Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites

Pulse-chase experiments have revealed that cyclin, the auxiliary protein of DNA polymerase-delta, is stable during the transition from growth to quiescence in 3T3 cells. Immunoblotting together with immunofluorescence analysis has shown that the amount of cyclin after 24 h of quiescence is 30-40% of that of growing cells and that it presents a nucleoplasmic staining. Immunofluorescence studies show the existence of two populations of cyclin during the S phase, one that is nucleoplasmic as in quiescent cells and is easily extracted by detergent, and another that is associated to specific nuclear structures. By using antibromodeoxyuridine immunofluorescence to detect the sites of DNA synthesis, it was shown that the staining patterns of the replicon clusters and their order of appearance throughout the S phase are identical to those observed for cyclin. Two-dimensional gel analysis of Triton-extracted cells show that 20-30% of cyclin remains associated with the replicon clusters. This population of cyclin could not be released from the nucleus using high-salt extractions. This demonstrates that cyclin is tightly associated to the sites of DNA replication and that it must have a fundamental role in DNA synthesis in eukaryotic cells.     

Heparin Dodecasaccharide Containing Two Antithrombin-binding Pentasaccharides: STRUCTURAL FEATURES AND BIOLOGICAL PROPERTIES

The antithrombin (AT) binding properties of heparin and low molecular weight heparins are strongly associated to the presence of the pentasaccharide sequence AGA*IA (A_NAc,6S-GlcUA-A_NS,3,6S-I_2S-A_NS,6S). By using the highly chemoselective depolymerization to prepare new ultra low molecular weight heparin and coupling it with the original separation techniques, it was possible to isolate a polysaccharide with a biosynthetically unexpected structure and excellent antithrombotic properties. It consisted of a dodecasaccharide containing an unsaturated uronate unit at the nonreducing end and two contiguous AT-binding sequences separated by a nonsulfated iduronate residue. This novel oligosaccharide was characterized by NMR spectroscopy, and its binding with AT was determined by fluorescence titration, NMR, and LC-MS. The dodecasaccharide displayed a significantly increased anti-FXa activity compared with those of the pentasaccharide, fondaparinux, and low molecular weight heparin enoxaparin.     

The stimulatory effect of human chorionic gonadotropin on amino acid uptake by amphibian follicles.

Human chorionic gonadotropin (hCG) stimulates the uptake of eight different amino acids and four nucleosides by Xenopus laevis ovarian follicles. This hormone also stimulates amino acid uptake in the follicles of another amphibian, Callyptocephallela caudiverbera. The stimulation of uptake is due to a reduction in the amino acid concentration required for half-maximal uptake velocity and not to an increment in V_max. The effect of hCG does not require protein synthesis but requires physiological conditions of temperature and pH. Incorporation of radioactive exogenous amino acid into proteins is also stimulated by the hormone, but high-resolution electrophoresis shows that there are no drastic qualitative changes in the pattern of proteins synthesized at early times after hCG treatment. The effect of hCG on the uptake of exogenous amino acids does not appear to be required for oocyte maturation because other hormones such as progesterone and testosterone which induce maturation do not increase amino acid uptake. Also the concentration of hCG required for oocyte maturation is significantly lower than that required for an effect on amino acid transport. Inhibitors of oocyte maturation such as theophylline and cycloheximide do not inhibit the action of hCG on amino acid uptake by the amphibian follicles.     

A single amino acid substitution within a coiled-coil motif changes the assembly of a 53-amino acid protein from two-dimensional sheets to filamentous structures

The bacteriophage phi29 replication protein p1 self-interacts in vitro, generating highly ordered structures. Specifically, the 53-amino acid protein p1DeltaN33, which retains the sequence of p1 spanning amino acids Met(34) to Lys(85), assembles into two-dimensional protofilament sheets. The region of protein p1 located between residues Glu(38) and Asn(65) presumably forms an alpha-helical coiled-coil structure. Here we have examined the role of this coiled-coil sequence in the formation of protofilament sheets. Using sedimentation assays and negative-stain electron microscopy analysis, we demonstrate that residues Leu(46), Met(53), and Leu(60), but not Leu(39), are essential for p1DeltaN33 assembly into sheets. Remarkably, replacement of Leu(46) by Val shifts the pathway of molecular assembly, leading to the formation of filamentous polymers approximately 10 nm in diameter. These results show, for the first time, that a short coiled-coil motif can mediate protein assembly into protofilament sheet structures.     

Substrate flow in catalases deduced from the crystal structures of active site variants of HPII from Escherichia coli.

The active site of heme catalases is buried deep inside a structurally highly conserved homotetramer. Channels leading to the active site have been identified as potential routes for substrate flow and product release, although evidence in support of this model is limited. To investigate further the role of protein structure and molecular channels in catalysis, the crystal structures of four active site variants of catalase HPII from Escherichia coli (His128Ala, His128Asn, Asn201Ala, and Asn201His) have been determined at approximately 2.0-A resolution. The solvent organization shows major rearrangements with respect to native HPII, not only in the vicinity of the replaced residues but also in the main molecular channel leading to the heme distal pocket. In the two inactive His128 variants, continuous chains of hydrogen bonded water molecules extend from the molecular surface to the heme distal pocket filling the main channel. The differences in continuity of solvent molecules between the native and variant structures illustrate how sensitive the solvent matrix is to subtle changes in structure. It is hypothesized that the slightly larger H(2)O(2) passing through the channel of the native enzyme will promote the formation of a continuous chain of solvent and peroxide. The structure of the His128Asn variant complexed with hydrogen peroxide has also been determined at 2.3-A resolution, revealing the existence of hydrogen peroxide binding sites both in the heme distal pocket and in the main channel. Unexpectedly, the largest changes in protein structure resulting from peroxide binding are clustered on the heme proximal side and mainly involve residues in only two subunits, leading to a departure from the 222-point group symmetry of the native enzyme. An active role for channels in the selective flow of substrates through the catalase molecule is proposed as an integral feature of the catalytic mechanism. The Asn201His variant of HPII was found to contain unoxidized heme b in combination with the proximal side His-Tyr bond suggesting that the mechanistic pathways of the two reactions can be uncoupled.     

Fanconi's anemia lymphocytes: effect of caffeine, adenosine and niacinamide during G2 prophase

In this investigation peripheral blood lymphocytes from 3 Fanconi's anemia (FA) patients, 2 FA heterozygotes and 4 normal subjects were treated with caffeine and/or adenosine, and/or niacinamide during G2 prophase. Caffeine dramatically increased breakage levels in homozygote and heterozygote cells. Niacinamide and adenosine decreased the amount of chromosomal aberrations detected in FA homozygote and heterozygote lymphocytes treated and untreated with caffeine during G2 prophase. Caffeine sensitivity of heterozygote lymphocytes is proposed as a new clinical test to explore heterozygosis in individuals of FA families.     

A STUDY OF THE SEXUAL REPRODUCTION AND DETERMINATION OF MATING TYPE OF GYMNODINIUM NOLLERI (DINOPHYCEAE) IN CULTURE1

Sexual reproduction of Gymnodinium nolleri ( Ellegaard & Moestrup 1999 ) was studied by intercrossing experiments in all combinations of six clonal strains and backcrossing of five clonal F1 offspring. The results indicated that the conjugation of G. nolleri responded to the existence of more than two sexual types (complex heterothallism) and that compatibility between progeny of one cyst (inbreeding) was the rule. Sexual fusion, planozygote formation and development, cyst formation, and germination and planomeiocyte division were followed using time‐lapse photography. This study revealed many similarities between the sexual stages and life cycle pattern of G. nolleri and the related G. catenatum and the existence under culture conditions of an alternative cycle between vegetative cells and zygotes without a hypnozygote stage. The fate of zygotes, division or encystment, was influenced by the nutritional status of the external medium. The division of G. nolleri planozygotes was promoted by high levels of external nutrients, whereas the maximum percentage of encystment was recorded when phosphates were reduced in the isolation medium. The division of zygotes might be different from both vegetative and planomeiocyte division because it resulted in two‐cell chains with the cells not oriented in parallel.     

Overexpression of ATP Sulfurylase in Indian Mustard Leads to Increased Selenate Uptake, Reduction, and Tolerance

In earlier studies, the assimilation of selenate by plants appeared to be limited by its reduction, a step that is thought to be mediated by ATP sulfurylase. Here, the Arabidopsis APS1 gene, encoding a plastidic ATP sulfurylase, was constitutively overexpressed in Indian mustard (Brassica juncea). Compared with that in untransformed plants, the ATP sulfurylase activity was 2- to 2.5-fold higher in shoots and roots of transgenic seedlings, and 1.5- to 2-fold higher in shoots but not roots of selenate-supplied mature ATP-sulfurylase-overexpressing (APS) plants. The APS plants showed increased selenate reduction: x-ray absorption spectroscopy showed that root and shoot tissues of mature APS plants contained mostly organic Se (possibly selenomethionine), whereas wild-type plants accumulated selenate. The APS plants were not able to reduce selenate when shoots were removed immediately before selenate was supplied. In addition, Se accumulation in APS plants was 2- to 3-fold higher in shoots and 1.5-fold higher in roots compared with wild-type plants, and Se tolerance was higher in both seedlings and mature APS plants. These studies show that ATP sulfurylase not only mediates selenate reduction in plants, but is also rate limiting for selenate uptake and assimilation.     

Cria alpaca body weight and perinatal survival in relation to age of the dam

The present study with alpacas determined effect of dam's age on body weight and survival of cria during the first week of life. Pregnant dams (n=424) and their crias were used in the study. Cria body weight (kg) was determined at time of placenta expulsion. Placenta weight and larger width were measured immediately after expulsion. Crias were monitored for the first week and a necropsy was performed if death occurred. Data were analyzed using analysis of variance. The body weight of crias at birth, the weight, and the largest width of placenta increased with age of the dam (P<0.05), reaching a peak at 9 years and then declined progressively. Placental efficiency also increased with the dam's age, and showed a bimodal shape, peaking at 6- and 11-year-old dams (P<0.05). Altogether, 398 crias survived and 26 died; of those 6 died of starvation, 5 of hypothermia, 4 were stillborn and the rest from other miscellaneous causes. More crias died from 2-year-old dams than from dams of any other age (P<0.05). In addition, dead crias had lesser body weights (6.4kg) than those of crias that survived (7.8kg, P<0.05). The weight and width of the placenta was similar for live and dead crias.