Rapid purification and properties of human glycodelin (endometrial α2-globulin)

The method presented can easily produce milligram amounts of glycodelin from pregnancy endometrium, with a 19% yield. It involves anion-exchange chromatography, gel permeation and chromatofocusing; it results in one stainable band at M_r 28 000 after sodium dodecyl sulphate–polyacrylamide electrophoresis, as well as after immunoblot analysis, performed using an affinity-purified IgG fraction from an antiserum against glycodelin. In spite of this, the corresponding gel isoelectric focusing pattern gives four stainable bands with pI values between 4.55 and 5.2. Western immunoblot analysis of tissue extracts indicates the presence of glycodelin epitopes associated with materials heavier than the native protein. Circular dichroism spectra of the highly purified protein in water solutions indicate a large amount of β-sheet conformation, whereas those obtained with different proportions of 2-propanol in water, show an increased proportion of α-helix conformation.     

Age-, cycle- and topographic dependency of human myometrial prostaglandin (6-keto-PGF1a, PGF2a)-synthesis in vitro

Specimens of human myometrium (isthmus and fundus) freshly obtained at hysterectomy were immediately transferred in ice cold Tyrode solution and placed in superfusion chambers. Spontaneous contractions were recorded, the effluent of the myometrium was analyzed for PGF2a and 6-keto-PGF1a by use of specific radioimmunoassay systems. Dating of the menstrual cycle was achieved by histological evaluation of the endometrium.The PG release rates expressed as ng/min/g wet weight were correlated to the patients age and to the phase of the menstrual cycle. The production rates of 6-keto-PGF1a were negatively correlated to the age of the patients and declined in fundus specimens from 2.89 ± 0.35 ng/min/g wet weight in 39–42 years old patients to 0.52 ± 0.17 ng/min/g wet weight in 48–52 years old women during the secretory phase (p< 0.001). Similar significant correlations were found in specimens obtained from the isthmus uteri.During the proliferative phase fundus specimes produced on average 1.61 ± 0.67 ng/min/g wet weight in 39–42 years old patients and0.49 ± 0.12 n/min/g weight 6-keto-PGF1a in 48–52 years old women respectively (p < 0.001).The PGF2a synthesis in myometrial specimens of fundus or isthmus origin was significantly lower than 6-keto-PGF1a and did not correlate to the age of the patients during the proliferative phase. However, PGF2a release rates during the secretory phase were significantly (p < 0.001) higher in younger women.These results suggest an age-, cycle- and topographic dependency of PGI2 synthesis in human myometrial tissue.     

Ploidy-dependent genomic stability in the tissue cultures of ornamental Phlox drummondii Hook

Comparisons of the chromosome numbers, 2C nuclear DNA amounts and karyomorphology were made in explant cultures of diploid (2n = 2x = 14) and autotetraploid (2n = 4x = 28) Phlox drummondii. In 6–36 week old calli derived from diploid internodal segment explants, and in cells of root tips regenerated from such callus, marked differences were observed in chromosome number. The chromosome numbers ranged from 2n = 14 to 2n = 100 and DNA amounts from 8.20 to 63.20 pg in the diploid derived callus, while the extent of variation was much reduced in the regenerated roots. In contrast, the autotetraploid cultures were characterised by the maintenance of the same chromosome number and DNA amounts as the mother plant. Modified chromosome structures were not apparent in any of the cultures. The possible reasons for the chromosomal instability at the diploid level and stability at the tetraploid level are discussed.     

Very old DNA

The verification of DNA sequences obtained from very old tissue sources as indeed ancient is a major point of discussion in the ancient DNA field. Proper controls and the use of the phylogenetic approach are the general methods employed for verification of the ancient DNA. Most studies have reported the recovery of extremely small amounts of nucleic acids which are sheared into rather small fragments. In addition, problems such as ‘PCR jumping’ can produce spurious sequence information. These observations suggest that random amplification techniques and the development of primers for highly informative but short target regions are essential for the further development of the ancient DNA field.     

Amplification of cox2 (～620 bp) from 2 mg of Up to 129 Years Old Herbarium Specimens,Comparing 19 Extraction Methods and 15 Polymerases

During the past years an increasing number of studies have focussed on the use of herbarium specimens for molecular phylogenetic investigations and several comparative studies have been published. However, in the studies reported so far usually rather large amounts of material (typically around 100 mg) were sampled for DNA extraction. This equals an amount roughly equivalent to 8 cm² of a medium thick leaf. For investigating the phylogeny of plant pathogens, such large amounts of tissue are usually not available or would irretrievably damage the specimens. Through systematic comparison of 19 DNA extraction protocols applied to only 2 mg of infected leaf tissue and testing 15 different DNA polymerases, we could successfully amplify a mitochondrial DNA region (cox2; ∼620 bp) from herbarium specimens well over a hundred years old. We conclude that DNA extraction and the choice of DNA polymerase are crucial factors for successful PCR amplification from small samples of historic herbarium specimens. Through a combination of suitable DNA extraction protocols and DNA polymerases, only a fraction of the preserved plant material commonly used is necessary for successful PCR amplification. This facilitates the potential use of a far larger number of preserved specimens for molecular phylogenetic investigation and provides access to a wealth of genetic information in preserved in specimens deposited in herbaria around the world without reducing their scientific or historical value.     

Absence of changes in fiber type with aging in avian muscles

Summary The anterior latissimus dorsi of the pigeon, a slow tonic muscle, and biventer cervicis, a mixed muscle, of two age groups (1–2 years old versus 6–8 years old) were compared with respect to percentages of fiber types and activities of adenosine triphosphatase (ATPase) and succinic dehydrogenase (SDH), as estimated histochemically, to determine whether these became altered with old age. These parameters did not change between the young and old birds for either type of muscle.This investigation was supported in part by research grants PCM 77-15960 and PCM 79-16540 from the National Science Foundation     

Age and growth estimates of the bull shark,Carcharhinus leucas,from the northern Gulf of Mexico

Synopsis Length at age and growth rates for 59 bull sharks, Carcharhinus leucas, collected from the northern Gulf of Mexico were estimated from the band patterns formed seasonally in the vertebral centra. The combined age at length data for both sexes were applied to a von Bertalanffy growth model producing parameter estimates of L = 285 cm TL, K = .076, t₀ = –3.0 yr. Lengths at age for males and females were similar except that males did not attain as great a length as females. Growth was apparently slow and varied among individuals, but in general, was estimated to be 15–20 cm yr^–1 for the first five years, 10 cm yr^–1 for years 6–10, 5–7 cm yr^–1 for years 11–16, and less than 4–5 cm yr^–1 thereafter. Males mature at 210–220 cm TL or 14–15 yr of age; females mature at>225 cm TL or 18+ yr of age. The largest male (245 cm TL) was 21.3 yr old; the largest female (268 cm TL) was 24.2 yr old.     

Growth and age structure of the swan mussel Anodonta cygnea (L.) at different depths in lake Mattsee (Salzburg, Austria)

Unionid clams were collected at 1–2 m, 3–4 m and 6–7 m depth in lake Mattsee, a moderately mesotrophic lake, to investigate the effect of depth on clam growth and age structure. No significant differences in age structure of Anodonta cygnea were found (p=0.65). Three and ten years old clams were present at all depths, but in different percentages. Whereas at 1–2 m 13.3% of the collected clams were <4 years old, this percentage was 4.4% at 6–7 m and 7.1% at 3–4 m. A greater percentage (6.7%) of older mussels (9, 10 years) were collected at 6–7 m than at 1–2 m (2.2%). Growth declined with depth. Total length at a given age of clams at 1–2 m and 3–4 m did not differ (p=0.54), whereas differences were significant between clams at 1–2 m and 6–7 m (p<0.05) as well as between 3–4 m and 6–7 m (p<0.05). The Growth constant k was highest at 1–2 m depth.     

Use of space and food by resident and migrant brown trout,Salmo trutta

Synopsis Parr and resident forms of brown trout,Salmo trutta, from Vangsvatnet Lake, Norway live in freshwater, while migrant forms live in coastal waters during summer and in freshwater during winter. About 80% of parr and residents live at depths <5 m, smolts and migrants are more confined to near-surface water. Brown trout partly segregate by size, age and sex from spring through autumn. More than 90% of parr and residents in the tributaries are 0–2 years old, 2–14 cm in length, in the littoral zone 0–3 years old, 7–24 cm in length, and in the pelagic zone 2–6 years old, 18–32 cm in length. The mean body length of equal-aged fish increases from tributaries through littoral to pelagic zones in the lake. Smolts (2–7 years, 14–29 cm) leave the lake from April through August and return during September–October. Migrants (2–11 years, 23–67 cm) leave the take in April–May and return during August–September; sexually mature fish return earlier than immatures. Female brown trout are less stream-dwelling, but more migrant and pelagic than males. Most individuals in the lake population spend the winter in the littoral zone. In the tributaries, diet differs significantly between age-groups of parr; young fish feed on smaller food items than do older fish. In the lake, parr and residents living in the same habitats feed on the same food items. Littoral brown trout feed mainly on insect larvae and chironomid pupae, pelagic brown trout feed on zooplankton and surface insects. Migrants feed little while staying in freshwater, except for matures which feed on young salmonids and surface arthropods during the 2 first months after they had returned from coastal waters. The results are discussed in relation to growth possibilities and mortality risks of the different habitats.Reprint request to B. Jonsson.     

Cytophotometric and autoradiographic evidence for functional apomixis in a gynogenetic fish,Poecilia formosa and its related,triploid unisexuals

Summary Amounts of DNA in individual Feulgen-stained nuclei from squash preparations of ovaries and testes from wild-caught and laboratory-reared stocks of Poecilia spp. were determined with an integrating microdensitometer. The DNA content of primary spermatocytes (4C) at zygotene, pachytene, or at metaphase I (3.3–3.4 pg) was approximately twice that found in secondary spermatocytes (2C) and four times that found for young spermatids (1C). Rarely, mature sperm were found with 2C DNA amounts. Nuclei from follicular epithelium and oogonia from both bisexual and diploid unisexual fish contained about 1.6–1.7 pg DNA; whereas, the DNA content of primary oocyte nuclei was about 3.5–3.7 pg DNA, indicating that just one cycle of chromosomal replication had occurred in these cells during the period of DNA synthesis before the visible onset of meiotic prophase. Similar results were obtained for triploid unisexuals whose 6C primary oocyte nuclei contained 5.0–5.1 pg DNA, which was twice the DNA content of 3C oogonia and follicular epithelial cells (2.4–2.5 pg DNA). Autoradiographic studies, designed to monitor the incorporation of ³H-thymidine by oogonia and primary oocytes in vivo and in vitro, also showed that there is no additional synthesis of DNA during the course of meiotic prophase in these unisexual fish. Therefore, we conclude that apomixis, not endoreduplication, is the cytological basis of reproduction in Poecilia formosa and its related, triploid biotypes.     

The extent of clonality in large stands of Lycopodium annotinum L.

In the Rothaargebirge (Northrhine-Westphalia) the interrupted club moss (Lycopodium annotinum L.) often grows in large (maximum size of patches observed: 18,000 m²) and dense (club moss cover 60–90%) clusters. By DNA amplification fingerprinting (DAF) analysis it was shown in three populations comprising a total of 69 samples that the clusters are of great genetical uniformity. Based on data from 47 polymorphic loci amplified with 13 primers 11 multilocus genotypes were identified, nine in one population and one per population in the other two. The data were subjected to similarity analysis, generating Jaccard′s similarity coefficients, on the basis of which cluster analysis was performed. Calculation of Nei's genetic diversity (1987) resulted in a mean value of 0.77 over all samples and a mean within populations diversity of 0.3. Corrected G′_ST value for the fraction of diversity among the populations was 0.7. Two out of three sites are most probably formed by one clone only. As the annual growth rate of the shoots is known, it has to be stated that the stands under investigation are at least 80–90 years old. Taking into consideration that annual accretion is virtually non-existent following forest thinning measures, it becomes very likely that the club moss plants are approximately as old as the trees of their forests (150–180 years). This hypothesis is supported by the observation that within the forests only large (i.e. old) stands, but no single shoots (i.e. young individuals) of the club moss exist. Thus one can conclude that in undisturbed forests no sexual reproduction of L. annotinum takes place.     

Pancreatic glucagon in human foetal stomach

Summary A combination of immunocytochemistry at light and electron microscopic levels, direct radioimmunoassay and measurement after gel chromatography have been used to identify and characterise a glucagon-like peptide detected in human foetal stomach. Immunocytochemistry, with region specific antisera, demonstrated that the glucagon-containing cells were indistinguishable from pancreatic A cells. Radioimmunoassay of tissue extracts confirmed the presence of significant quantities of glucagon, mean 21 pmol/g wet weight (range 14–29) in 16–26 week old foetuses, increasing to 41 pmol/g wet weight (range 31–52) in 33–30 week old foetuses and after gel chromatography the peptide was found to elute at the same position as standard porcine glucagon. It is apparent, therefore, that the human foetal fundus contains significant quantities of true pancreatic-type glucagon.DeccasedPreliminary details of this work were presented at the Society for Endocrinology Meeting, London, November 1981     

Recovery of conservation values in Central African rain forest after logging and shifting cultivation

Secondary forests in Central Africa are increasing in importance for biodiversity conservation as old growth forests outside the few protected areas are disappearing rapidly. We examined vegetation recovery in a lowland rain forest area in Cameroon based on a detailed botanical survey of old growth forest and different-aged logging gaps (5–27 years) and shifting cultivation fields (10–60 years). Our analysis focuses on the long-term recovery of botanical conservation values by analysing trends in vegetation structure, species composition, species diversity and levels of endemism and rarity. In the total survey (4.25 ha), we recorded 834 species of which 23% were endemic to the Lower Guinea forest region. The proportion of endemic species was high in shrubs and low in herbs. Geographic range and (local) rarity were not significantly associated. The proportion of rare species (relative frequency <10%) was high in woody climbers and low in trees. In logging gaps, recovery of all vegetation characteristics was relatively quick (5–14 years). Recovery in shifting cultivation sites took longer (30–60 years). Endemic species were found to be highly sensitive to shifting cultivation practices and even after 50–60 years the level of endemism was still significantly lower compared to old growth forest. The proportion of rare species was not significantly different between disturbed sites and old growth forest. We conclude that secondary forests can contribute to biodiversity conservation, e.g. as buffer zones around protected areas. However, this contribution should be assessed differently between land use types and widespread versus endemic species.     

Micronuclei and Ageing in a Sample of Yugoslavian Population

Objective: instability in the organization and expression of the genetic material has been hypothesized as the basic mechanism of ageing. Aim of this study was to quantify the effect of ageing on spontaneous micronuclei (MN) frequency in peripheral blood lymphocytes. Method: analysis of Yugoslavian population sample (164 tested individuals, age 0–62 years) has performed by application of cytokinesis-block technique (CB). Results: there was an increase of MN frequency with age, from newborns to 40-year-old persons. The total average of MN frequency per 1000 analyzed binuclear cells amounts to 8.03 ± 0.42, with variation from 0 to 26 MNs. In a sample of 29 newborns the recorded average MN frequency was 6.91 ± 0.81, while among 69 persons 25–39 years old, the MN frequency was 9.16 ± 1.00. The lowest average MN frequency, however, was noted in the sample of 34 tested individuals 40 to 62 years of age. Conclusion: an increase with age in MN frequency has been observed in samples of studied individuals from newborns to 40-year-old persons. A decrease of MN frequency in older groups could be explained by a gradual decrease of proliferative cell capacities.     

Purification to Homogeneity of Candida albicans Glucosamine-6-phosphate Synthase Overexpressed in Escherichia coli

The Candida albicans GFA1 gene encoding glucosamine-6-phosphate synthase, an enzyme of cell wall biosynthesis pathway in fungi and bacteria, recently an object of interest as a target for the chemotherapy of systemic mycoses, was PCR amplified and cloned to an Escherichia coli expression vector pET23b. The activity of the enzyme in the lysates from the overproducing E. coli strain was approximately 50–100 times higher than in the lysates from the control E. coli strain. This abundant overproduction allows to purify milligram amounts of the enzyme to homogeneity.     

Blood Lead Levels in Children Aged 0–6 Years Old in Hunan Province,China from 2009–2013

Objectives

The aim of this study is to describe blood lead levels (BLLs) and the prevalence of elevated blood lead levels (EBLLs) in children aged 0–6 years old and to analyze the BLL trend in children from 2009 to 2013 in China.

Methods

A total of 124,376 children aged 0–6 years old were recruited for this study from January 1^st 2009 to December 31^st 2013. Their blood lead levels were analyzed using atomic absorption spectrometry.

Results

The median BLL was 64.3 μg/L (IQR: 49.6–81.0), and the range was 4.3–799.0 μg/L. Blood lead levels were significantly higher in boys (66.0 μg/L) than in girls (61.9 μg/L) (P<0.001). The overall prevalence of BLLs≥100 μg/L was 10.54% in children aged 0–6 years in Hunan Province. Between 2009 and 2013, the prevalence of EBLLs (≥100 μg/L) decreased from 18.31% to 4.26% in children aged 0–6 years and increased with age. The prevalence of EBLLs has dramatically decreased in two stages (2009–2010 and 2012–2013), with a slight fluctuation in 2010 and 2011.

Conclusions

Both BLLs and the prevalence of EBLLs in children aged 0–6 years old declined substantially from 2009 to 2013 in Hunan Province; however, both remain at unacceptably high levels compared to developed countries. Comprehensive strategies are required to further reduce blood lead levels in children.     

Satellite DNA sequences in the neotropical marmoset Callimico goeldii (Primates,Platyrrhini)

Two families of tandemly repeated satellite DNAs were isolated from the neotropical primate Callimico goeldii (Goeldi's marmoset). One satellite, CgoA, is over 70% A+T and has a monomer length of 338 bp. The other satellite, CgoB, is 50% A+T and has a monomer length of 916 bp. Both CgoA and CgoB hybridize strongly with Callimico DNA, but not with the DNA of other new and old world primates. Based upon a neutral substitution rate of 1.5×10^–9/site per year for primates, sequence data from 15 CgoA monomers indicate that the tandem array is at least 30 million years old. Since no other neotropical primate has amplified CgoA sequences, the data suggest that the ancestor of Callimico separated from the other neotropical primates at least 30 million years ago. This value is about fourfold larger than the value of 7–9 million years derived from immunological data by Sarich and Cronin (1980). Possible reasons for this discrepancy are discussed. On leave from: Genetics Section, Instituto Nacional do Cancer, Rio de Janeiro/Department of Genetics, Universidade Federal do Rio de Janeiro, Brazil     

Liquid chromatography–tandem mass spectrometric quantitation of cyclophosphamide and its hydroxy metabolite in plasma and tissue for determination of tissue distribution

Cyclophosphamide (CP) and its metabolite, hydroxycyclophosphamide (OH-CP) have been quantitated in mouse plasma and tissue by derivatization combined with liquid chromatography–tandem mass spectrometry (LC–MS–MS). The derivatization was conducted immediately upon sample collection, to trap the OH-CP metabolite intermediate prior to further conversion to phosphoramide mustard or other reaction products. This simple and straightforward derivatization procedure, combined with sample extraction via protein precipitation, allowed quantitation of CP and the oxime derivative of OH-CP in plasma for concentrations ranging from approximately 12.5–3333 ng/ml, and in spleen tissue for concentrations of 1250–50 000 ng/g. The short cycle time (2.5 min) of the LC–MS–MS method allowed high throughput analysis with minimal matrix interference. Mouse plasma levels were quantitated for doses of 40, 65 and 120 mg/kg; spleen concentrations were determined for mice dosed at 120 mg/kg. The CP and oxime plasma levels correlated well with dose amounts. The CP levels in the spleen and plasma were similar while the oxime levels in the spleen were significantly lower than the plasma.     

A microassay for detection of DNA and RNA in small numbers of plant cells

Summary A microtechnique for the detection of DNA or RNA in small numbers of plant cells (1–50) has been developed using cauliflower mosaic virus (CaMV) infection of turnip as a model system. Both DNA and RNA extracted from 10 mesophyll protoplasts from CaMV-infected plants can be detected by hybridization using a radioactive probe made from cloned CaMV DNA (pCaMV10). No hybridization above background was detected in extracts of protoplasts from uninfected plants. At least 0.15 pg (11 000 molecules) of purified pCaMV10 DNA can be detected. This method is superior to existing macro techniques for nucleic acid detection as smaller amounts of tissue are required and the detection is approximately 100-fold more sensitive. re]19850326 rv]19850530 ac]19850611