Reproductive isolation and patterns of genetic differentiation in a cryptic butterfly species complex.

Molecular studies of natural populations are often designed to detect and categorize hidden layers of cryptic diversity, and an emerging pattern suggests that cryptic species are more common and more widely distributed than previously thought. However, these studies are often decoupled from ecological and behavioural studies of species divergence. Thus, the mechanisms by which the cryptic diversity is distributed and maintained across large spatial scales are often unknown. In 1988, it was discovered that the common Eurasian Wood White butterfly consisted of two species (Leptidea sinapis and Leptidea reali), and the pair became an emerging model for the study of speciation and chromosomal evolution. In 2011, the existence of a third cryptic species (Leptidea juvernica) was proposed. This unexpected discovery raises questions about the mechanisms preventing gene flow and about the potential existence of additional species hidden in the complex. Here, we compare patterns of genetic divergence across western Eurasia in an extensive data set of mitochondrial and nuclear DNA sequences with behavioural data on inter- and intraspecific reproductive isolation in courtship experiments. We show that three species exist in accordance with both the phylogenetic and biological species concepts and that additional hidden diversity is unlikely to occur in Europe. The Leptidea species are now the best studied cryptic complex of butterflies in Europe and a promising model system for understanding the formation of cryptic species and the roles of local processes, colonization patterns and heterospecific interactions for ecological and evolutionary divergence.

Multi-omics for studying and understanding polar life.

Polar ecosystems are experiencing amongst the most rapid rates of regional warming on Earth. Here, we discuss 'omics' approaches to investigate polar biodiversity, including the current state of the art, future perspectives and recommendations. We propose a community road map to generate and more fully exploit multi-omics data from polar organisms. These data are needed for the comprehensive evaluation of polar biodiversity and to reveal how life evolved and adapted to permanently cold environments with extreme seasonality. We argue that concerted action is required to mitigate the impact of warming on polar ecosystems via conservation efforts, to sustainably manage these unique habitats and their ecosystem services, and for the sustainable bioprospecting of novel genes and compounds for societal gain.

Pituitary regulation of postnatal small intestinal ontogeny in the rat: differential regulation of digestive hydrolase maturation by thyroxine and growth hormone.

During the third week of postnatal life, dramatic ontogenic changes occur in the morphology and enzymology of the small intestine of the infant rat, enabling the animal to make the transition from milk to solid food. To investigate the roles of T4 and GH in regulation of these changes, infant rats were hypophysectomized on day 6 of life by the transauricular technique. Hypophysectomy resulted in diminution of somatic and intestinal growth as well as abnormal maturation of the disaccharidases lactase, sucrase, and maltase when measured on day 25. Administration of either T4 or GH to hypophysectomized animals resulted in moderately increased intestinal growth, while complete restoration of small intestinal growth resulted from administration of the combination of both hormones. Although T4, GH, or the combination of hormones reduced lactase activities, T4 alone produced normal maturation of sucrase and maltase. Neither hypophysectomy nor hormone replacement affected aminooligopeptidase. The molecular structure of lactase, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was not altered to a major degree in hypophysectomized animals or animals that received hormone replacement, but minor alterations were evident in sucrase structure in hypophysectomy. These studies indicate that 1) T4 and GH actively participate in postnatal regulation of small intestinal ontogeny; 2) thyroid hormones act directly on developing intestinal tissues to independently produce the normal maturation of the disaccharidases by mechanisms that are not likely to involve alterations in processing of the enzyme-protein; and 3) maturation of aminooligopeptidase is not regulated by pituitary hormones, in distinct contrast to the disaccharidases.

Genetic association of Ctla-4 to myasthenia gravis with thymoma.

Cytotoxic T lymphocyte associated antigen-4 (CTLA-4) plays a pivotal role in downregulating both the cellular and the humoral response by suppressing ongoing responses of activated T cells. Our earlier study showed that genetic variations in interleukin-1 genes confer susceptibility to myasthenia gravis, especially in patients having the lowest risk from major histocompatibility complex genes. Here we describe an association of Ctla-4 gene to the disease with thymoma and a higher prevalence of CTLA-4 gene polymorphism allele 104 in patients positive for IL-1beta TaqI allele 2, an IL-1beta 'high secretor' phenotype. There was no association in patients with hyperplasia and normal thymic histology. These results further advocate that MG is a polygenetic disease and suggest that co-stimulators such as CTLA-4 and CD28 might have an important role in the pathogenesis of the disease.

Methylsulfonyl metabolites of PCBs and DDE in human tissues.

Methylsulfonyl metabolites of chlorinated biphenyls (MeSO2-CBs) and p,p'-DDE (MeSO2-DDEs) were determined in human adipose and liver tissues obtained at autopsy of seven Swedish individuals 47-80 years of age. Twenty MeSO2-CBs and two MeSO2-DDEs were found in the analyzed samples. In adipose tissue, most of the 4-MeSO2-CBs were found at higher concentrations than the corresponding 3-MeSO2-CBs and, in all samples of adipose tissue, 4-MeSO2-2,2',3',4',5-pentaCB (4-87) and 4-MeSO2-2,2',3,4',5',6-hexaCB (4-149) occurred at higher concentrations than other MeSO2-CBs. In the liver, 3-MeSO2-2,2',3',4',5,6-hexaCB (3-132) was by far the most abundant MeSO2-CB, contributing to 61-82% of the sum of MeSO2-CBs. In this tissue, most of the other 3-MeSO2-CBs were also found at higher concentrations than the corresponding 4-MeSO2-CBs. The ratios of the sum of MeSO2-CBs to the sum of determined chlorinated biphenyls (CBs) were 1/250 and 1/28 in adipose tissue and the liver, respectively, calculated from the median values. The concentration of 2-MeSO2-DDE was lower than that of 3-MeSO2-DDE in both adipose tissue and liver, except in the liver from one of the individuals. The concentration ratios of 2-MeSO2-DDE to 3-MeSO2-DDE were about 10 times higher in liver than in adipose tissue. The ratios of the sum of MeSO2-DDEs to p,p'-DDE were 1/455 and 1/61 in adipose tissue and liver, respectively, calculated from the median values. MeSO2-CBs and MeSO2-DDEs were also determined in lung tissue from one of the individuals. In this sample, the profiles of MeSO2-CBs and MeSO2-DDEs were similar to the profiles of these compounds in adipose tissue.

Methylsulfonyl metabolites of PCBs and DDE in human milk in Sweden, 1972-1992.

A multicomponent method used for analysis of organochlorine pesticides, polychlorinated biphenyls (PCBs), naphthalenes, dibenzo-p-dioxins, and dibenzofurans was adapted for the analysis of methylsulfonyl metabolites of chlorinated biphenyls (MeSO2-CBs) and of p,p'-DDE (MeSO2-DDE) in human milk. The extraction and initial purification was made by liquid-gel partitioning. Additional purification and separation steps were achieved by adsorption and gel permeation chromatography. The mean recoveries of 23 MeSO2-CBs and MeSO2-DDE standards, added to the milk before extraction, were 80-97%. Human milk sampled in Stockholm during 1972, 1976, 1980, 1984/85, 1990, 1991, and 1992 was analyzed by GC-MS. During the time course studied, the concentrations of MeSO2-CBs decreased from approximately 9 to 2 ng/g lipids and of MeSO2-DDE from 5 to 0.4 ng/g lipids. The concentrations of MeSO2-CBs and MeSO2-DDE correlated to the levels of total PCB and p,p'-DDE, respectively. 3-MeSO2-DDE was the major isomer of the aryl methyl sulfones studied in the milk. PCB methyl sulfones with five and six chlorine atoms in the molecule were predominant among the PCB methyl sulfones Generally, the concentrations of 4-MeSO2-CBs were higher than the corresponding 3-MeSO2-CB compound. The major MeSO2-CBs in the milk were 4-MeSO2-2,5,2',3',4'-pentaCB (4-87) and 4-MeSO2-2,3,6,2',4',5'-hexaCB (4-149).

Polychlorinated naphthalenes and other organochlorine contaminants in human adipose and liver tissue.

Chlorinated naphthalenes (CNs) and chlorinated biphenyls (CBs), hexachlorobenzene (HCB), and p,p'-DDE were determined in human adipose and liver tissues collected at autopsy of 5 men and 2 women (Swedish), 47-80 yr of age. In paired adipose tissue and liver samples, the differences of the distribution of CNs, CBs, HCB, and p,p'-DDE were small, but the concentrations of the compounds (lipid weight basis) varied between the subjects. Generally, the profiles of the contaminants were similar in the subjects. However, in one of the male subjects (80 yr of age) the concentrations of 1,2,3,5,7/1,2,4,6,7-pentaCN and 1,2,3,4,6,7/1,2,3,5,6,7-hexaCN were much higher in the liver: 6 and 20 times higher than in adipose tissue, respectively. The concentration of the toxic coplanar 3,3',4,4',5-pentaCB (CB-126) was also high in the liver from this individual, three times higher concentration than in adipose tissue. It is suggested that the multiplicity of contaminants found in humans should be taken into consideration in risk assessment and that further studies are needed on the distribution of contaminants in different tissues.

Analysis of polybrominated diphenyl ethers in Swedish human milk. A time-related trend study, 1972-1997.

A previously described method for analysis of organochlorine compounds in human milk was adopted for analysis of brominated diphenyl ethers (BDEs) substituted with three to six bromine atoms. Analytes were extracted from human milk with the lipophilic gel Lipidex 5000. Further purifications were performed on partly deactivated aluminum oxide and silica gel columns, followed by gel permeation chromatography. The concentrations of BDEs were determined by gas chromatography/mass spectrometry (GC/MS). The average recoveries of 2,2',4-triBDE (BDE-17), 2,4,4'-triBDE (BDE-28), 2,2',4,4'-tetraBDE (BDE-47), 2,3',4,4'-tetraBDE (BDE-66), 2,2,3,4,4'-pentaBDE (BDE-85), 2,2',4,4',5-pentaBDE (BDE-99), 2,2',4,4',6-pentaBDE (BDE-100), 2,2',4,4',5,5'-hexaBDE (BDE-153), and 2,2',4,4',5,6'-hexaBDE (BDE-154) added to the samples before extraction ranged from 86% to 102%. Pooled samples of breast milk, collected at eight time periods between 1972 and 1997, were analyzed for PBDEs. BDE-47 was the most abundant PBDE congener in all samples. In total, eight PBDE congeners were identified in the milk. The sum of the concentrations of BDE congeners in human milk increased from 0.07 to 4.02 ng/g lipids during the 25-yr period studied.

Contemporary and retrospective investigations of human milk in the trend studies of organochlorine contaminants in Sweden.

Milk from the Mothers' Milk Centre in Stockholm has been analysed for organochlorine contaminants in different time periods between 1967 and 1989. The contemporary investigations showed a decrease of the levels of certain pesticides and polychlorinated biphenyls (PCBs). The changes were related to the prohibitions and restrictions applied on the usage of the compounds. Milk samples, remaining from the contemporary studies, were archived from 1972 and onward. These samples were later reanalyzed and the investigations were extended to also include polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and specific congeners of PCBs, including non-ortho and mono-ortho coplanar PCBs. These retrospective investigations, of the same milk, showed a time related decrease in the concentrations also for these compounds. Calculating the levels by toxic equivalency factors relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) it was found that PCBs contributed the major part of the toxic equivalents in milk.

Certain organochlorine and organobromine contaminants in Swedish human milk in perspective of past 20-30 years.

The investigations of organochlorine compounds in breast milk from women living in the Stockholm region started in 1967. The present study summarises the investigations of polychlorinated biphenyls (PCBs), naphthalenes (PCNs), dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), polybrominated diphenyl ethers (PBDEs) and pesticides (DDT, DDE, hexachlorobenzene, dieldrin) as well as methylsulfonyl metabolites of PCBs and DDE in human milk sampled during different periods up to 1997. During the course of 20-30 yr the levels of organochlorine compounds in human milk have decreased to various extent. A decrease to the half of the original concentration was attained in the range of 4-17 yr periods. On the contrary to the organochlorine compounds, the concentrations of PBDEs have increased during the period 1972-1997, indicating a doubling of the levels by 5 yr. The levels reflect the environmental contamination and background levels in the population. The accumulation and ongoing increase in the levels of PBDEs calls for immediate measures to stop the environmental pollution and human exposure to PBDEs.

Occupational exposure: Organochlorine compounds in blood plasma from potentially exposed workers. PCB, PCN, PCDD/PCDF, HCB and methylsulphonyl metabolites of PCB.

Concentrations of polychlorinated biphenyls (PCB), polychlorinated naphthalenes (PCN), polychlorinated dibenzo-p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF), hexachlorobenzene and methylsulphonyl metabolites of PCB were determined in blood plasma from potentially exposed workers and controls. Three of the potentially exposed subjects had worked with cable incineration and two were electricians. Extraction of the organochlorine compounds and lipids were performed using the lipophilic gel Lipidex. Different adsorbents and gel permeation chromatography were applied for further purification of the samples and separation of analytes. Determinations of the chlorinated compounds were made by using gas chromatography with electron-capture detection and gas chromatography-mass spectrometry. Only small differences in the concentrations of organochlorine compounds were found in the plasma from the three subject groups. Thus, specific exposure of the workers could not be confirmed.

Levels of organochlorine contaminants in human milk in relation to the dietary habits of the mothers.

The levels of p,p'-DDT, p,p'-DDE, dieldrin, alpha- and beta-hexachlorocyclohexane (alpha-HCH, beta-HCH), hexachlorobenzene and polychlorinated biphenyls (PCBs) were determined in milk fat from mothers with different dietary habits. The categories studied comprised mothers eating a lacto-vegetarian diet (18 milk samples), a mixed diet (20 milk samples) and a mixed diet which regularly included fatty fish from the Baltic (11 milk samples). The lowest levels of p,p'-DDT+p,p'-DDE and PCBs were found in milk from lacto-vegetarians and the highest levels in milk from mothers who regularly consumed fatty fish from the Baltic. Factors other than the diets which could influence the levels of the organochlorine compounds are discussed, e.g. mother's age, amount of milk produced and mother's weight loss.

Organochlorine contaminants in Swedish human milk from the Stockholm region.

The levels of fat-soluble organochlorine contaminants in human milk in the Stockholm region are decreasing. For p,p'-DDT, p,p'-DDE, and dieldrin a reduction has taken place during the last decade, but the levels of polychlorinated biphenyls (PCBs) started declining only from about 1977. Generally, the pooled samples from the Mothers' Milk Centre in Stockholm in 1980 contained lower levels of organochlorine contaminants than the current Swedish maximum acceptable limits for milk. In 2% of the samples the total DDT levels slightly exceeded the limit of 0.05 mg DDT + DDD + DDE/kg. Milk from three mothers was analysed during the nursing of two or three consecutive infants. In each case, the levels of organochlorine contaminants declined.

Analysis of pentachlorophenol in water and urine by enrichment with Lipidex 5000.

A method using the lipophilic gel Lipidex 5000 for sorption of pentachlorophenol (PCP) from water and urine is described. The procedure for water gives a simultaneous clean-up from lipophilic contaminants and also offers the possibility for determination of less polar compounds, such as 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane (p,p'-DDT) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), collected in a separate fraction. The extracted PCP is derivatized to pentachlorophenyl acetate, which is determined by electron-capture gas chromatography. The average recovery of 0.01-0.15 ng PCP per ml water was 96% and of labelled p,p'-DDT (ca. 5 ng/ml) and TCDD (ca. 0.001 ng/ml) 95 and 92%, respectively. A similar method for enrichment of PCP was applied to acidified urine and to urine hydrolysed with hydrochloric acid or with digestive juice from Helix pomatia. Recoveries of 0.1-2.5 ng PCP per ml non-hydrolysed urine were on an average 92% and of 2.5-10 ng PCP per ml hydrolysed urine 96%. The analyses indicate that PCP in urine from non-occupationally exposed persons is originally conjugated and to some extent liberated when stored. The contamination of organic solvents and laboratory environments with PCP is discussed.

Liquid-gel partitioning using Lipidex in the determination of polychlorinated biphenyls in cod liver oil.

A technique was developed for transfer of fat and polychlorinated biphenyls from cod liver oil into the lipophilic gel Lipidex 5000. Subsequent elution of the gel separated about 60% of the fat from the sample. Following further purification on aluminium oxide and silica gel, toxic non-ortho- and mono-ortho-PCB congeners were isolated in two separate fractions on charcoal. Recoveries were studied by addition of twelve different PCB congeners to 0.2 g of fat. The non-ortho-PCBs were labelled with 13C. The recoveries of 5-50 ng of the unlabelled compounds were 80-100% and those of 50-100 pg of the labelled compounds were 76-106%.

Analysis of organochlorine pesticides, polychlorinated biphenyls, dibenzo-p-dioxins and dibenzofurans in human milk by extraction with the lipophilic gel Lipidex 5000.

A method for multicomponent determination of organochlorine contaminants in human milk is described. The lipophilic gel Lipidex 5000 was used for extraction of lipids and organochlorine compounds. Further purification and separation was achieved by chromatography on partly deactivated aluminium oxide, Lipidex, silica gel and active basic and acidic aluminium oxide. The concentrations of pesticides and polychlorinated biphenyls (PCBs) were determined by electron-capture gas chromatography. Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) were determined by selected-ion monitoring capillary column gas chromatography-mass spectrometry at a resolution of 8000-9000. The levels of 2,2-bis(4-chlorophenyl)-1,1,1-trichloroethane, 1,1-bis(4-chlorophenyl)-2,2-dichloroethene, hexachlorobenzene, alpha-hexachlorocyclohexane, beta-hexachlorocyclohexane, dieldrin, trans-nonachlor, oxychlordane and PCBs found by this method agreed with the levels determined using a previous solvent-extraction method. Recovery experiments were performed by addition of pentachlorophenol and certain PCDDs and PCDFs. The average recovery of 0.5-2.0 ng pentachlorophenol per ml milk was 92%. Ten PCDDs and PCDFs reported to occur in milk were added at levels between 0.5 and 50 pg per ml milk. Recoveries of these compounds were on an average 79-91%.

Polychlorinated naphthalenes and other organochlorine contaminants in Swedish human milk, 1972-1992.

The concentrations of polychlorinated naphthalenes (PCNs) were determined together with polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), 1,1-bis-(4-chlorophenyl)-2,2,2-trichloroethane (p,p'-DDT), 2,2-bis(4-chlorophenyl)-1,1-dichloroethylene (p,p'-DDE) and hexachlorobenzene (HCB) in milk, sampled in the course of 1972-92 from mothers living in Stockholm. A previously developed method for multicomponent analysis of organochlorine environmental contaminants was adapted for simultaneous analysis of PCNs. The mean recoveries of seven chlorinated naphthalene (CN) congeners added to milk prior to extraction were 76-99%. Similar recoveries were obtained for the commercial PCN product Halowax 1014. The pattern of PCNs in milk differed to a great extent from that in the commercial PCN products. The dominating congeners in breast milk were 1,2,3,5,7-pentachloronaphthalene (CN-52), 1,2,3,4,6,7- and/or 1,2,3,5,6,7-hexachloronaphthalene (CN-66/ CN-67) and one unidentified tetrachloronaphthalene. There was a notable decrease in the concentrations of PCNs as was of the other organochlorine contaminants in milk from 1972 to 1992. During this time period the sum of CN congeners decreased from 3,081 to 483 pg/g milk fat and the sum of toxic equivalents of dioxin and dioxin-like compounds decreased from 100 to 39 pg/g milk fat.

Construction of high-complexity combinatorial phage display peptide libraries.

Random peptide libraries displayed on the surface of filamentous bacteriophage are widely used as tools for the discovery of ligands for biologically relevant macromolecules, including antibodies, enzymes, and cell surface receptors. Phage display results in linkage of an affinity-selectable function (the displayed peptide) to the DNA encoding that function, allowing selection of individual binding clones by iterative cycles of in vitro panning and in vivo amplification. Critical to the success of a panning experiment is the complexity of the library: the greater the diversity of clones within the library, the more likely the library contains sequences that will bind a given target with useful affinity. A method for construction of high-complexity (> or = 10(9) independent clones) random peptide libraries is presented. The key steps are highly efficient binary ligation under conditions where the vector is relatively dilute, with only a modest molar excess of insert, followed by efficient electrotransformation into Escherichia coli. Library design strategies and a protocol for rapid sequence characterization are also presented.

Distribution of PCB congeners, DDE, hexachlorobenzene, and methylsulfonyl metabolites of PCB and DDE among various fractions of human blood plasma.

The concentrations of chlorinated biphenyls (CBs), 1, 1-bis(4-chlorophenyl)-2,2-dichloroethene (DDE), hexachlorobenzene (HCB), and the methylsulfonyl metabolites of CBs (MeSO(2)-CBs) and DDE (MeSO(2)-DDE) were determined in human plasma samples and in the fractions obtained by ultracentrifugation of plasma into very-low-density (VLDL), low-density (LDL), high-density (HDL) lipoprotein and lipoprotein depleted (LPDP) fractions (containing primarily albumin). The concentrations of triacylglycerols, cholesterol, phospholipids, and apolipoprotein B (apoB) were determined. The organochlorine compounds were associated with all fractions, but predominantly with the LPDP fraction. On an average 44% of CBs, 61% of MeSO(2)-CBs, 73% of DDE, 77% of MeSO(2)-DDE, and 45% of HCB were distributed in the LPDP fraction. A tendency to greater association of 3-methylsulfonyl substituted than of corresponding 4-methylsulfonyl substituted chlorobiphenyls to the LPDP fraction was noticed. Among the lipoprotein fractions, LDL was the main carrier of HCB, DDE and CBs. MeSO(2)-DDE was predominantly found in HDL and MeSO(2)-CBs were distributed equally among the LDL and HDL fractions. Calculating the concentrations of organochlorine compounds in relation to the content of apoB, the levels were about 10 times higher in VLDL than in LDL.