Reconstructing the reproductive history of female polar bears using cementum patterns of premolar teeth

Premolar teeth collected from 220 adult female polar bears (Ursus maritimus) from western Hudson Bay, Canada, were examined to determine whether past reproductive events are recorded in cementum. The widths of annular cementum growth layer groups (GLGs) were measured and compared as proportional width index (PWI) values to correct for age and body size bias. Known reproductive states (pregnant, with cubs, or with yearlings) were used to confirm and calibrate cementum annuli. Significant differences in PWI were observed between GLGs formed the year females were pregnant versus when accompanied by cubs or yearlings. The probability of a female having produced a cub in adulthood was determined by fitting a logistic regression model between the ΔPWI of females when pregnant and with their cubs. Logistic regression of ΔPWI (β₀ = −0.229, β₁ = −13.465, G ² = 46.55, df = 1, P < 0.001) correctly classified the presence or absence of cubs in 72% of GLGs. Cementum width did not vary between different litter sizes. Observations of females with early litter loss suggests that longer periods of lactation contributes to decreased cementum width and therefore cementum may record a minimum age of litter survival. Predictions of litter production rate (0.43 litters/female/year) derived from cementum were similar to field observations; however, age at first parturition was underestimated by 1 year. We conclude that patterns of cementum deposition may be useful to determine individual reproductive histories and establish course estimates of reproductive parameters when regular field observations are not feasible. We also conclude that reproductive parameters derived from cementum are not adequate on their own for monitoring populations which are in decline or under stress and field observation should not be replaced under these conditions.     

Sugar-induced increases in trehalose 6-phosphate are correlated with redox activation of ADPglucose pyrophosphorylase and higher rates of starch synthesis in Arabidopsis thaliana

Tre6P (trehalose 6-phosphate) is implicated in sugar-signalling pathways in plants, but its exact functions in vivo are uncertain. One of the main obstacles to discovering these functions is the difficulty of measuring the amount of Tre6P in plant tissues. We have developed a highly specific assay, using liquid chromatography coupled to MS-Q3 (triple quadrupole MS), to measure Tre6P in the femto-picomole range. The Tre6P content of sucrose-starved Arabidopsis thaliana seedlings in axenic culture increased from 18 to 482 pmol x g(-1) FW (fresh weight) after adding sucrose. Leaves from soil-grown plants contained 67 pmol x g(-1) FW at the end of the night, which rose to 108 pmol x g(-1)FW after 4 h of illumination. Even greater changes in Tre6P content were seen after a 6 h extension of the dark period, and in the starchless mutant, pgm. The intracellular concentration of Tre6P in wild-type leaves was estimated to range from 1 to 15 microM. It has recently been reported that the addition of Tre6P to isolated chloroplasts leads to redox activation of AGPase (ADPglucose pyrophosphorylase) [Kolbe, Tiessen, Schluepmann, Paul, Ulrich and Geigenberger (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 11118-11123]. Using the new assay for Tre6P, we found that rising sugar levels in plants are accompanied by increases in the level of Tre6P, redox activation of AGPase and the stimulation of starch synthesis in vivo. These results indicate that Tre6P acts as a signalling metabolite of sugar status in plants, and support the proposal that Tre6P mediates sucrose-induced changes in the rate of starch synthesis.     

Mutational analysis of conserved glycine residues 142, 143 and 146 reveals Gly(142) is critical for tetramerization of CTP synthase from Escherichia coli

CTPS (cytidine 5'-triphosphate synthase) catalyses the ATP-dependent formation of CTP from UTP using either ammonia or L-glutamine as the nitrogen source. Binding of the substrates ATP and UTP, or the product CTP, promotes oligomerization of CTPS from inactive dimers to active tetramers. In the present study, site-directed mutagenesis was used to replace the fully conserved glycine residues 142 and 143 within the UTP-binding site and 146 within the CTP-binding site of Escherchia coli CTPS. CD spectral analyses of wild-type CTPS and the glycine mutants showed a slight reduction of approximately 15% in alpha-helical content for G142A and G143A relative to G146A and wild-type CTPS, suggesting some local alterations in structure. Relative to wild-type CTPS, the values of k(cat)/K(m) for ammonia-dependent and glutamine-dependent CTP formation catalysed by G143A were reduced 22- and 16-fold respectively, whereas the corresponding values for G146A were reduced only 1.4- and 1.8-fold respectively. The glutaminase activity (k(cat)) of G146A was similar to that exhibited by the wild-type enzyme, whereas that of G143A was reduced 7.5-fold. G146A exhibited substrate inhibition at high concentrations of ammonia and a partial uncoupling of glutamine hydrolysis from CTP production. Although the apparent affinity (1/[S](0.5)) of G143A and G146A for UTP was reduced approximately 4-fold, G146A exhibited increased co-operativity with respect to UTP. Thus mutations in the CTP-binding site can affect UTP-dependent activity. Surprisingly, G142A was inactive with both ammonia and glutamine as substrates. Gel-filtration HPLC experiments revealed that both G143A and G146A were able to form active tetramers in the presence of ATP and UTP; however, nucleotide-dependent tetramerization of G142A was significantly impaired. Our observations highlight the sensitivity of the structure of CTPS to mutations in the UTP- and CTP-binding sites, with Gly(142) being critical for nucleotide-dependent oligomerization of CTPS to active tetramers. This 'structural sensitivity' may limit the number and/or types of mutations that could be selected for during the development of resistance to cytotoxic pyrimidine nucleotide analogues.     

定量电子束显微分析中生物薄样品的质量损失

对多种生物薄样品和标样进行电子探针X射线能谱显微定量分析,分别以电子束轰击后样品的O Kα峰计数和介于4.2-6.2keV区间的连续X－射线计数变化监测质量损失,结果显示样品O Kα峰计数减少幅度大于连续X－射线计数减少幅度,在相同的分析条件下,各样品质量损失程度不相同（P＜0.05）。培养肝癌细胞冷冻干燥超薄切片、明胶冷冻干燥超薄切片、BSA薄膜、氨基塑料超薄切片、红细胞冷冻干燥超薄切片和卵黄高磷蛋白薄膜样品的质量损失分别为33％、28％、26％、18％、13％和13％,以上结果提示：以O Kα峰计数的减少监测样品的质量损失较敏感,在进行生物薄试样定量EPMA时应对各样品的质量损失进行相应校正。     

A point mutation that decreases the thermal stability of human interferon gamma.

We have identified a mutation of human gamma-interferon (IFN gamma) causing a temperature-sensitive phenotype. We used a randomized oligonucleotide to mutagenize a synthetic human IFN gamma gene, then screened the resulting mutants produced in Escherichia coli for proteins with altered biological activity. One mutant protein selected for detailed characterization exhibited less than 0.3% of the specific biological activity of native IFN gamma in an antiviral activity assay performed at 37 degrees C. However, the protein bound the human IFN gamma receptor with native efficiency at 4 degrees C. Sequencing the plasmid DNA encoding this protein showed that the mutation changed the lysine residue at amino acid 43 to glutamic acid (IFN gamma/K43E). Site-specific mutagenesis at amino acid 43 showed that this protein's phenotype resulted from positioning a negative charge at position 43. Structural characterization of IFN gamma/K43E using CD demonstrated that the protein had native conformation at 25 degrees C, but assumed an altered conformation at 37 degrees C. IFN gamma/K43E in this altered conformation bound poorly to the IFN gamma receptor at 37 degrees C, providing a rationale for the mutant's decreased antiviral activity.     

Evidence of adoption,monozygotic twinning,and low inbreeding rates in a large genetic pedigree of polar bears

Multigenerational pedigrees have been developed for free-ranging populations of many species, are frequently used to describe mating systems, and are used in studies of quantitative genetics. Here, we document the development of a 4449-individual pedigree for the Western Hudson Bay subpopulation of polar bears (Ursus maritimus), created from relationships inferred from field and genetic data collected over six generations of bears sampled between 1966 and 2011. Microsatellite genotypes for 22–25 loci were obtained for 2945 individuals, and parentage analysis was performed using the program FRANz, including additional offspring–dam associations known only from capture data. Parentage assignments for a subset of 859 individuals were confirmed using an independent medium-density set of single nucleotide polymorphisms. To account for unsampled males in our population, we performed half-sib–full-sib analysis to reconstruct males using the program COLONY, resulting in a final pedigree containing 2957 assigned maternities and 1861 assigned paternities with only one observed case of inbreeding between close relatives. During genotyping, we identified two independently captured 2-year-old males with identical genotypes at all 25 loci, showing—for the first time—a case of monozygotic twinning among polar bears. In addition, we documented six new cases of cub adoption, which we attribute to cub misidentification or misdirected maternal care by a female bereaved of her young. Importantly, none of these adoptions could be attributed to reduced female vigilance caused by immobilization to facilitate scientific handling, as has previously been suggested.     

An erythropoietin fusion protein comprised of identical repeating domains exhibits enhanced biological properties.

The hematopoietic growth factor erythropoietin (Epo) initiates its intracellular signaling cascade by binding to and inducing the homodimerization of two identical receptor molecules. We have now constructed and expressed in COS cells a cDNA encoding a fusion protein consisting of two complete human Epo domains linked in tandem by a 17-amino acid flexible peptide. On SDS-polyacrylamide gel electrophoresis, the Epo-Epo fusion protein migrated as a broad band with an average apparent molecular mass of 76 kDa, slightly more than twice the average apparent molecular mass of Epo, 37 kDa. Enzymatic N-deglycosylation resulted in an Epo-Epo species that migrated on SDS-polyacrylamide gel electrophoresis as a narrow band with an average apparent molecular mass of 39 kDa. The specific activity of the Epo-Epo fusion protein in vitro (1,007 IU/microgram; 76 IU/pmol) was significantly greater than that of Epo (352 IU/microgram; 13 IU/pmol). Moreover, secretion of Epo-Epo by COS cells was 8-fold greater than that of Epo. Subcutaneous administration of a single dose of Epo-Epo to mice resulted in a significant increase in red blood cell production within 7 days. In contrast, administration of an equivalent dose of conventional recombinant Epo was without effect. The pharmacokinetic behavior of Epo-Epo differed significantly from that of Epo. The results suggest that Epo-Epo may have important biological and therapeutic advantages.