Failure of cell cleavage induces senescence in tetraploid primary cells

Tetraploidy can arise from various mitotic or cleavage defects in mammalian cells, and inheritance of multiple centrosomes induces aneuploidy when tetraploid cells continue to cycle. Arrest of the tetraploid cell cycle is therefore potentially a critical cellular control. We report here that primary rat embryo fibroblasts (REF52) and human foreskin fibroblasts become senescent in tetraploid G1 after drug- or small interfering RNA (siRNA)-induced failure of cell cleavage. In contrast, T-antigen–transformed REF52 and p53+/+ HCT116 tumor cells rapidly become aneuploid by continuing to cycle after cleavage failure. Tetraploid primary cells quickly become quiescent, as determined by loss of the Ki-67 proliferation marker and of the fluorescent ubiquitination-based cell cycle indicator/late cell cycle marker geminin. Arrest is not due to DNA damage, as the γ-H2AX DNA damage marker remains at control levels after tetraploidy induction. Arrested tetraploid cells finally become senescent, as determined by SA-β-galactosidase activity. Tetraploid arrest is dependent on p16INK4a expression, as siRNA suppression of p16INK4a bypasses tetraploid arrest, permitting primary cells to become aneuploid. We conclude that tetraploid primary cells can become senescent without DNA damage and that induction of senescence is critical to tetraploidy arrest.     

Relationship between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related proteins.

Based on the recently determined X-ray structures of Torpedo californica acetylcholinesterase and Geotrichum candidum lipase and on their three-dimensional superposition, an improved alignment of a collection of 32 related amino acid sequences of other esterases, lipases, and related proteins was obtained. On the basis of this alignment, 24 residues are found to be invariant in 29 sequences of hydrolytic enzymes, and an additional 49 are well conserved. The conservation in the three remaining sequences is somewhat lower. The conserved residues include the active site, disulfide bridges, salt bridges, and residues in the core of the proteins. Most invariant residues are located at the edges of secondary structural elements. A clear structural basis for the preservation of many of these residues can be determined from comparison of the two X-ray structures.     

The plasma membrane H+-ATPase gene family in Arabidopsis: genomic sequence of AHA10 which is expressed primarily in developing seeds

The plasma membrane H+-ATPases in Arabidopsis thaliana represent the largest family of cation translocating P-type ATPases identified in plants or animals. We report here seven new isoforms, which were identified by polymerase chain reaction (PCR) amplification of genomic DNA. Amplifications were performed with degenerate primers corresponding to two short conserved sequence motifs (CSDK and GDGV) found in most P-type ATPases. A comparison was made of three CSDK-side primers, which were used either as totally degenerate mixtures or rendered less degenerate by substitution with deoxyinosine or fluorodeoxyuridine. Amplified genomic fragments were cloned, partially sequenced and shown to correspond to Arabidopsis genes by Southern blot analysis with gene-specific probes. One newly identified isoform, AHA10, was isolated as a cosmid clone and sequenced. The 5 and 3 ends of the gene were determined by comparison with the AHA10 cDNA sequence. AHA10 is the most divergent isoform characterized in the Arabidopsis family. AHA10 appears to be expressed primarily in developing seeds, as indicated by Northern blot analysis of AHA10 mRNA and by the analysis of transgenic plants expressing a -glucuronidase (GUS) reporter gene fused to an AHA10 promoter. Our results indicate that one function of this unusually large H⁺-ATPase gene family is to allow for expression of different isoforms in different cell types.     

A comparison of salivary pH in sympatric wild lemurs (Lemur catta and Propithecus verreauxi) at Beza Mahafaly Special Reserve, Madagascar

Chemical deterioration of teeth is common among modern humans, and has been suggested for some extinct primates. Dental erosion caused by acidic foods may also obscure microwear signals of mechanical food properties. Ring-tailed lemurs at the Beza Mahafaly Special Reserve (BMSR), Madagascar, display frequent severe tooth wear and subsequent tooth loss. In contrast, sympatric Verreaux's sifaka display far less tooth wear and infrequent tooth loss, despite both species regularly consuming acidic tamarind fruit. We investigated the potential impact of dietary acidity on tooth wear, collecting data on salivary pH from both species, as well as salivary pH from ring-tailed lemurs at Tsimanampesotse National Park, Madagascar. We also collected salivary pH data from ring-tailed lemurs at the Indianapolis Zoo, none of which had eaten for at least 12 hr before data collection. Mean salivary pH for the BMSR ring-tailed lemurs (8.098, n=41, SD=0.550) was significantly more alkaline than Verreaux's sifaka (7.481, n=26, SD=0.458). The mean salivary pH of BMSR (8.098) and Tsimanampesotse (8.080, n=25, SD=0.746) ring-tailed lemurs did not differ significantly. Salivary pH for the Indianapolis Zoo sample (8.125, n=16, SD=0.289) did not differ significantly from either the BMSR or Tsimanampesotse ring-tailed lemurs, but was significantly more alkaline than the BMSR Verreaux's sifaka sample. Regardless of the time between feeding and collection of pH data (from several minutes to nearly 1 hr), salivary pH for each wild lemur was above the "critical" pH of 5.5, below which enamel demineralization occurs. Thus, the high pH of lemur saliva suggests a strong buffering capacity, indicating the impact of acidic foods on dental wear is short-lived, likely having a limited effect. However, tannins in tamarind fruit may increase friction between teeth, thereby increasing attrition and wear in lemurs. These data also suggest that salivary pH varies between lemur species, corresponding to broad dietary categories.     

Conformational instability of the N- and C-terminal lobes of porcine pepsin in neutral and alkaline solutions.

Pepsin contains, in a single chain, two conformationally homologous lobes that are thought to have been evolutionarily derived by gene duplication and fusion. We have demonstrated that the individual recombinant lobes are capable of independent folding and reconstitution into a two-chain pepsin or a two-chain pepsinogen (Lin, X., et al., 1992, J. Biol. Chem. 267, 17257-17263). Pepsin spontaneously inactivates in neutral or alkaline solutions. We have shown in this study that the enzymic activity of the alkaline-inactivated pepsin was regenerated by the addition of the recombinant N-terminal lobe but not by the C-terminal lobe. These results indicate that alkaline inactivation of pepsin is due to a selective denaturation of its N-terminal lobe. A complex between recombinant N-terminal lobe of pepsinogen and alkaline-denatured pepsin has been isolated. This complex is structurally similar to a two-chain pepsinogen, but it contains an extension of a denatured pepsin N-terminal lobe. Acidification of the complex is accompanied by a cleavage in the pro region and proteolysis of the denatured N-terminal lobe. The structural components that are responsible for the alkaline instability of the N-terminal lobe are likely to be carboxyl groups with abnormally high pKa values. The electrostatic potentials of 23 net carboxyl groups in the N-terminal domain (as compared to 19 in the C-terminal domain) of pepsin were calculated based on the energetics of interacting charges in the tertiary structure of the domain. The groups most probably causing the alkaline denaturation are Asp11, Asp159, Glu4, Glu13, and Asp118.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of Bax and caspase-3 in doppel-induced apoptosis of cerebellar granule cells

Doppel (Dpl) protein is a paralog of the prion protein (PrP) that shares 25% sequence similarity with the C-terminus of PrP, a common N-glycosylation site and a C-terminal signal peptide for attachment of a glycosylphophatidyl inositol anchor. Whereas PrP^C is highly expressed in the central nervous system (CNS), Dpl is detected mostly in testes and its ectopic expression in the CNS leads to ataxia as well as Purkinje and granule cell degeneration in the cerebellum. The mechanism through which Dpl induces neurotoxicity is still debated. In the present work, primary neuronal cultures derived from postnatal cerebellar granule cells of wild-type and PrP-knockout FVB mice were used in order to investigate the molecular events that occur upon exposure to Dpl. Treatment of cultured cerebellar neurons with recombinant Dpl produced apoptosis that could be prevented by PrP co-incubation. When primary neuronal cultures from Bax-deficient mice were incubated with Dpl, no apoptosis was observed, suggesting an important role of Bax in triggering neurodegeneration. Similarly, cell survival increased when recDpl-treated cells were incubated with an inhibitor of caspase-3, which mediates apoptosis in mammalian cells. Together, our findings raise the possibility that Bax and caspase-3 feature in Dpl-mediated apoptosis.     

Electrooptical measurements demonstrate a large permanent dipole moment associated with acetylcholinesterase.

Acetylcholinesterase (AChE) from krait (Bungarus fasciatus) venom is a soluble, nonamphiphilic monomer of 72 kDa. This snake venom AChE has been analyzed by measurements of the stationary and the transient electric dichroism at different field strengths. The stationary values of the dichroism are consistent with the orientation function for permanent dipoles and are not consistent with the orientation function for induced dipoles. The permanent dipole moment obtained by least-squares fits for a buffer containing 5 mM MES is 1000 D, after correction for the internal directing field, assuming a spherical shape of the protein. The dipole moment decreases with increasing buffer concentration to 880 D at 10 mM MES and 770 D at 20 mM MES. The dichroism decay time constant is 90 ns (+/- 10%) which is clearly larger than the value expected from the size/shape of the protein and indicates contributions from sugar residues attached to the protein. The dichroism rise times observed at low field strengths are larger than the decay times and, thus, support the assignment of a permanent dipole moment, although it has not been possible to approach the limit where the energy of the dipole in the electric field is sufficiently low compared to kT. The experimental value of the permanent dipole moment is similar to that calculated for a model structure of Bungarus fasciatus AChE, which has been constructed from its amino and acid sequence, in analogy to the crystal structure of AChE from Torpedo californica.     

X-ray structure of human acid-beta-glucosidase,the defective enzyme in Gaucher disease

Gaucher disease, the most common lysosomal storage disease, is caused by mutations in the gene that encodes acid-β-glucosidase (GlcCerase). Type 1 is characterized by hepatosplenomegaly, and types 2 and 3 by early or chronic onset of severe neurological symptoms. No clear correlation exists between the ~200 GlcCerase mutations and disease severity, although homozygosity for the common mutations N370S and L444P is associated with non- neuronopathic and neuronopathic disease, respectively. We report the X-ray structure of GlcCerase at 2.0 Å resolution. The catalytic domain consists of a (β/α)₈ TIM barrel, as expected for a member of the glucosidase hydrolase A clan. The distance between the catalytic residues E235 and E340 is consistent with a catalytic mechanism of retention. N370 is located on the longest α-helix (helix 7), which has several other mutations of residues that point into the TIM barrel. Helix 7 is at the interface between the TIM barrel and a separate immunoglobulin-like domain on which L444 is located, suggesting an important regulatory or structural role for this non-catalytic domain. The structure provides the possibility of engineering improved GlcCerase for enzyme-replacement therapy, and for designing structure-based drugs aimed at restoring the activity of defective GlcCerase.     

A role for PI 3-kinase and PKB activity in the G2/M phase of the cell cycle

The role of the PI 3-kinase cascade in regulation of cell growth is well established [1]. PKB (protein kinase B) is a key downstream effector of the PI 3-kinase pathway and is best known for its antiapoptotic effects [2,3] and the role it plays in initiation of S phase [4]. Here, we show that PKB activity is high in the G2/M phase of the cell cycle in epithelial cells. Inhibition of the PI 3-kinase pathway in MDCK cells induces apoptosis at the G2/M transition, prevents activation of cyclin B-associated kinase, and prohibits entry of the surviving cells into mitosis. All of these consequences of the inhibition of PI 3-kinase are relieved by expression of a constitutively active form of PKB (caPKB), indicating that PKB plays a role in regulation of the G2/M phase. Inhibition of PI 3-kinase results in activation of Chk1, whereas caPKB inhibits the ability of Chk1 to become activated in response to treatment with hydroxyurea. Preliminary data show that PKB phosphorylates the Chk1 polypeptide in vitro on serine 280. These results not only implicate PKB activity in transition through the G2/M stage of the cell cycle, but they also suggest the existence of crosstalk between the PI 3-kinase pathway and the key regulators of the DNA damage checkpoint machinery.