Study of cyclin proteolysis in anaphase-promoting complex (APC) mutant cells reveals the requirement for APC function in the final steps of the fission yeast septation initiation network.

Cytokinesis in eukaryotic cells requires the inactivation of mitotic cyclin-dependent kinase complexes. An apparent exception to this relationship is found in Schizosaccharomyces pombe mutants with mutations of the anaphase-promoting complex (APC). These conditional lethal mutants arrest with unsegregated chromosomes because they cannot degrade the securin, Cut2p. Although failing at nuclear division, these mutants septate and divide. Since septation requires Cdc2p inactivation in wild-type S. pombe, it has been suggested that Cdc2p inactivation occurs in these mutants by a mechanism independent of cyclin degradation. In contrast to this prediction, we show that Cdc2p kinase activity fluctuates in APC cut mutants due to Cdc13/cyclin B destruction. In APC-null mutants, however, septation and cutting do not occur and Cdc13p is stable. We conclude that APC cut mutants are hypomorphic with respect to Cdc13p degradation. Indeed, overproduction of nondestructible Cdc13p prevents septation in APC cut mutants and the normal reorganization of septation initiation network components during anaphase.

Coordination between fission yeast glucan formation and growth requires a sphingolipase activity.

css1 mutants display a novel defect in Schizosaccharomyces pombe cell wall formation. The mutant cells are temperature-sensitive and accumulate large deposits of material that stain with calcofluor and aniline blue in their periplasmic space. Biochemical analyses of this material indicate that it consists of alpha- and beta-glucans in the same ratio as found in cell walls of wild-type S. pombe. Strikingly, the glucan deposits in css1 mutant cells do not affect their overall morphology. The cells remain rod shaped, and the thickness of their walls is unaltered. Css1p is an essential protein related to mammalian neutral sphingomyelinase and is responsible for the inositolphosphosphingolipid-phospholipase C activity observed in S. pombe membranes. Furthermore, expression of css1(+) can compensate for loss of ISC1, the enzyme responsible for this activity in Saccharomyces cerevisiae membranes. Css1p localizes to the entire plasma membrane and secretory pathway; a C-terminal fragment of Css1p, predicted to encode a single membrane-spanning segment, is sufficient to direct membrane localization of the heterologous protein, GFP. Our results predict the existence of an enzyme(s) or process(es) essential for the coordination of S. pombe cell wall formation and division that is, in turn, regulated by a sphingolipid metabolite.

Identification and characterization of Schizosaccharomyces pombe asp1(+), a gene that interacts with mutations in the Arp2/3 complex and actin.

The Arp2/3 complex is an essential component of the actin cytoskeleton in yeast and is required for the movement of actin patches. In an attempt to identify proteins that interact with this complex in the fission yeast Schizosaccharomyces pombe, we sought high-copy suppressors of the S. pombe arp3-c1 mutant, and have identified one, which we have termed asp1(+). The asp1(+) open reading frame (ORF) predicts a highly conserved protein of 921 amino acids with a molecular mass of 106 kD that does not contain motifs of known function. Neither asp1(+) nor its apparent Saccharomyces cerevisiae ortholog, VIP1, are essential genes. However, disruption of asp1(+) leads to altered morphology and growth properties at elevated temperatures and defects in polarized growth. The asp1 disruption strain also is hypersensitive to Ca+ ions and to low pH conditions. Although Asp1p is not stably associated with the Arp2/3 complex nor localized in any discrete structure within the cytoplasm, the asp1 disruption mutant was synthetically lethal with mutations in components of the Arp2/3 complex, arp3-c1 and sop2-1, as well as with a mutation in actin, act1-48. Moreover, the vip1 disruption strain showed a negative genetic interaction with a las17Delta strain. We conclude that Asp1p/Vip1p is important for the function of the cortical actin cytoskeleton.

Phosphorylation of the myosin-II light chain does not regulate the timing of cytokinesis in fission yeast.

Proper coordination of cytokinesis with chromosome separation during mitosis is crucial to ensure that each daughter cell inherits an equivalent set of chromosomes. It has been proposed that one mechanism by which this is achieved is through temporally regulated myosin regulatory light chain (RLC) phosphorylation (Satterwhite, L. L., and Pollard, T. D. (1992) Curr. Opin. Cell Biol. 4, 43-52). A variety of evidence is consistent with this model. A direct test of the importance of RLC phosphorylation in vivo has been done only in Dictyostelium and Drosophila; phosphorylation of the RLC is essential in Drosophila (Jordan, P., and Karess, R. (1997) J. Cell Biol. 139, 1805-1819) but not essential in Dictyostelium (Ostrow, B. D., Chen, P., and Chisholm, R. L. (1994) J. Cell Biol. 127, 1945-1955). The Schizosaccharomyces pombe myosin light chain Cdc4p is essential for cytokinesis, but it was unknown whether phosphorylation played a role in its regulation. Here we show that the S. pombe myosin light chain Cdc4p is phosphorylated in vivo on either serine 2 or 6 but not both. Mutation of either or both of these sites to alanine did not effect the ability of Cdc4p to bind the type II myosin Myo2p, and cells expressing only these mutated versions of Cdc4p grew and divided normally. Similarly, mutation of Ser-2, Ser-6, or both residues to aspartic acid did not affect growth or division of cells. Thus we conclude that phosphorylation of Cdc4p is not essential in vivo for the function of the protein.

The schizosaccharomyces pombe dim1(+) gene interacts with the anaphase-promoting complex or cyclosome (APC/C) component lid1(+) and is required for APC/C function.

The Schizosaccharomyces pombe dim1(+) gene is required for entry into mitosis and for chromosome segregation during mitosis. To further understand dim1p function, we undertook a synthetic lethal screen with the temperature-sensitive dim1-35 mutant and isolated lid (for lethal in dim1-35) mutants. Here, we describe the temperature-sensitive lid1-6 mutant. At the restrictive temperature of 36 degrees C, lid1-6 mutant cells arrest with a "cut" phenotype similar to that of cut4 and cut9 mutants. An epitope-tagged version of lid1p is a component of a multiprotein approximately 20S complex; the presence of lid1p in this complex depends upon functional cut9(+). lid1p-myc coimmunoprecipitates with several other proteins, including cut9p and nuc2p, and the presence of cut9p in a 20S complex depends upon the activity of lid1(+). Further, lid1(+) function is required for the multiubiquitination of cut2p, an anaphase-promoting complex or cyclosome (APC/C) target. Thus, lid1p is a component of the S. pombe APC/C. In dim1 mutants, the abundances of lid1p and the APC/C complex decline significantly, and the ubiquitination of an APC/C target is abolished. These data suggest that at least one role of dim1p is to maintain or establish the steady-state level of the APC/C.

A phosphorylation site mutant of Schizosaccharomyces pombe cdc2p fails to promote the metaphase to anaphase transition.

The protein kinase cdc2p is a key regulator of the G1-S and G2-M cell cycle transitions in the yeast Schizosaccharomyces pombe. Activation of cdc2p is regulated by its phosphorylation state and by interaction with other proteins. We have analyzed the consequences for cell cycle progression of altering the conserved threonine phosphorylation site, within the activation loop of cdc2p, to glutamic acid. This mutant, T167 E, promotes entry into mitosis, as judged by the accumulation of mitotic spindles and condensed chromosomes, despite the fact that it lacks demonstrable kinase activity both in vitro and in vivo. However, T167 E cannot promote the metaphase-anaphase transition. Since a component of the anaphase-promoting complex (APC) in S. pombe, cut9p, remains hypophosphorylated at the T167 E arrest point, the cell cycle block might be due to the inability of T167 E to activate the APC. T167 E is lethal when overexpressed, and overproduction also causes a mitotic arrest. Multicopy suppressors of the dominant negative phenotype were isolated, and identified as cdc13+ and suc1+. Overexpression of suc1+ suppresses the effects of T167 E overproduction by restoring sufficient amounts of suc1p to the cell to allow passage through mitosis.

[Development of a method of studying actual nutrition according to analysis of the frequency of consumption of food products: creation of a questionnaire and general evaluation of the reliability of the method]. / Razrabotka metoda issledovaniia fakticheskogo pitaniia po analizu chastoty potrebleniia pishchevykh produktov: sozdanie voprosnika i obshchaia otsenka dostovernosti metoda.

The method of actual feeding evaluation on frequency of food-stuffs consumption was developed. The method of 24-hour reproduction of a feed and the method of consumed food registration in a diary by testing person himself. The main results of study testified to reliability of data, received by a developed method.

Fission yeast cdc24(+) encodes a novel replication factor required for chromosome integrity.

A mutation within the Schizosaccharomyces pombe cdc24(+) gene was identified previously in a screen for cell division cycle mutants and the cdc24(+) gene was determined to be essential for S phase in this yeast. We have isolated the cdc24(+) gene by complementation of a new temperature-sensitive allele of the gene, cdc24-G1. The DNA sequence predicts the presence of an open reading frame punctuated by six introns which encodes a pioneer protein of 58 kD. A cdc24 null mutant was generated by homologous recombination. Haploid cells lacking cdc24(+) are inviable, indicating that cdc24(+) is an essential gene. The transcript of cdc24(+) is present at constant levels throughout the cell cycle. Cells lacking cdc24(+) function show a checkpoint-dependent arrest with a 2N DNA content, indicating a block late in S phase. Arrest is accompanied by a rapid loss of viability and chromosome breakage. An S. pombe homolog of the replicative DNA helicase DNA2 of S. cerevisiae suppresses cdc24. These results suggest that Cdc24p plays a role in the progression of normal DNA replication and is required to maintain genomic integrity.

[Dietary intake of different forms of iron and other factors of anemia epidemiology in women of childbearing age in S. Petersburg]. / Potrebleniie razlichnykh form zheleza s pishchei i drugie faktory rasprostraneniia anemii u zhenshchin detorodnogo vozrasta Sankt Peterburga.

Actual nutritional status and dietary intake of different iron forms of the women in Sankt-Peterburg were investigated. The concentration of blood hemoglobin was studied. Signs of deficiency of nutrition of the investigated women were not found both by analysis of foodstuffs and energy intake and by anthropometric estimation of nutritional status.

Myb-related Schizosaccharomyces pombe cdc5p is structurally and functionally conserved in eukaryotes.

Schizosaccharomyces pombe cdc5p is a Myb-related protein that is essential for G2/M progression. To explore the structural and functional conservation of Cdc5 throughout evolution, we isolated Cdc5-related genes and cDNAs from Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and Homo sapiens. Supporting the notion that these Cdc5 gene family members are functionally homologous to S. pombe cdc5(+), human and fly Cdc5 cDNAs are capable of complementing the temperature-sensitive lethality of the S. pombe cdc5-120 mutant. Furthermore, S. cerevisiae CEF1 (S. cerevisiae homolog of cdc5(+)), like S. pombe cdc5(+), is essential during G2/M. The location of the cdc5-120 mutation, as well as mutational analyses of Cef1p, indicate that the Myb repeats of cdc5p and Cef1p are important for their function in vivo. However, we found that unlike in c-Myb, single residue substitutions of glycines for hydrophobic residues within the Myb repeats of Cef1p, which are essential for maintaining structure of the Myb domain, did not impair Cef1p function in vivo. Rather, multiple W-to-G substitutions were required to inactivate Cef1p, and many of the substitution mutants were found to confer temperature sensitivity. Although it is possible that Cef1p acts as a transcriptional activator, we have demonstrated that Cef1p is not involved in transcriptional activation of a class of G2/M-regulated genes typified by SWI5. Collectively, these results suggest that Cdc5 family members participate in a novel pathway to regulate G2/M progression.

[Monitoring food consumption and nutritional status of Moscow schoolchildren in 1992-1994. 2. Anthropometric evaluation of nutritional status, effect of social factors on the character and status of nutrition]. / Monitoring potrebleniia pishchi i sostoianiia pitaniia detei-shkol'nikov Moskvy v 1992-1994 gg.

In this part of the study the nutritional status of Moscow's schoolchildren was assessed by height and weight. The anthropometric data were compared with the CDC/WHO international growth references standards by ANTHRO version 1.01 software. The prevalence of low weight-for-age (Z-score < -2) was more frequent in boys of 15 years of age. Low height-for-age was more prevalent in the group of boys of 15 and girls of 10 years of age. In the period under study there was a slight decrease in height and weight of schoolchildren, but this was within the limits of expected normal variation. A few selected socio-economic variables such as parental education, family size, participation in organized sports and use of school breakfast/lunch options were included. The size of the family or whether it is a one- or two-parent household does not seem to influence energy or nutrient intake, nor are the anthropometric variables significantly affected. The more size of family and the lower the father's education than lower the children's total fat intake and intake of energy from fat.

The Schizosaccharomyces pombe actin-related protein, Arp3, is a component of the cortical actin cytoskeleton and interacts with profilin.

The gene encoding the actin-related protein Arp3 was first identified in the fission yeast Schizosaccharomyces pombe and is a member of an evolutionarily conserved family of actin-related proteins. Here we present several key findings that define an essential role for Arp3p in the functioning of the cortical actin cytoskeleton. First, mutants in arp3 interact specifically with profilin and actin mutants. Second, Arp3 localizes to cortical actin patches which are required for polarized cell growth. Third, the arp3 gene is required for the reorganization of the actin cytoskeleton during the cell cycle. Finally, the Arp3 protein is present in a large protein complex. We believe that this complex may mediate the cortical functions of profilin at actin patches in S. pombe.

Fission yeast Sop2p: a novel and evolutionarily conserved protein that interacts with Arp3p and modulates profilin function.

Profilins bind to monomeric actin and also interact with ligands such as phosphoinositide 4,5-bisphosphate, the proline-rich protein VASP and a complex of four to six polypeptides identified in Acanthamoeba that includes two actin-related proteins. Here, we report the identification and characterization of an essential gene from Schizosaccharomyces pombe, sop2+, a mutation in which rescues the temperature-sensitive lethality of a profilin mutation, cdc3-124. The sop2-1 mutant is defective for cell elongation and septation, suggesting that it is involved in multiple cortical actin-requiring processes. Consistent with a role in actin cytoskeletal function, negative interactions have been identified between sop2-1 and act1-48, a mutant allele of actin. Sop2p is a novel 377 amino acid polypeptide with similarity to proteins of the beta-transducin repeat family. Sop2p-related proteins have been identified by sequencing projects in diverse species, and we have isolated a human cDNA highly related to sop2+, SOP2 Hs, which functionally complements the sop2-1 mutation. Sop2p proteins from all species contain peptide sequences identical or highly similar to two peptide sequences from an Acanthamoeba beta-transducin repeat protein present in the profilin binding complex. Biochemical analyses demonstrate that Sop2p is present in a complex which also contains the actin-related protein, Arp3p. Immunofluorescence studies reveal the presence of Sop2p in (i) punctate structures distributed throughout the cell, (ii) cables that extend the length of the cell, and (iii) a medial band in a small percentage of septating cells. Collectively these data demonstrate the interaction of Sop2p with Arp3p, profilin and actin.

Characterization of novel mutations at the Schizosaccharomyces pombe cdc2 regulatory phosphorylation site, tyrosine 15.

The cdc2 protein kinase family is regulated negatively by phosphorylation in the glycine ATP-binding loop at a conserved tyrosine residue, Y15, alone or in combination with T14 phosphorylation. In Schizosaccharomyces pombe and other systems, substitution of these residues with structurally similar but nonphosphorylatable amino acids has generated proteins (Y15F or T14AY15F) that behave as constitutively tyrosine-dephosphorylated proteins or threonine and tyrosine-dephosphorylated proteins. Here we report the characteristics of three additional mutants at Y15--Y15E, Y15S, and Y15T--in S. pombe cdc2p. All three mutant proteins are active in in vitro kinase assays, but are unable to functionally complement cdc2 loss-of-function mutations in vivo. Additionally, all three mutants are dominant negatives. A more detailed analysis of the Y15T mutant indicates that it can initiate chromosome condensation and F-actin contractile ring formation, but is unable to drive the reorganization of microtubules into a mitotic spindle.

Construction of vectors and a genomic library for use with his3-deficient strains of Schizosaccharomyces pombe.

The construction of vectors for use in Schizosaccharomyces pombe using the his3+ gene as a selectable marker is described. In addition, we report the construction of a genomic library in a his3(+)-containing shuttle vector to facilitate the cloning of genes by complementation of mutant function in strains defective for His3 activity.

Schizosaccharomyces pombe skp1+ encodes a protein kinase related to mammalian glycogen synthase kinase 3 and complements a cdc14 cytokinesis mutant.

We report the cloning of the skp1+ gene, a Schizosaccharomyces pombe homolog of the glycogen synthase kinase 3 (GSK-3) family whose members in higher eukaryotes are involved in cell fate determination, nuclear signalling, and hormonal regulation. skp1 is 67% identical to mammalian GSK-3 beta and displays similar biochemical properties in vitro. Like GSK-3 beta, skp1 is phosphorylated on a conserved tyrosine residue, and this phosphorylation is required for efficient activity. skp1 is also phosphorylated at a serine which has been identified as S-335. Phosphorylation at this site is likely to inhibit its function. Unlike the mammalian enzyme, skp1 both tyrosine autophosphorylates in yeast cells and can phosphorylate other proteins on tyrosine in bacteria. The skp1+ gene is not essential. However, cells with deletions in skp1+ are sensitive to heat shock and exhibit defects in sporulation. Overexpression of wild-type skp1+ specifically complements cdc14-118, one of several mutations causing a defect in cytokinesis. In addition, certain phosphorylation site mutants induce a delay or block in cytokinesis when overexpressed. Together, these data identify novel interactions of a fission yeast GSK-3 homolog with elements of the cytokinesis machinery.

[Monitoring of dietary intake and nutritional status of Moscow's school children, 1992-1994. 1. Methodology of the study. Energy and nutrient intakes]. / Monitoring potrebleniia pishchi i sostoianiia pitaniia detei-shkol'nikov Moskvy v 1992-1994 gg. 1. Metodologiia issledovaniia. Potreblenie énergii i pishchevykh veshchestv.

Surveys of dietary intakes and nutritional status of schoolchildren aged 10 and 15 years in Moscow were made during a period of rapid economic transformation, 1992-1994. It was part of multicentre study of schoolchildren dietary intake evaluation sponsored by WHO/UNICEF. Information on food intake was collected using two 24-hour recall interviews. The design of study was carefully elaborated by international group of nutritional epidemiologists. There was a slight change in food pattern with age, and some differences between boys and girls of 15 years old. Nutrient intakes in the groups studied did not change significantly during the period of study. Protein contributed about 12% and fat 29-32% of the dietary energy, and total energy intake was overall on a satisfactory level. 70% of the subjects had low intake levels of vitamin B2 and calcium compared with the chosen reference values. Milk and dairy products, fruits, juices and vegetables were consumed in small quantities and relatively infrequently. The consumption of bread and bread products, porridge, confectionery, meat and meat products were consumed more frequently and in larger quantities.

Overexpression of the neurotrophic cytokine S100 beta in human temporal lobe epilepsy.

Neuritic sprouting and disturbances of calcium homeostasis are well described in epilepsy. S100 beta is an astrocyte-derived cytokine that promotes neurite growth and induces increases in levels of intracellular calcium in neurons. In sections of neocortex of surgically resected temporal lobe tissue from patients with intractable epilepsy, we found that the number of S100 beta-immunoreactive astrocytes was approximately threefold higher than that found in control patients (p < 0.001). These astrocytes were activated, i.e., enlarged, and had prominent processes. Temporal lobe tissue levels of S100 beta were shown by ELISA to be fivefold higher in 21 epileptics than in 12 controls (p < 0.001). The expression of the astrocyte intermediate filament protein, glial fibrillary acidic protein, was not significantly elevated in epileptics, suggesting a selective up-regulation of S100 beta expression. Our findings, together with established functions of S100 beta, suggest that this neurotrophic cytokine may be involved in the pathophysiology of epilepsy.

[Nutritional status of native and non-native population of Russia's Extreme North and Far East]. / Fakticheskoe pitanie korennogo i nekorennogo naseleniia raionov Krainevo Severa i Dal'nevo Vostoka.

Daily diets studied in various groups of native and non-native population living in the Far North and East of Russia (Kamchatka, Chukotka, Sakhalin) are shown to be inadequate for the adult capable population. Chemical composition of food provides insufficient supply of vegetable oil, calcium, vitamins, magnesium.