Biosynthesis of peptide precursors and protease inhibitors using new constitutive and inducible eukaryotic expression vectors

AbstractA series of expression vectors has been constructed as based on the pML derivative of pBR322. The eukaryotic transcription units employ various promoters followed by polycloning sites for 3–9 commonly used restriction enzymes and are completed by the SV40 polyadenylation sequence. In 4 of the vectors, designed for co-transfection or transient expression studies, only a single transcription unit containing either a constitutive or an inducible promoter was incorporated. The human ubiquitin (UbC) promoter was used as a strong constitutive promoter, while the mouse metallothionein promoter and the promoter of the long terminal repeats of the mouse mammary tumor virus were used as inducible promoters. Another vector contained an additional transcription unit encoding a eukaryotic selection marker, the neomycin resistance encoding gene. The vectors were used in CHO cells and in neuroendocrine CA77 cells to synthesize peptide precursors, protease inhibitors and a protease. It is shown that these vectors are very efficient for the constitutive and inducible expression of nucleotide sequences in both transient and stable transfections of eukaryotic cells

Complete androgen insensitivity in an Icelandic family caused by mutation in the steroid binding region of the androgen receptor

Neðst á síðunni er hægt að nálgast greinina í heild sinni með því að smella á hlekkinn View/OpenIntroduction: Androgen insensitivity syndrome (AIS) is a X-linked rescessive disorder characterized by impairment of the androgen-dependant male sexual differentiation. The cause of AIS is in most cases a mutation in the gene of the androgen receptor on the X chromosome. In this study we describe an Icelandic family with two girls with AIS. A search for mutations in the androgen receptor gene was performed in order to identify the genetical and molecular basis for AIS in this family. Material and methods: Genomic DNA was isolated from two girls with complete AIS and their close relatives. PCR was used to amplify all eight exons of the androgen receptor gene of the two AIS girls and SSCP used to screen for mutations. DNA fragments showing abnormal SSCP pattern were subjected to nucleotide sequencing. PCR based diagnostic method was developed and used to detect the mutation causing AIS in the family. Results and conclusions: Using SSCP and DNA sequencing a CGA to CAA missense mutation in exon 5 at codon 752 was identified. The mutation causes in an Arg to Gln amino acid substitution (R752Q mutation) in the ligand binding domain of the androgen receptor and a complete androgen insensitivity. Members of the family were genotyped using a PCR based method for identification of the mutant allele. The results strongly indicated a de novo mutation in a germ cell of the maternal grandmother, as the mutation was not found in her blood leucocytes. The diagnostic test provided a basis for genetic counselling for the family.Inngangur: Heilkenni andrógenónæmis (androgen insensitivity syndrome, AIS) er sjúkdómur, sem erfist kynbundið víkjandi, og hefur í för með sér truflun á eðlilegri kynþróun hjá karlfóstri, sem á sér stað fyrir tilstilli testósteróns. Orsök heilkennisins er í flestum tilvikum stökkbreyting í geni andrógenviðtækis á X-litningi. Í þessari rannsókn er íslenskri fjölskyldu lýst, þar sem fundist hafa tvær stúlkur með heilkenni andrógenónæmis. Leitað var að stökkbreytingum í geni andrógenviðtækisins í fjölskyldunni. Efniviður og aðferðir: Erfðaefni var einangað úr blóði tveggja stúlkna með algjört andrógenónæmi svo og nánustu ættingja þeirra. Fjölliðunarhvarf var notað til að fjölfalda allar átta útraðirnar í geni andrógenviðtækis tilfella með AIS. SSCP-aðferðin var notuð til að skima fyrir stökkbreytingum í geninu. Niturbasaröð þeirrar útraðar sem gaf óeðlilegt SSCP mynstur var síðan ákvörðuð. Greiningaraðferð, sem byggir á fjölliðunarhvarfi og skerðibútabreytileika, var þróuð og notuð til að finna stökkbreytta samsætu meðal fjölskyldumeðlima. Niðurstöður og ályktun: Með notkun SSCP og niturbasaraðgreiningar fannst sama stökkbreytingin í útröð 5 í geni andrógenviðtækis stúlknanna tveggja með AIS. Niturbasaröðin CGA var stökkbreytt í CAA, sem hefur í för með sér að í stað amínósýrunnar argeníns í stöðu 752 kemur glútamín (R752Q samsæta). Þessi stökkbreyting er staðsett í sterabindistað andrógenviðtækisins og hefur í för með sér að það getur ekki bundið testósterón. Niðurstöður rannsókna á fjölskyldumeðlimum með sérstakri erfðagreiningaraðferð fyrir samsætuna R752Q sýndu að um var að ræða nýja (de novo) stökkbreytingu í kynfrumum móðurömmu, þar sem ekki var hægt að greina samsætuna í erfðaefni hvítra blóðkorna hennar. Erfðagreiningin skapaði grundvöll til erfðaráðgjafar hjá fjölskyldunni

Sex-specific survival and tumor mutational burden in early stage melanoma

Introduction Tumor mutational burden (TMB) is a promising biomarker of clinical response to immune checkpoint inhibitors in metastatic cancers, and melanoma-specific survival. There are also significant gender-specific differences in TMB with men having consistently higher TMB than women. This relationship is provocative given the well-documented female melanoma survival advantage, and has not been investigated in early-stage primary tumors naïve to treatment. Approach Here we present preliminary findings on sex, survival, and tumor mutational burden from Stages II and III primary melanoma tumors, none of which have received immunotherapy using the MSK IMPACT™ next generation sequencing assay. Our team evaluated survival in 581 primary melanoma tumors procured by the parent P01 grant; 251 from patients who died with melanoma within five years (median survival, 2.4 years), and 330 from individuals who have lived at least five years (median follow up 8.5 years). Preliminary Results In the full dataset, we found the expected female survival advantage (log rank test P=0.049). After controlling for multiple comparisons using maximally selected ranked statistics7 the protective effect of high TMB on survival disappeared (HR=0.43, 95% CI=0.19 to 0.97, P=0.037). When stratified by sex, high TMB was associated with significantly improved melanoma specific survival among men (p=0.024), but not women (P=0.9). Broader Impacts Our study is the first to investigate the relationship between sex, tumor mutational burden, and mortality in an early stage primary cohort that has not received immunotherapy. In our small sample, we observed the expected protective effect of TMB on survival, but no evidence of gender differences in TMB or survival, despite the robust, consistent, and well-documented female survival advantage 5,6. Our results are an important first step to increasing our understanding of the relationship between mutational burden, survival, and biological sex. Limitations These results are exploratory and have not been adjusted for potential confounding factors such as stage, Breslow score, gender, or age

Seven new taxa from the butterfly subtribe Euptychiina (Lepidoptera: Nymphalidae: Satyrinae) with revisional notes on \u3ci\u3eHarjesia\u3c/i\u3e Forster, 1964 and \u3ci\u3ePseudeuptychia\u3c/i\u3e Forster, 1964

Seven new euptychiine (Lepidoptera: Nymphalidae: Satyrinae) taxa are described and named herein, namely Harjesia argentata Nakahara, Zacca and Lamas, n. sp., Orotaygetis Nakahara and Zacca, n. gen., O. surui Nakahara, Zacca and Lamas, n. sp., Euptychoides sanmarcos Nakahara and Lamas, n. sp., Pseudeuptychia cuzquenya Nakahara and Lamas, n. sp., P. languida austrina Nakahara and Lamas, n. ssp., and Godartiana astronesthes Lamas and Nakahara, n. sp. A revisional note is provided for Harjesia Forster, 1964 and Pseudeuptychia Forster, 1964, and as a result, Taygetis vrazi Kheil, 1896 is removed from Harjesia and a new taxonomic arrangement, Pseudodebis vrazi n. comb., is proposed based on both morphology and molecular data

Maternal arsenic exposure and impaired glucose tolerance during pregnancy

Background: Accumulating evidence has shown an increased risk of type 2 diabetes in general populations exposed to arsenic, but little is known about exposures during pregnancy and the association with gestational diabetes (GD). Objectives: We studied 532 women living proximate to the Tar Creek Superfund Site to investigate whether arsenic exposure is associated with impaired glucose tolerance during pregnancy. Methods: Blood glucose was measured between 24 and 28 weeks gestation after a 1-hr oral glucose tolerance test (GTT) as part of routine prenatal care. Blood and hair were collected at delivery and analyzed for arsenic using inductively coupled plasma mass spectrometry with dynamic reaction cell. Results: Arsenic concentrations ranged from 0.2 to 24.1 μg/L (ppb) (mean ± SD, 1.7 ±1.5) and 1.1 to 724.4 ng/g (ppb) (mean ± SD, 27.4 ± 61.6) in blood and hair, respectively. One-hour glucose levels ranged from 40 to 284 mg/dL (mean ± SD, 108.7 ± 29.5); impaired glucose tolerance was observed in 11.9% of women when using standard screening criterion (> 140 mg/dL). Adjusting for age, Native-American race, prepregnancy body mass index, Medicaid use, and marital status, women in the highest quartile of blood arsenic exposure had 2.8 higher odds of impaired GTT than women in the lowest quartile of exposure (95% confidence interval, 1.1–6.9) (p-trend = 0.008). Conclusions: Among this population of pregnant women, arsenic exposure was associated with increased risk of impaired GTT at 24–28 weeks gestation and therefore may be associated with increased risk of GD

Variants in Iron Metabolism Genes Predict Higher Blood Lead Levels in Young Children

Background: Given the association between iron deficiency and lead absorption, we hypothesized that variants in iron metabolism genes would predict higher blood lead levels in young children. Objective: We examined the association between common missense variants in the hemochromatosis (HFE) and transferrin (TF) genes and blood lead levels in 422 Mexican children. Methods: Archived umbilical cord blood samples were genotyped for HFE (H63D and C282Y) and TF (P570S) variants. Blood lead was measured at 24, 30, 36, 42, and 48 months of age. A total of 341 subjects had at least one follow-up blood lead level available and data available on covariates of interest for inclusion in the longitudinal analyses. We used random-effects models to examine the associations between genotype (HFE, TF, and combined HFE + TF) and repeated measures of blood lead, adjusting for maternal blood lead at delivery and child’s concurrent anemia status. Results: Of 422 children genotyped, 17.7, 3.3, and 18.9% carried the HFE H63D, HFE C282Y, and TF P570S variants, respectively. One percent of children carried both the HFE C282Y and TF P570S variants, and 3% of children carried both the HFE H63D and TF P570S variants. On average, carriers of either the HFE (β = 0.11, p = 0.04) or TF (β = 0.10, p = 0.08) variant had blood lead levels that were 11% and 10% higher, respectively, than wild-type subjects. In models examining the dose effect, subjects carrying both variants (β = 0.41, p = 0.006) had blood lead 50% higher than wild-type subjects and a significantly higher odds of having a blood lead level > 10 μg/dL (odds ratio = 18.3; 95% confidence interval, 1.9–177.1). Conclusions: Iron metabolism gene variants modify lead metabolism such that HFE variants are associated with increased blood lead levels in young children. The joint presence of variant alleles in the HFE and TF genes showed the greatest effect, suggesting a gene-by-gene-by-environment interaction