Ca2+-calmodulin promotes survival of pheromone-induced growth arrest by activation of calcineurin and Ca2+-calmodulin-dependent protein kinase.

The cmd1-6 allele contains three mutations that block Ca2+ binding to calmodulin from Saccharomyces cerevisiae. We find that strains containing cmd1-6 lose viability during cell cycle arrest induced by the mating pheromone alpha-factor. The 50% lethal dose (LD50) of alpha-factor for the calmodulin mutant is almost fivefold below the LD50 for a wild-type strain. The calmodulin mutants are not more sensitive to alpha-factor, as measured by activation of a pheromone-responsive reporter gene. Two observations indicate that activation of the Ca2+-calmodulin-dependent protein phosphatase calcineurin contributes to survival of pheromone-induced arrest. First, deletion of the gene encoding the calcineurin regulatory B subunit, CNB1, from a wild-type strain decreases the LD50 of alpha-factor but has no further effect on a cmd1-6 strain. Second, a dominant constitutive calcineurin mutant partially restores the ability of the cmd1-6 strain to survive exposure to alpha-factor. Activation of the Ca2+-calmodulin-dependent protein kinase (CaMK) also contributes to survival, thus revealing a new function for this enzyme. Deletion of the CMK1 and CMK2 genes, which encode CaMK, decreases the LD50 of pheromone compared with that for a wild-type strain but again has no effect in a cmd1-6 strain. Furthermore, the LD50 of alpha-factor for a mutant in which the calcineurin and CaMK genes have been deleted is the same as that for the calmodulin mutant. Finally, the CaMK and calcineurin pathways appear to be independent since the ability of constitutive calcineurin to rescue a cmd1-6 strain is not blocked by deletion of the CaMK genes.

The essential mitotic target of calmodulin is the 110-kilodalton component of the spindle pole body in Saccharomyces cerevisiae.

Two independent methods identified the spindle pole body component Nuf1p/Spc110p as the essential mitotic target of calmodulin. Extragenic suppressors of cmd1-1 were isolated and found to define three loci, XCM1, XCM2, and XCM3 (extragenic suppressor of cmd1-1). The gene encoding a dominant suppressor allele of XCM1 was cloned. On the basis of DNA sequence analysis, genetic cosegregation, and mutational analysis, XCM1 was identified as NUF1/SPC110. Independently, a C-terminal portion of Nuf1p/Spc110p, amino acid residues 828 to 944, was isolated as a calmodulin-binding protein by the two-hybrid system. As assayed by the two-hybrid system, Nuf1p/Spc110p interacts with wild-type calmodulin and triple-mutant calmodulins defective in binding Ca2+ but not with two mutant calmodulins that confer a temperature-sensitive phenotype. Deletion analysis by the two-hybrid system mapped the calmodulin-binding site of Nuf1p/Spc110p to amino acid residues 900 to 927. Direct binding between calmodulin and Nuf1p/Spc110p was demonstrated by a modified gel overlay assay. Furthermore, indirect immunofluorescence with fixation procedures known to aid visualization of spindle pole body components localized calmodulin to the spindle pole body. Sequence analysis of five suppressor alleles of NUF1/SPC110 indicated that suppression of cmd1-1 occurs by C-terminal truncation of Nuf1p/Spc110p at amino acid residues 856, 863, or 881, thereby removing the calmodulin-binding site.

Ribosomal protein genes are overexpressed in colorectal cancer: isolation of a cDNA clone encoding the human S3 ribosomal protein.

We have isolated a cDNA clone encoding the human S3 ribosomal protein from a normal human colon cDNA library. The clone was identified as one of many that detected genes whose level of expression was increased in adenocarcinoma of the colon relative to normal colonic mucosa. Increased levels of the S3 transcript were present in the tumors of all eight patients examined. Moreover, the S3 mRNA was also more abundant in 7 of 10 adenomatous polyps, the presumed precursor of carcinoma. Additional studies demonstrated that increased levels of mRNAs encoding several other ribosomal proteins, including S6, S8, S12, L5, and P0, were present in colorectal tumors and polyps. These results suggest that there is increased synthesis of ribosomes in colorectal tumors and that this increase is an early event in colon neoplasia.

The yeast dynactin complex is involved in partitioning the mitotic spindle between mother and daughter cells during anaphase B.

Although vertebrate cytoplasmic dynein can move to the minus ends of microtubules in vitro, its ability to translocate purified vesicles on microtubules depends on the presence of an accessory complex known as dynactin. We have cloned and characterized a novel gene, NIP100, which encodes the yeast homologue of the vertebrate dynactin complex protein p150(glued). Like strains lacking the cytoplasmic dynein heavy chain Dyn1p or the centractin homologue Act5p, nip100Delta strains are viable but undergo a significant number of failed mitoses in which the mitotic spindle does not properly partition into the daughter cell. Analysis of spindle dynamics by time-lapse digital microscopy indicates that the precise role of Nip100p during anaphase is to promote the translocation of the partially elongated mitotic spindle through the bud neck. Consistent with the presence of a true dynactin complex in yeast, Nip100p exists in a stable complex with Act5p as well as Jnm1p, another protein required for proper spindle partitioning during anaphase. Moreover, genetic depletion experiments indicate that the binding of Nip100p to Act5p is dependent on the presence of Jnm1p. Finally, we find that a fusion of Nip100p to the green fluorescent protein localizes to the spindle poles throughout the cell cycle. Taken together, these results suggest that the yeast dynactin complex and cytoplasmic dynein together define a physiological pathway that is responsible for spindle translocation late in anaphase.

Saccharomyces cerevisiae genes required in the absence of the CIN8-encoded spindle motor act in functionally diverse mitotic pathways.

Kinesin-related Cin8p is the most important spindle-pole-separating motor in Saccharomyces cerevisiae but is not essential for cell viability. We identified 20 genes whose products are specifically required by cell deficient for Cin8p. All are associated with mitotic roles and represent at least four different functional pathways. These include genes whose products act in two spindle motor pathways that overlap in function with Cin8p, the kinesin-related Kip1p pathway and the cytoplasmic dynein pathway. In addition, genes required for mitotic spindle checkpoint function and for normal microtubule stability were recovered. Mutant alleles of eight genes caused phenotypes similar to dyn1 (encodes the dynein heavy chain), including a spindle-positioning defect. We provide evidence that the products of these genes function in concept with dynein. Among the dynein pathway gene products, we found homologues of the cytoplasmic dynein intermediate chain, the p150Glued subunit of the dynactin complex, and human LIS-1, required for normal brain development. These findings illustrate the complex cellular interactions exhibited by Cin8p, a member of a conserved spindle motor family.

Expression of the myc gene family in different stages of human colorectal cancer.

Colorectal carcinoma serves as a model system for the study of changes in gene expression and structure relating to tumorigenesis. In this study, the levels of c-myc, N-myc and L-myc mRNAs were assessed in normal human colonic mucosa in 33 cases representing different stages of adenocarcinoma and in 36 adenomatous polyps, the presumed premalignant stage. Consistent with the findings of Erisman et al. (1985), we found that the c-myc gene was overexpressed (3-24-fold) in approximately two-thirds of the tumors examined. Amplification of the gene (3-4-fold) was observed in 2 of 12 tumors examined and did not correlate with the level of expression. Greater amounts of c-myc-specific mRNA than occur in normal tissue was also found in about two-thirds of the polyps examined demonstrating that premalignant lesions also overexpress the gene. N-myc and L-myc specific transcripts can be detected at low abundance in normal colonic mucosa. These genes were found to be frequently overexpressed in tumors and polyps, but in most cases the level of overexpression was modest. A single case of adenocarcinoma showed an approximately 30-fold increase in the level of N-myc mRNA without gene amplification. Adenomatous polyps more frequently overexpressed the L-myc gene than tumors.

Saccharomyces cerevisiae PAC2 functions with CIN1, 2 and 4 in a pathway leading to normal microtubule stability.

The products of the Saccharomyces cerevisiae CIN1, CIN2 and CIN4 genes participate in a nonessential pathway required for normal microtubule function. In this article, we demonstrate that the product of PAC2 also functions in this pathway. PAC2 deletion mutants displayed phenotypes and genetic interactions similar to those caused by cin1 delta, cin2 delta and cin4 delta. These include cold-sensitive microtubule structures and sensitivity to the microtubule depolymerizing agent benomyl. Involvement in a common functional pathway is indicated by the observation that all double mutant recombinations are viable and no more affected than any single mutant. In addition, extra copies of CIN1 were found to suppress the benomyl sensitivity of pac2 delta, cin2 delta and cin4 delta, but not that caused by other mutations that affect microtubule function. Cin1p and Pac2p were found to be related in sequence to mammalian proteins that aid in the folding of beta-tubulin into an assembly-competent state. Alleles of CIN1 were identified that could suppress the benomyl sensitivity of cin4-4 in a highly specific fashion. Our findings suggest that the guanine nucleotide-binding Cin4p interacts with Cin1p and regulates its tubulin folding activity.

Effect of RBC shape and deformability on pulmonary O2 diffusing capacity and resistance to flow in rabbit lungs.

Isolated rabbit lungs were perfused with washed and resuspended human red blood cells (RBCs) in the presence of drugs known to change the shape and deformability of RBCs. With sodium salicylate (0.5-2 g/l), which causes echinocytosis and increases RBC deformability, lung diffusing capacity for O2 (DLO2) increased by 21%. When chlorpromazine, which induces stomatocytosis and stiffens RBCs, was given (50 mg/l), DLO2 decreased by 18%. With sodium salicylate, the mean pulmonary artery pressure dropped by 14% from control values, whereas it increased by 18% under chlorpromazine. Comparative experiments with hemoglobin solutions did not reveal any effect of those two drugs either on DLO2 or on pulmonary arterial pressure, which indicates that the effects of sodium salicylate and chlorpromazine were due to changes in RBC shape and deformability. It is concluded that RBC shape and deformability affect pulmonary artery pressure and oxygen diffusing capacity, which may have an influence on oxygen transfer to tissue and hence be of clinical relevance.

Molecular adaptations in human skeletal muscle to endurance training under simulated hypoxic conditions.

This study was performed to explore changes in gene expression as a consequence of exercise training at two levels of intensity under normoxic and normobaric hypoxic conditions (corresponding to an altitude of 3,850 m). Four groups of human subjects trained five times a week for a total of 6 wk on a bicycle ergometer. Muscle biopsies were taken, and performance tests were carried out before and after the training period. Similar increases in maximal O(2) uptake (8.3-13.1%) and maximal power output (11.4-20.8%) were found in all groups. RT-PCR revealed elevated mRNA concentrations of the alpha-subunit of hypoxia-inducible factor 1 (HIF-1) after both high- (+82.4%) and low (+78.4%)-intensity training under hypoxic conditions. The mRNA of HIF-1alpha(736), a splice variant of HIF-1alpha newly detected in human skeletal muscle, was shown to be changed in a similar pattern as HIF-1alpha. Increased mRNA contents of myoglobin (+72.2%) and vascular endothelial growth factor (+52.4%) were evoked only after high-intensity training in hypoxia. Augmented mRNA levels of oxidative enzymes, phosphofructokinase, and heat shock protein 70 were found after high-intensity training under both hypoxic and normoxic conditions. Our findings suggest that HIF-1 is specifically involved in the regulation of muscle adaptations after hypoxia training. Fine-tuning of the training response is recognized at the molecular level, and with less sensitivity also at the structural level, but not at global functional responses like maximal O(2) uptake or maximal power output.

Resistance of mammalian red blood cells of different size to hypertonic milieu.

1. The resistance of different mammalian red blood cells (RBCs) to hyperosmotic environments was studied. RBCs of six mammalian species were exposed to 10 increasingly hyperosmotic NaCl solutions for 24 hr at 5 degrees C. 2. The osmolality at which the amount of liberated haemoglobin reached a preset level (e.g. 3-4% of the total haemoglobin) showed a linear correlation with negative slope with RBC volume. This indicates that small RBCs are more resistant to hyperosmotic milieu than large ones. 3. A similar relation can be found from literature data when maximal urinary tonicities are plotted as a function of RBC volume, i.e. animals with the ability to produce highly concentrated urine have small RBCs. 4. RBC volume and maximal urinary tonicity in mammals are therefore tightly linked. Future research will have to show whether this correlation is fortuitous or not and whether, as can be speculated, RBC size is directly or indirectly regulated by the kidney.

Genetic analysis of the mitotic spindle.

Much of our understanding of the molecular basis of mitotic spindle function has been achieved within the past decade. Studies utilizing genetically tractable organisms have made important contributions to this field and these studies form the basis of this review. We focus upon three areas of spindle research: spindle poles, centromeres, and spindle motors. The structure and duplication mechanisms of spindle poles are considered as well as their roles in organizing spindle microtubules. Centromeres vary considerably in their size and complexity. We describe recent progress in our understanding of the relatively simple centromeres of the yeast Saccharomyces cerevisiae and the complex centromeres that are more typical of eukaryotic cells. Microtubule-based motor proteins that generate the characteristic spindle movements have been identified in recent years and can be grouped into families defined by conserved primary sequence and mitotic function.

Simultaneous O2 and CO diffusing capacity estimates from assumed lognormal VA, Q and DL distributions.

O2 and CO pulmonary transfer data obtained in dogs under steady-state conditions in hypoxia by Savoy et al. (Respir. Physiol. 42: 43-59, 1980) have been submitted to reevaluation and have yielded new estimates of the lung diffusing capacity, DL. For the proposed DL computation it has been assumed that functional inhomogeneity can be considered as resulting from lognormal distributions of the VA/Q and VA/DL ratios. The standard deviation, sigma, of the VA/Q distribution is computed from the measured (PA -Pa)CO2, and the same sigma value is assumed to prevail for the VA/DL distributions. This is equivalent to assume constant DL/Q ratios in the entire lung. With the so defined distributions, DL values, called D sigma O2 and D sigma CO, were sought, for which the model calculations yielded O2 partial pressures and CO fluxes equal to those measured. Compared with DL estimates computed with conventional procedures, these results show that D sigma O2 is twice as large as DLO2 computed with ideal alveolar PO2 and that D sigma CO lies between DLCO computed with the mean alveolar PCO and that computed with the ideal alveolar PCO. The D sigma O2/D sigma CO ratio was on the average 1.2, a value which, unlike the ratios obtained with conventional DLO2 and DLCO estimates, is in good agreement with the characteristics of diffusion and of chemical association of O2 and CO with blood.

Expression of the mouse metallothionein-I and -II genes provides a reproductive advantage during maternal dietary zinc deficiency.

The function of metallothionein in zinc homeostasis was examined by using mice homozygous for knockout (KO) of the metallothionein-I or -II (MT-I and MT-II) genes. Pregnant MT-I/II KO mice or control mice were fed a zinc-deficient (1 microg/g or 5 microg/g) diet or a zinc-adequate (50 microg/g) diet during specific periods of pregnancy, and the effects on morphogenesis of the embryos were determined at day 14 of pregnancy (day 1 = vaginal plug). In the homozygous MT-I/II KO, as well as in the nontransgenic control mice, severe dietary zinc deficiency (1 microg/g) beginning on day 1 of pregnancy was embryotoxic and teratogenic, and the majority of the embryos in both strains were dead by mid-gestation. However, 53% of the surviving embryos in the MT-I/II KO mice were morphologically abnormal compared to only 32% of the embryos in the control mice. In subsequent experiments, moderate dietary zinc deficiency (5 microg/g beginning on day 1 of pregnancy or 1 microg/g dietary zinc beginning on day 8 of pregnancy) exerted teratogenic, but not embryotoxic effects. Embryos in the MT-I/II KO mice were 260 to 290% as likely to develop abnormally than were embryos in the control mice fed these same diets. These results demonstrate that the expression of the MT-I and -II genes in pregnant females improves reproductive success during maternal dietary zinc deficiency.

Pulmonary O2 diffusing capacity estimates from assumed log-normal VA/Q distributions.

Steady-state pulmonary gas exchange has been measured in hypoxia in 33 mongrel dogs with the aim of comparing DLO2 estimates obtained with three procedures differing by the models assumed for functional inhomogeneity. In the first procedure the lung was assumed to be homogeneous and the corresponding DLO2 estimate was 15 mumol . min-1. Torr-1 . kg-1. In the second procedure, which is the one commonly used in respiratory physiology, alveolar dead space was considered as the unique form of functional inhomogeneity and the corresponding DLO2 estimate was 31 mumol . min-1. Torr-1 . kg-1. In the third procedure, which has been specially worked out for this study, functional inhomogeneity was represented by a log-normal distribution of the VA/Q ratios and the corresponding DLO2 estimate was 50 mumol . min-1 . Torr-1 . kg-1. The relation between the DLO2 estimates by the second and by the third procedures was found to depend upon the blood pH. This could be explained on the basis of the effects of acidosis on the blood capacitances for O2 and for CO2. Analysis suggests that in hypoxia where normally the O2 capacitance is about half the CO2 one, the third procedure yields DLO2 estimates about twice as large as those obtained by the second one.

Closed system venography in the evaluation of upper extremity hemangiomas.

Closed system venography is a technically simple radiographic technique of minimal patient morbidity and inconvenience that has proven to be useful in evaluating the extent of upper extremity hemangiomata and in planning its treatment. The only apparent contraindication to the technique is allergy to the radiopaque dye. In a recent series of cases, this technique of venography has yielded more information than has arteriography.

Gas transfer in isolated lungs perfused with red cell suspension or hemoglobin solution.

Rapid mixing experiments have shown that the reaction between oxygen and hemoglobin is faster in hemoglobin solutions than in red cell suspensions. In this study we tested whether this observation can also be made in the lung. Excised rabbit lungs were perfused either with washed human red cell suspensions or with hemoglobin solutions, each with 50 g hemoglobin/L, and steady-state diffusing capacities (DLO2) for oxygen elimination measured. Mean settings were a temperature of 29.5 degrees C of the inflowing and outflowing perfusate of the lung, a total ventilation of 1.7 L.min-1, and a perfusion rate of 116 ml.min-1. Under those conditions resulted a DLO2 with hemoglobin solution of 0.68 +/- 0.18 ml.min-1.mm Hg-1, and a significantly lower value of 0.50 +/- 0.06 ml.min-1.mm Hg-1 with red cell suspension (P less than 0.01). An extraerythrocytic diffusing resistance, formed by a plasma layer and/or arising from a dynamic diffusion boundary layer, which is also known as unstirred layer, could explain the lower value with red cell suspensions.

Lung diffusing capacity and red blood cell volume.

The aim of this study was to test whether a large surface to volume ratio of the red blood cells is advantageous for O2 transfer in lungs. Suspensions of washed large (human) and small (sheep) red blood cells were used to perfuse in random order isolated cat lungs (n = 7). The lungs were kept at 27 degrees C, ventilated with 1.0 l/min and perfused with 110 ml/min. Hemoglobin concentration was 70 g/l. Assuming an inhomogeneous lung with an alveolar dead space compartment, pulmonary diffusing capacity for O2 (DLO2) was 0.53 +/- 0.10 ml.min-1.mmHg-1 (mean +/- SD) when perfusing with sheep red cells and 0.52 +/- 0.13 with human red cells. On the basis of a lung model with a low VA/Q and a high VA/Q compartment the values were 0.86 and 0.93. Thus, DLO2 was not larger with smaller red blood cells, i.e. with higher red cell surface area per blood volume. Possible explanations for this unexpected result are discussed.