Experience with using aclarubicin in the treatment of acute leukemia and blast crisis of chronic myeloid leukemia]

The clinical efficacy of aclarubicin, an anthracycline antibiotic, was studied in 48 patients with leukemia. The antibiotic was used in the following combinations with cytarabine: "7 + 7", "5 + 5" and "7 & 3". A complete remission was stated in 14 (42.4 per cent) out of 33 patients with acute nonlymphoid leukemia, 6 (43 per cent) out of the 14 patients having relapses. The combined therapy was effective in 4 out of 5 pre-resistant patients. The "7 + 3" scheme was the most beneficial. The most common adverse reactions were nausea and vomiting.     

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

H+-pyrophosphatase of Rhodospirillum rubrum. High yield expression in Escherichia coli and identification of the Cys residues responsible for inactivation my mersalyl

H(+)-translocating pyrophosphatase (H(+)-PPase) of the photosynthetic bacterium Rhodospirillum rubrum was expressed in Escherichia coli C43(DE3) cells. Recombinant H(+)-PPase was observed in inner membrane vesicles, where it catalyzed both PP(i) hydrolysis coupled with H(+) transport into the vesicles and PP(i) synthesis. The hydrolytic activity of H(+)-PPase in E. coli vesicles was eight times greater than that in R. rubrum chromatophores but exhibited similar sensitivity to the H(+)-PPase inhibitor, aminomethylenediphosphonate, and insensitivity to the soluble PPase inhibitor, fluoride. Using this expression system, we showed that substitution of Cys(185), Cys(222), or Cys(573) with aliphatic residues had no effect on the activity of H(+)-PPase but decreased its sensitivity to the sulfhydryl modifying reagent, mersalyl. H(+)-PPase lacking all three Cys residues was completely resistant to the effects of mersalyl. Mg(2+) and MgPP(i) protected Cys(185) and Cys(573) from modification by this agent but not Cys(222). Phylogenetic analyses of 23 nonredundant H(+)-PPase sequences led to classification into two subfamilies. One subfamily invariably contains Cys(222) and includes all known K(+)-independent H(+)-PPases, whereas the other incorporates a conserved Cys(573) but lacks Cys(222) and includes all known K(+)-dependent H(+)-PPases. These data suggest a specific link between the incidence of Cys at positions 222 and 573 and the K(+) dependence of H(+)-PPase.     

Stt7-dependent Phosphorylation during State Transitions in the Green Alga Chlamydomonas reinhardtii

Photosynthetic organisms are able to adapt to changes in light conditions by balancing the light excitation energy between the light-harvesting systems of photosystem (PS) II and photosystem I to optimize the photosynthetic yield. A key component in this process, called state transitions, is the chloroplast protein kinase Stt7/STN7, which senses the redox state of the plastoquinone pool. Upon preferential excitation of photosystem II, this kinase is activated through the cytochrome b₆f complex and required for the phosphorylation of the light-harvesting system of photosystem II, a portion of which migrates to photosystem I (state 2). Preferential excitation of photosystem I leads to the inactivation of the kinase and to dephosphorylation of light-harvesting complex (LHC) II and its return to photosystem II (state 1). Here we compared the thylakoid phosphoproteome of the wild-type strain and the stt7 mutant of Chlamydomonas under state 1 and state 2 conditions. This analysis revealed that under state 2 conditions several Stt7-dependent phosphorylations of specific Thr residues occur in Lhcbm1/Lhcbm10, Lhcbm4/Lhcbm6/Lhcbm8/Lhcbm9, Lhcbm3, Lhcbm5, and CP29 located at the interface between PSII and its light-harvesting system. Among the two phosphorylation sites detected specifically in CP29 under state 2, one is Stt7-dependent. This phosphorylation may play a crucial role in the dissociation of CP29 from PSII and/or in its association to PSI where it serves as a docking site for LHCII in state 2. Moreover, Stt7 was required for the phosphorylation of the thylakoid protein kinase Stl1 under state 2 conditions, suggesting the existence of a thylakoid protein kinase cascade. Stt7 itself is phosphorylated at Ser⁵³³ in state 2, but analysis of mutants with a S533A/D change indicated that this phosphorylation is not required for state transitions. Moreover, we also identified phosphorylation sites that are redox (state 2)-dependent but independent of Stt7 and additional phosphorylation sites that are redox-independent.The primary photochemical reactions of photosynthesis are catalyzed by the pigment-protein complexes photosystem II (PSII)¹ and PSI (PSI), which are linked in series through the plastoquinone pool, the cytochrome b₆f complex, and plastocyanin in the thylakoid membranes. Upon light absorption by the antenna systems of PSII and PSI, charge separations occur across the membrane that lead to the oxidation of water by PSII and electron flow to PSI and ultimately to the reduction of NADP⁺. Because the antenna systems of PSII and PSI have different pigment composition, they are differentially sensitized upon changes in light quality and quantity. However, photosynthetic organisms have the ability to adapt to changes in light. They balance energy input and consumption in the short term through dissipation of excess absorbed light energy into heat through non-photochemical quenching and regulate absorption of excitation energy between PSII and PSI through state transitions (supplemental Fig. 1). This reversible redistribution leads to an overall increase in photosynthetic quantum yield. State transitions occur when preferential excitation of PSII reduces the plastoquinone pool. This leads to the activation of a thylakoid protein kinase as a result of the docking of plastoquinol to the Qo site of the cytochrome b₆f complex (1, 2) and to the phosphorylation of the polypeptides of the light-harvesting complex II (LHCII), a part of which migrates to PSI (state 2) (3–5). The process is reversible as preferential excitation of PSI inactivates the kinase and allows for dephosphorylation of LHCII and its return to PSII (state 1) (3, 6). In the green alga Chlamydomonas reinhardtii, the LHCII protein set consists of Type I (Lhcbm3, Lhcbm4, Lhcbm6, Lhcbm8, and Lhcbm9), Type II (Lhcbm5), Type III (Lhcbm2 and Lhcbm7), and Type IV (Lhcbm1 and Lhcbm10) proteins and of Lhcb7, CP26, and CP29 (7). Because of their nearly identical sequences and sizes, several of these Lhcbm proteins cannot be distinguished by SDS-PAGE. Most of them fractionate into four bands called P11 and P13 (Type I), P16 (Type IV), and P17 (Type III). Whereas P16 is not phosphorylated, phosphorylation events occur on P11, P13, and P17 (7, 8).The association of the mobile part of LHCII to PSI during a transition from state 1 to state 2 requires the PsaH subunit (9) and CP29, which also moves to PSI and is essential for docking LHCII to PSI (10–12). The lateral displacement of LHCII from the PSII-rich grana to the PSI-rich lamellar thylakoid regions results in transfer to PSI of about 80% of the excitation energy absorbed by LHCII in C. reinhardtii (13), a considerably higher amount than in land plants in which only 15–20% of LHCII is mobile (3). In C. reinhardtii, state transitions are associated with a reorganization of the photosynthetic electron transfer chain with a switch from linear to cyclic electron flow during a transition from state 1 to state 2 (14, 15). Thus, cells produce ATP and NADPH in state 1 but only ATP in state 2. It appears that the major function of state transitions in this alga is to adjust the level of ATP and the ATP/NADPH ratio to cellular demands (5).Thylakoid membranes contain appressed grana and nonappressed stromal domains in which PSII and PSI are enriched, respectively. Because LHCII is a major stabilizer of the grana structure (16), the movement of LHCII from PSII to PSI is expected to lead to major rearrangements of these membranes during state transitions. Indeed, based on extensive electron microscope studies, it was proposed that fusion and fission events occur at the interface between the grana and stroma lamellar domains that lead to a remodeling of the membranes (17).Mapping of in vivo protein phosphorylation sites in photosynthetic membranes of Chlamydomonas revealed a total of 19 sites corresponding to 15 genes (18). It was shown that the major changes are clustered at the interface between the PSII core and the associated LHCII proteins during state transitions. Phosphorylation of the PSII core subunits D2 and PsbR and multiple phosphorylations of the minor LHCII antenna subunit CP29 were detected as well as phosphorylation of Lhcbm1, which belongs to the major LHCII complex (18).Although the phosphorylation of LHCII was observed many years ago (6), it is only recently that kinases involved in this process were uncovered. Fleischmann et al. (19) and Kruse et al. (20) used a genetic approach in C. reinhardtii with the aim of dissecting the signal transduction chain of state transitions. Two allelic mutants blocked in state 1 were identified that are affected in the Stt7 gene encoding a thylakoid Ser-Thr protein kinase that is required for LHCII phosphorylation during a transition from state 1 to state 2 (21). This Stt7 kinase is conserved in land plants and has an ortholog, STN7, in Arabidopsis (22).The 754-amino acid Stt7 kinase has a catalytic domain characteristic of Ser-Thr kinases (21). It contains a putative 41-amino acid transit peptide at its N-terminal end, and the protein is localized on the thylakoid membrane. Stt7 is associated with photosynthetic complexes including LHCII, PSI, and the cytochrome b₆f complex (23). Stt7 also contains a transmembrane region that separates its catalytic kinase domain on the stromal side from its N-terminal end in the thylakoid lumen with two conserved Cys residues that are critical for its activity and state transitions (23). Moreover, the level of Stt7 decreases considerably under state 1 conditions, and the kinase acts in catalytic amounts (23). However, it is not yet known whether this kinase directly phosphorylates LHCII or whether it is part of a kinase cascade involved in the signaling pathway of state transitions.In this work, we used a mass spectrometry-based approach (24) to map the in vivo Stt7-dependent protein phosphorylation sites within thylakoid membranes isolated from the green alga C. reinhardtii subjected to state 1 and state 2 conditions. In contrast with the earlier studies via direct MS/MS sequencing of the IMAC-enriched phosphorylated peptides from thylakoid proteins (18, 25), we performed additional LC-MS/MS-based analyses using alternating collision-induced dissociation and electron transfer dissociation of peptide ions. This approach revealed novel phosphorylation sites in LHCII polypeptides, in several other membrane and membrane-associated proteins, and in the thylakoid protein kinases Stt7 and Stl1, suggesting the existence of a thylakoid protein kinase cascade. Relative quantification of phosphorylated peptides labeled with stable isotopes determined the specific Stt7-dependent phosphorylation site in CP29 linker protein under state 2. Moreover, we also identified phosphorylation sites that are redox-dependent but independent of Stt7 and additional phosphorylation sites that are redox-independent. This mapping provides new insights into the regulatory network of protein phosphorylation in algal photosynthetic membranes during state transitions.     

Soluble CD44 interacts with intermediate filament protein vimentin on endothelial cell surface

CD44 is a cell surface glycoprotein that functions as hyaluronan receptor. Mouse and human serum contain substantial amounts of soluble CD44, generated either by shedding or alternative splicing. During inflammation and in cancer patients serum levels of soluble CD44 are significantly increased. Experimentally, soluble CD44 overexpression blocks cancer cell adhesion to HA. We have previously found that recombinant CD44 hyaluronan binding domain (CD44HABD) and its non-HA-binding mutant inhibited tumor xenograft growth, angiogenesis, and endothelial cell proliferation. These data suggested an additional target other than HA for CD44HABD. By using non-HA-binding CD44HABD Arg41Ala, Arg78Ser, and Tyr79Ser-triple mutant (CD443MUT) we have identified intermediate filament protein vimentin as a novel interaction partner of CD44. We found that vimentin is expressed on the cell surface of human umbilical vein endothelial cells (HUVEC). Endogenous CD44 and vimentin coprecipitate from HUVECs, and when overexpressed in vimentin-negative MCF-7 cells. By using deletion mutants, we found that CD44HABD and CD443MUT bind vimentin N-terminal head domain. CD443MUT binds vimentin in solution with a Kd in range of 12-37 nM, and immobilised vimentin with Kd of 74 nM. CD443MUT binds to HUVEC and recombinant vimentin displaces CD443MUT from its binding sites. CD44HABD and CD443MUT were internalized by wild-type endothelial cells, but not by lung endothelial cells isolated from vimentin knock-out mice. Together, these data suggest that vimentin provides a specific binding site for soluble CD44 on endothelial cells.     

The transit peptide of CP29 thylakoid protein in Chlamydomonas reinhardtii is not removed but undergoes acetylation and phosphorylation

The surface-exposed peptides were cleaved by trypsin from the photosynthetic thylakoid membranes isolated from the green alga Chlamydomonas reinhardtii. Two phosphorylated peptides, enriched from the peptide mixture and sequenced by nanospray quadrupole time-of-flight mass spectrometry, revealed overlapping sequences corresponding to the N-terminus of a nuclear-encoded chlorophyll a/b-binding protein CP29. In contrast to all known nuclear-encoded thylakoid proteins, the transit peptide in the mature algal CP29 was not removed but processed by methionine excision, N-terminal acetylation and phosphorylation on threonine 6. The importance of this phosphorylation site is proposed as the reason of the unique transit peptide retention.     

From recommendation to action: psychosocial factors influencing physician intention to use Health Technology Assessment (HTA) recommendations

Background

The Evidence-Based Practice (EBP) Project has been investigating the implementation of evidence-based mental health practices (Assertive Community Treatment, Family Psychoeducation, Integrated Dual Diagnosis Treatment, Illness Management and Recovery, and Supported Employment) in state public mental health systems in the United States since 2001. To date, Project findings have yielded valuable insights into implementation strategy characteristics and effectiveness. This paper reports results of an effort to identify and classify state-level implementation activities and strategies employed across the eight states participating in the Project.

Methods

Content analysis and Greenhalgh et al's (2004) definition of innovation were used to identify and classify state-level activities employed during three phases of EBP implementation: Pre-Implementation, Initial Implementation and Sustainability Planning. Activities were coded from site visit reports created from documents and notes from key informant interviews conducted during two periods, Fall 2002 – Spring 2003, and Spring 2004. Frequency counts and rank-order analyses were used to examine patterns of implementation activities and strategies employed across the three phases of implementation.

Results

One hundred and six discreet implementation activities and strategies were identified as innovative and were classified into five categories: 1) state infrastructure building and commitment, 2) stakeholder relationship building and communications, 3) financing, 4) continuous quality management, and 5) service delivery practices and training. Implementation activities from different categories were employed at different phases of implementation.

Conclusion

Insights into effective strategies for implementing EBPs in mental health and other health sectors require qualitative and quantitative research that seeks to: a) empirically test the effects of tools and methods used to implement EBPs, and b) establish a stronger evidence-base from which to plan, implement and sustain such efforts. This paper offers a classification scheme and list of innovative implementation activities and strategies. The classification scheme offers potential value for future studies that seek to assess the effects of various implementation processes, and helps establish widely accepted standards and criteria that can be used to assess the value of innovative activities and strategies.     

An invisible ubiquitin conformation is required for efficient phosphorylation by PINK1

The Ser/Thr protein kinase PINK1 phosphorylates the well‐folded, globular protein ubiquitin (Ub) at a relatively protected site, Ser65. We previously showed that Ser65 phosphorylation results in a conformational change in which Ub adopts a dynamic equilibrium between the known, common Ub conformation and a distinct, second conformation wherein the last β‐strand is retracted to extend the Ser65 loop and shorten the C‐terminal tail. We show using chemical exchange saturation transfer (CEST) nuclear magnetic resonance experiments that a similar, C‐terminally retracted (Ub‐CR) conformation also exists at low population in wild‐type Ub. Point mutations in the moving β5 and neighbouring β‐strands shift the Ub/Ub‐CR equilibrium. This enabled functional studies of the two states, and we show that while the Ub‐CR conformation is defective for conjugation, it demonstrates improved binding to PINK1 through its extended Ser65 loop, and is a superior PINK1 substrate. Together our data suggest that PINK1 utilises a lowly populated yet more suitable Ub‐CR conformation of Ub for efficient phosphorylation. Our findings could be relevant for many kinases that phosphorylate residues in folded protein domains.