Altered longevity‐assurance activity of p53:p44 in the mouse causes memory loss,neurodegeneration and premature death

The longevity‐assurance activity of the tumor suppressor p53 depends on the levels of Δ40p53 (p44), a short and naturally occurring isoform of the p53 gene. As such, increased dosage of p44 in the mouse leads to accelerated aging and short lifespan. Here we show that mice homozygous for a transgene encoding p44 (p44^+/+) display cognitive decline and synaptic impairment early in life. The synaptic deficits are attributed to hyperactivation of insulin‐like growth factor 1 receptor (IGF‐1R) signaling and altered metabolism of the microtubule‐binding protein tau. In fact, they were rescued by either Igf1r or Mapt haploinsufficiency. When expressing a human or a ‘humanized’ form of the amyloid precursor protein (APP), p44^+/+ animals developed a selective degeneration of memory‐forming and ‐retrieving areas of the brain, and died prematurely. Mechanistically, the neurodegeneration was caused by both paraptosis‐ and autophagy‐like cell deaths. These results indicate that altered longevity‐assurance activity of p53:p44 causes memory loss and neurodegeneration by affecting IGF‐1R signaling. Importantly, Igf1r haploinsufficiency was also able to correct the synaptic deficits of APP_695/swe mice, a model of Alzheimer’s disease.     

Disease-associated mutations in the extracytoplasmic loops of cystic fibrosis transmembrane conductance regulator do not impede biosynthetic processing but impair chloride channel stability

Consistent with its function as a chloride channel regulated entirely from the cytoplasmic side of the plasma membrane, the cystic fibrosis transmembrane conductance regulator (CFTR) glycoprotein exposes little of its mass on the exterior surface of cells. The first and fourth extracytoplasmic loops (ELs) contain approximately 15 and 30 residues, respectively; the other four ELs are extremely short. To examine the influence of missense mutants in ELs detected in patients with cystic fibrosis, we have expressed them in mammalian (baby hamster kidney (BHK21)) cells and assessed their biosynthetic processing and chloride channel activity. In contrast to previous findings that 18 of 30 disease-associated missense mutations in cytoplasmic loops caused retention of the nascent polypeptides in the endoplasmic reticulum, all the EL mutants studied matured and were transported to the cell surface. This pronounced asymmetry is consistent with the notion that endoplasmic reticulum quality control of nascent CFTR is exerted primarily on the cytoplasmic side of the membrane. Although this set of EL mutations has little effect on CFTR maturation, most of them seriously compromise its chloride channel activity. Substitutions at six different positions in EL1 and single positions in EL2 and EL4 all destabilized the open state, some of them severely, indicating that the ELs contribute to the stability of the CFTR ion pore.     

The PDZ-binding chloride channel ClC-3B localizes to the Golgi and associates with cystic fibrosis transmembrane conductance regulator-interacting PDZ proteins

ClC chloride channels are widely distributed in organisms across the evolutionary spectrum, and members of the mammalian family play crucial roles in cellular function and are mutated in several human diseases (Jentsch, T. J., Stein, V., Weinreich, F., and Zdebik, A. A. (2002) Physiol. Rev. 82, 503-568). Within the ClC-3, -4, -5 branch of the family that are intracellular channels, two alternatively spliced ClC-3 isoforms were recognized recently (Ogura, T., Furukawa, T., Toyozaki, T., Yamada, K., Zheng, Y. J., Katayama, Y., Nakaya, H., and Inagaki, N. (2002) FASEB J. 16, 863-865). ClC-3A resides in late endosomes where it serves as an anion shunt during acidification. We show here that the ClC-3B PDZ-binding isoform resides in the Golgi where it co-localizes with a small amount of the other known PDZ-binding chloride channel, CFTR (cystic fibrosis transmembrane conductance regulator). Both channel proteins bind the Golgi PDZ protein, GOPC (Golgi-associated PDZ and coiled-coil motif-containing protein). Interestingly, however, when overexpressed, GOPC, which is thought to influence traffic in the endocytic/secretory pathway, causes a large reduction in the amounts of both channels, probably by leading them to the degradative end of this pathway. ClC-3B as well as CFTR also binds EBP50 (ERM-binding phosphoprotein 50) and PDZK1, which are concentrated at the plasma membrane. However, only PDZK1 was found to promote interaction between the two channels, perhaps because they were able to bind to two different PDZ domains in PDZK1. Thus while small portions of the populations of ClC-3B and CFTR may associate and co-localize, the bulk of the two populations reside in different organelles of cells where they are expressed heterologously or endogenously, and therefore their cellular functions are likely to be distinct and not primarily related.     

Binding of [3H]cytochalasin B and [3H]colchicine to isolated liver plasma membranes

The binding to isolated hepatocyte plasma membranes of radioactively labelled inhibitors of microfilamentous and microtubular protein function ([³H]cytochalasin B and [³H]colchicine, respectively) was studied as one means of assessing the degree of association of these proteins with cell surface membranes. [³H]Cytochalasin B which behaved identically to the unlabelled compound with respect to binding to these membranes was prepared by reduction of cytochalasin A with NaB³H₄. The binding was rapid, readily reversible, proportional to the amount of membrane and relatively insentive to changes of pH or ionic strength. At 10^?6 M [³H]cytochalasin B, glucose or p-chloromercuribenzoate, an inhibitor of glucose transport inhibited binding by about 20%; treatment of membranes with 0.6 M KI which depolymerizes F actin to G actin caused about 60% inhibition of binding. These two types of inhibition were additive indicating two separate classes of binding sites, one associated with sugar transport and one with microfilaments. Filamentous structures with the diameter of microfilaments (50 Å) were seen in electron micrographs of thin sections of the membranes. At concentrations greater than 10^?5 M [³H]cytochalasin B, binding was proportional to drug concentration, characteristic of non-specific adsorption or partitioning. Intracellular membranes of the hepatocyte also bound [³H]cytochalasin B, those of the smooth endoplasmic reticulum to a greater extent than plasma membranes.[³H]Colchicine bound to plasma membranes in proportion to the amount of membrane and at a rate compatible with binding to tubulin. However, other properties of the binding including effects of temperature, drug concentration and antisera against tubulin were different from those of binding to tubulin. Hence, no evidence was obtained for association of microtubular elements with these membranes. Despite this there appeared to be an interdependence between microtubule and microfilament inhibitors: vinblastine sulfate stimulated [³H]cytochalasin B binding and cytochalasin B stimulated ³H colchicine binding. [³H]Colchicine also bound to intracellular membranes, especially smooth microsomes.     

Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR).

Circumstantial evidence has accumulated suggesting that CFTR is a regulated low-conductance Cl- channel. To test this postulate directly, we have purified to homogeneity a recombinant CFTR protein from a high-level baculovirus-infected insect cell line. Evidence of purity included one- and two-dimensional gel electrophoresis, N-terminal peptide sequence, and quantitative amino acid analysis. Reconstitution into proteoliposomes at less than one molecule per vesicle was accomplished by established procedures. Nystatin and ergosterol were included in these vesicles, so that nystatin conductance could serve as a quantitative marker of vesicle fusion with a planar lipid bilayer. Upon incorporation, purified CFTR exhibited regulated chloride channel activity, providing evidence that the protein itself is the channel. This activity exhibited the basic biophysical and regulatory properties of the type of Cl- channel found exclusively in CFTR-expressing cell types and believed to underlie cAMP-evoked secretion in epithelial cells.     

Chromosome analysis in chorionic villi samples from the first trimester elective terminations

Chorionic villi sampling (CVS) has become a first trimester alternative to amniocentesis for prenatal diagnosis. The cytogenetic findings in 150 experimental samples are presented. The ages of the mothers ranged from 12 to 35 years, but the majority of them were 18 and 19 years of age. Various parameters of culturing and processing the samples in order to improve the method, were investigated. Short term incubation for 48 h was the method routinely employed in processing the biopsies for cytogenetic analysis. In the first series of 100 cases one mosaic case (46,XX/45,X), one Robertsonian translocation (13;14), one marker chromosome and one fragment were found. The foetal tissues were not analysed for chromosomes. In the second series of 50 samples, one case of mosaicism was found in the chorionic villi (46,XX/47,XX, 18q-), but this abnormality was absent in the foetal tissue. One variant inv(9) was observed in the foetal tissue as well as in the chorionic villi. In all other cases the karyotypes from the chorionic villi samples matched those of the corresponding foetal samples. There was no maternal contamination in this series of 50 samples. The discrepancies in the cytogenetic results from other investigators are discussed.     

A molecular and ultrastructural description of Spathidiopsis buddenbrocki and the phylogenetic position of the family Placidae (Ciliophora)

Spathidiopsis and Placus are the only two genera within the family Placidae. The family has been placed in the class Prostomatea and order Prorodontida because its members have somatic monokinetids with a radial transverse ribbon, a straight non‐overlapping postciliary ribbon, and anteriorly directed non‐overlapping kinetodesmal fibril, an apical cytostome lacking specialized oral cilia, a brosse, and toxicysts. To confirm the stability of this placement, ultrastructural morphology and small subunit rRNA gene sequences of Spathidiopsis socialis, Spathidiopsis buddenbrocki, and Placus striatus were determined. These data were combined with information from other ciliates, and phylogenetic trees were generated using maximum‐likelihood and maximum‐parsimony methods. The analyses confirmed the family Placidae to be a monophyletic group in the Prostomatea with the Placidae a sister group to a Cryptocaryon + Coleps + Prorodon clade.