Acute myeloid leukemia: treatment over 60

Undertreatment of older patients with acute myeloid leukemia (AML) can explain, in part, their inferior outcome when compared to that of younger patients. In agreement with the benefit seen by patients under age 60 from high-dose cytosine arabinoside (Ara-C), there are dose effects in the over 60s, in particular for daunorubicin, in induction treatment and for the duration of postremission treatment. The use of these effects can partly overcome the mostly unfavorable disease biology in older age AML, as expressed by the absence of favorable and the over-representation of adverse chromosomal abnormalities as well as the expression of drug resistance. We recommend an adequate dosage of 60 mg/m2 daunorubicin on 3 days in combination with standard dose Ara-C and 6-thioguanine given for induction and consolidation, and followed by a prolonged monthly maintenance chemotherapy for at least 1 year's duration. Further improvements in supportive care may help to deliver additional antileukemic cytotoxicity. As a novel approach, nonmyeloablative preparative regimens may open up the possibility of allogeneic transplantation for older patients with AML. Other new options like multidrug resistance modulators, antibody targeted therapies and molecular targeting are under clinical investigation. A questionnaire study in patients with AML showed that, according to patients' self-assessment, intensive and prolonged treatment did not result in a diminished quality of life. This finding did not vary by age, under or over 60 years. As the median age in this disease is more than 60 years, the adequate management of AML in older patients remains the major challenge.     

Rad54: the Swiss Army knife of homologous recombination?

Homologous recombination (HR) is a ubiquitous cellular pathway that mediates transfer of genetic information between homologous or near homologous (homeologous) DNA sequences. During meiosis it ensures proper chromosome segregation in the first division. Moreover, HR is critical for the tolerance and repair of DNA damage, as well as in the recovery of stalled and broken replication forks. Together these functions preserve genomic stability and assure high fidelity transmission of the genetic material in the mitotic and meiotic cell divisions. This review will focus on the Rad54 protein, a member of the Snf2-family of SF2 helicases, which translocates on dsDNA but does not display strand displacement activity typical for a helicase. A wealth of genetic, cytological, biochemical and structural data suggests that Rad54 is a core factor of HR, possibly acting at multiple stages during HR in concert with the central homologous pairing protein Rad51.     

The chaperone binding domain of SopE inhibits transport via flagellar and SPI-1 TTSS in the absence of InvB

Type III secretion systems (TTSS) are used by many Gram-negative pathogens for transporting effector proteins into eukaryotic host cells. Two modes of type III effector protein transport can be distinguished: transport into the surrounding medium (secretion) and cell-contact induced injection of effector proteins directly into the host cell cytosol (translocation). Two domains within the N-terminal regions of effector proteins determine the mode of transport. The amino terminal approximately 20 amino acids (N-terminal secretion signal, NSS) mediate secretion. In contrast, translocation generally requires the NSS, the adjacent approximately 100 amino acids (chaperone binding domain, CBD) and binding of the cognate chaperone to this CBD. TTSS are phylogenetically related to flagellar systems. Because both systems are expressed in Salmonella Typhimurium, correct effector protein transport involves at least two decisions: transport via the Salmonella pathogenicity island 1 (SPI-1) but not the flagellar TTSS (= specificity) and translocation into the host cell instead of secretion into the surrounding media (= transport mode). The mechanisms guiding these decisions are poorly understood. We have studied the S. Typhimurium effector protein SopE, which is specifically transported via the SPI-1 TTSS. Secretion and translocation strictly require the cognate chaperone InvB. Alanine replacement of amino acids 30-42 (and to some extent 44-54) abolished tight InvB binding, abolished translocation into the host cell and led to secretion of SopE via both, the flagellar and the SPI-1 TTSS. In clear contrast to wild-type SopE, secretion of SopE(Ala30-42) and SopE(Ala44-54) via the SPI-1 and the flagellar export system did not require InvB. These data reveal a novel function of the CBD: the CBD inhibits secretion of wild-type SopE via the flagellar and the SPI-1 TTSS in the absence of the chaperone InvB. Our data provide new insights into mechanisms ensuring specific effector protein transport by TTSS.     

Salmonella type III secretion effectors: pulling the host cell's strings

The enteric pathogen Salmonella employs type III secretion systems to transport a cocktail of effector proteins directly into its host cell. These effectors act in concert to control a variety of host cell processes to successfully invade intestinal cells and to establish an intracellular, replication-permissive niche. Recent studies reveal new insights into the molecular mechanisms that underlie effector protein injection, host cell invasion, and manipulation of vesicle trafficking induced by the interplay between multiple effectors and host systems. These findings corroborate the importance of spatio-temporal regulation of effector protein function for fine-tuned modulation of the host cell machinery.     

Identification of functional domains in the Sep1 protein (= Kem1, Xrn1), which is required for transition through meiotic prophase in Saccharomyces cerevisiae

The Sep1 (also known as Kem1, Xrn1, Rar5, DST2/Stp) protein of Saccharomyces cerevisiae is an Mr 175,000 multifunctional exonuclease with suspected roles in RNA turnover and in the microtubular cytoskeleton as well as in DNA recombination and DNA replication. The most striking phenotype of SEP1 null mutations is quantitative arrest during meiotic prophase at the pachytene stage. We have constructed a set of N- and C-terminal as well as internal deletions of the large SEP1 gene. Analysis of these deletion mutations on plasmids in a host carrying a null allele (sep1) revealed that at least 270 amino acids from the C-terminus of the wildtype protein were dispensable for complementing the slow growth and benomyl hypersensitivity of a null mutant. In contrast, any deletion at the N-terminus abrogated complementing activity for these phenotypes. The sequences essential for function correspond remarkably well with the regions of Sep1 that are homologous to its Schizosaccharomyces pombe counterpart Exo2. In addition, these experiments showed that, despite the high intracellular levels of Sep1, over-expression of this protein above these levels is detrimental to the cell. We discuss the potential cellular roles of the Sep1 protein as a microtubule-nucleic acid interface protein linking its suspected function in the microtubular cytoskeleton with its role as a nucleic acid binding protein.     

Preparation and regeneration of protoplasts and spheroplasts for fusion and transformation ofSchizosaccharomyces pombe

Preparation and regeneration of protoplasts is essential for somatic hybridization and transformation of yeasts. We present conditions that were found to be optimal for preparing and regeneratingSchizosaccharomyces pombe protoplasts for cell fusion. In contrast to these conditions, genetic transformation ofS. pombe requires spheroplasts that are osmotically sensitive, but still have some wall material attached to the cell. The main finding were as follows: (a) For protoplast formation with Novozym SP234, 0.9M sorbitol was found to be the optimal osmotic milieu and -mercaptoethanol is not necessary. (b) Embedding in soft agar yields considerably better regeneration frequencies than direct plating. (c) Cell fusion is optimal when both fusion partners are fully protoplasted, although considerable fusion occurs between spheroplasted cells as well. (d)Schizosaccharomyces pombe transformation frequencies are much higher with spheroplasts than with protoplasts. Inclusion of -mercaptoethanol did not enhance transformation frequency.     

Meiotic recombination in RAD54 mutants of Saccharomyces cerevisiae

The Rad54 protein is an important component of the recombinational DNA repair pathway in vegetative Saccharomyces cerevisiae cells. Unlike those in other members of the RAD52 group, the meiotic defect in rad54 is rather mild, reducing spore viability only to 26%–65%. A consistently greater requirement for Rad54p during meiosis was observed in hybrid strains, suggesting that Rad54p has a certain role in interhomolog interactions. Such a role is probably minor as no recombination defects were found in the surviving gametes in three genetic intervals on chromosome V. Also, the spore viability pattern in tetrads did not reflect an increase in nondisjunction at meiosis I indicative of a meiotic recombination defect. We suggest that the meiotic defect of rad54 cells lies in the failure to repair meiosis-specific double-strand breaks outside the context of the highly differentiated pathway leading to interhomolog joint molecules and meiotic crossovers that ensure accurate segregation at meiosis I. Received: 15 November 1999; in revised form: 11 January 2000 / Accepted: 11 January 2000     

Reconciling classical and molecular phylogenies in the stichotrichines (Ciliophora, Spirotrichea), including new sequences from some rare species

We performed a comparative morphological and molecular study on oxytrichid and urostylid stichotrichs (=part of the former hypotrichs). Included are new small subunit (18S) ribosomal RNA (rRNA) gene sequences from five rare oxytrichids (Gonostomum namibiense, Cyrtohymena citrina, Hemiurosoma terricola, Onychodromopsis flexilis, Orthoamphisiella breviseries) and published sequences, based on cultures provided by the senior author, of two key stichotrichid genera, viz., Gastrostyla and Engelmanniella. These and other sequences, altogether 27 species representing 23 genera, were used to analyze how 18S rRNA-based phylogenetic trees can be reconciled with the morphological and ontogenetical data. In 18S rRNA trees, the oligotrichine family Halteriidae invariably clusters within the oxytrichid clade, usually near Oxytricha granulifera, type species of the genus. This position is hardly supported by morphological and ecological evidence and, especially, it contradicts the current ontogenetic findings; possibly, it is an artifact caused by taxa undersampling and/or special molecular evolutionary events. In contrast, most morphological and DNA sequence data of the stichotrichs can be harmonized with the CEUU (Convergent Evolution of Urostylids and Uroleptids) hypothesis which suggests that the urostylid midventral pattern evolved from an oxytrichine ancestor, developing a second time within the Oxytrichidae. The systematic position of one of the two key genera could be clarified with the 18S rRNA sequences: Gastrostyla is a stylonychine oxytrichid. Based on the molecular data and a reassessment of ontogenesis, a new genus, Styxophrya nov. gen., is established for Onychodromus quadricornutus Foissner, Schlegel & Prescott, 1987.