrane fractions one or several polypeptides with an apparent size of 45 to 55 kD cross-reacted with antibodies raised against a microsomal NADH-ferricyanide reductase on western blots.

Methodological aspects of DNA image cytometry in normal bone marrow smears.

The possibility of using DNA image cytometry in hematological material was evaluated in 27 Feulgen stained normal bone marrow smears. The 95% normal limits of DNA index (DI) determined separately for each cell population in bone marrow were 0.89-1.15, 0.93-1.09 and 0.92-1.12 for blasts, granulocytes and lymphocytes, respectively. The mean DI for blasts, granulocytes and lymphocytes was 1.02, 1.01 and 1.02, respectively. In the blasts population the mean percentage of cells in S+G2/M phase was 38.1% (S.D. 12.9%, range 14.3-65.6%), whereas the corresponding values for granulocytes and lymphocytes were below 1%. Calculation of DI and G0/G1 C.V. obtained in bone marrow smears subjected to Feulgen hydrolysis (5 N HCl, 22 degrees C) for different periods of time revealed the presence of a plateau of results between 60 and 120 min for all of the cell types. The intraobserver and interobserver reproducibility was confirmed by duplicate measurements of each subpopulation (P > 0.05). A comparison of DI and G0/G1 C.V. using smears from the same donors stained with the Feulgen method either straight away or after destaining from May-Grunwald-Giemsa confirmed that destained smears can be used, provided applying appropriately prepared reference cells. We conclude that image cytometry can be used reliably to analyze DNA content in hematological material.

Variation in DNA content of immature normal bone marrow cells.

A variability in DNA content detected was found with image cytometry, in immature bone marrow cells from 13 healthy donors (median age 31 yr). The mean coefficient of variation (C.V.) of the DNA content was found to be significantly (P = 0.0002) higher in immature blasts and promyelocytes than in mature granulocytes, lymphocytes, and monocytes. The finding was not due to high DNA contents secondary to DNA-synthesis in immature cells, since the percentage of such cells with a reduced DNA content was also significantly (P = 0.0424-0.0002) increased. The error of the method expressed as the variance of multiple measurements has been found to be 0.0002-0.0004. A staining error has been found for some hydrolysis times, but not for times between 60 and 120 minutes. A measuring error was found for an average of 1.38 to 3.38% of the cells. If many normal cells with DNA-aneuploidy are sterile, this would explain the present findings as well as previous ones about intra-marrow cell death, and also the fact that the variability in DNA content could not be detected with cytogenetic or flow cytometric methods.

Comparison of acute myeloid leukemia occurring de novo or preceded by a myelodysplastic stage: differences in cellular DNA content.

The DNA content of bone marrow cells in patients with acute leukemia preceded by a myelodysplastic stage (MDS-AML) was compared to that in patients with de novo AML. We studied granulocytes, lymphocytes, monocytes, and blasts/promyelocytes from Feulgen-stained bone marrow smears of 11 patients with de novo AML, ten patients with MDS-AML, and 13 apparently healthy controls. The mean amount of DNA per cell (DNA index; DI) in each cell population was determined using a digital video-based image-analyzing system (CAS-100). Analysis of variance (F test) showed a significant difference in the DNA content between de novo AML on one hand and MDS-AML and controls on the other as regards to blasts/promyelocytes (P < 0.01), lymphocytes (P < 0.05), and monocytes (P < 0.01), respectively. In three of 11 (27%) patients with de novo AML, a lower than normal limit DI was found both in immature and mature bone marrow cells. Patients with MDS-AML had those of DI values similar to normal controls. In consequence, a significantly reduced mean DI was found in patients with de novo AML in blasts/promyelocytes (P < 0.01), and monocytes (P < 0.05) compared to both normal controls and MDS-AML. Together with data published separately, suggesting differences in granulocyte morphology, clonality, and HLA-DR expression, these data suggest biological differences between the two diseases.

DNA image analysis in childhood acute lymphoblastic leukemia.

DNA index (DI) and percentages of cells in S and G2/M phase were determined in Feulgen stained nuclei of blasts from 31 cases of childhood ALL at diagnosis. In 6 cases the results of DNA analysis and cytogenetics were concordant showing hyperdiploidy. Two other cases with normal karyotype were revealed as DNA aneuploid with image analysis. Cases with cytogenetic abnormalities like translocation, deletion or presence of single or double supernumerary chromosomes had DI within normal ranges. Nine ALL cases (29%) were found to be DNA aneuploid--8 hyperdiploid and 1 hypodiploid. The percentages of cells in S and G2/M phase for blasts from bone marrow (mean 17.6%) were significantly higher than those estimated in the peripheral blood (mean 1.57%). We conclude that analysis by image cytometry can detect aneuploid DNA content even in cases, which showed a normal karyotype and provides new information concerning the biological aspects of leukemic blasts.

Dysplastic peripheral blood polymorphs link acute myeloblastic leukaemia in elderly to the myelodysplastic syndromes.

We studied dysplastic features in peripheral blood polymorphs from 80 patients with acute leukaemia. Thirty-seven patients with de novo acute myeloblastic leukaemia (AML) were compared to 26 patients with AML that had developed after a myelodysplastic phase (MDS-AML), and 17 cases of acute lymphoblastic leukaemia (ALL). Cytoplasmic hypogranulation in neutrophils, measured as a score value (G-score; normal range: 255-300), and the percentage of pelgeroid polymorphs (ppp; normal range: 0.5%) were studied retrospectively by reviewing the diagnostic peripheral blood smears. The mean G-score was decreased in MDS-AML (178 +/- 67.9), and in de novo AML (212 +/- 65.1), but not in ALL (275 +/- 24.3). When de novo AML patients were divided by age, the elderly (greater than 60 yr) had significantly (p = 0.0001) lower mean G-score than the younger (less than 45 yr) ones; 156 +/- 64.8 v 243 +/- 41.4. This age-related difference became accentuated when only patients with extreme hypogranulation (G-score less than 150) were studied. Elderly de novo AML patients also had significantly (p = 0.0057) higher mean ppp. By studying the degree of polymorph dysplasia in the peripheral blood, it seems possible to identify a subset of dysplastic elderly AML patients, who might have passed a (preleukaemic) MDS phase unnoticed.

Bone marrow cells in the DNA-synthesis-phase in the myelodysplastic syndrome and lymphome.

The bromodeoxyuridine (BRDU) labelling of bone marrow cells was studied in 46 subjects. The labelling in 14 patients, mostly untreated, with the myelodysplastic syndrome (MDS) and four lymphoma patients was significantly (p = 0.043) higher (11.38 +/- SE 2.3% S-phase cells) than that of marrow cells (7.18 +/- SE 1.04%) from 14 apparently healthy normal controls and from nine patients with non hematologic disease. Six iron deficiency had numerically but not significantly increased values. Bone marrow samples from MDS-patients showing the highest numbers of cells in the DNA-synthesis phase had the lowest numbers of colonies and clusters in the CFU-C assay (p < 0.03). The data suggest that the DNA-synthesis period is longer in MDS than in controls.

Sealed inside-out and right-side-out plasma membrane vesicles : optimal conditions for formation and separation.

Plasma membrane preparations of high purity (about 95%) are easily obtained by partitioning in aqueous polymer two-phase systems. These preparations, however, mainly contain sealed right-side-out (apoplastic side out) vesicles. Part of these vesicles have been turned inside-out by freezing and thawing, and sealed inside-out and right-side-out vesicles subsequently separated by repeating the phase partition step. Increasing the KCI concentration in the freeze/thaw medium as well as increasing the number of freeze/thaw cycles significantly increased the yield of inside-out vesicles. At optimal conditions, 15 to 25% of total plasma membrane protein was recovered as inside-out vesicles, corresponding to 5 to 10 milligrams of protein from 500 grams of sugar beet (Beta vulgaris L.) leaves. Based on enzyme latency, trypsin inhibition of NADH-cytochrome c reductase, and H(+) pumping capacity, a cross-contamination of about 20% between the two fractions of oppositely oriented vesicles was estimated. Thus, preparations containing about 80% inside-out and 80% right-side-out vesicles, respectively, were obtained. ATPase activity and H(+) pumping were both completely inhibited by vanadate (K(i) approximately 10 micromolar), indicating that the fractions were completely free from nonplasma membrane ATPases. Furthermore, the polypeptide patterns of the two fractions were close to identical, which shows that the vesicles differed in sidedness only. Thus, preparations of both inside-out and right-side-out plasma membrane vesicles are now available. This permits studies on transport, signal transduction mechanisms, enzyme topology, etc., using plasma membrane vesicles of either orientation.