Synthesis of a human insulin gene. VII. Synthesis of preproinsulin-like human DNA, its cloning and expression in M13 bacteriophage

A 74-bp DNA sequence coding for the pre sequence of human preproinsulin and containing EcoRI termini was synthesized by the chemical enzymatic method, joined with previously synthesized proinsulin DNA, and cloned in the M 13mp8 vector. A clone pNB82 -121 was identified by DNA sequence which confirmed the correct orientation of the pre sequence to the proinsulin DNA. The EcoRI site at the junction of pre- and proinsulin DNA was eliminated by removing a triplet ATT using a synthetic 19-mer primer. To simplify preproinsulin isolation and to study its expression in the M 13 system, a 25-bp affinity leader sequence coding for (glu)7 was inserted at the remaining EcoRI site; this put the preproinsulin DNA in a correct reading frame with the AUG initiation codon of beta-galactosidase. Preproinsulin was expressed under lac promoter control as analyzed by a radioimmunoassay (RIA) against C-peptide.     

Rat plasma 25-hydroxyvitamin D3 binding protein: An inhibitor of the 25-hydroxyvitamin D3-1 α-hydroxylase

An antibody was prepared from serum of rabbits injected with a pure inhibitor protein obtained from rat serum for chick renal 25-hydroxyvitamin D₃-1α-hydroxylase. The antibody was separated from the endogenous inhibitor in rabbit serum. The antibody shows a single precipitin line with the rat serum antigen and with crude calf serum. Furthermore, the antibody removes the 4.0 S 25-hydroxyvitamin D₃ binding protein from rat serum. The removal of the 25-hydroxyvitamin D₃ binding protein from rat serum with antibody brings about a proportionate removal of inhibitor of the 25-hydroxyvitamin D₃-1α-hydroxylase. The pure inhibitor binds 25-hydroxyvitamin D₃, as demonstrated by sucrose density gradient sedimentation, and shows specificity of binding identical to the serum transport globulin for 25-hydroxyvitamin D₃. Thus, the previously reported inhibitor of the 25-hydroxyvitamin D₃-1α-hydroxylase in rat preparations is the serum 25-hydroxyvitamin D₃ transport protein or some derivative thereof. The antibody added to rat renal mitochondrial preparations does increase the activity of the 1- and 24-hydroxylases slightly but not markedly.     

Role of oxidants on calcium and sodium movement in healthy and diseased cardiac myocytes

In this review article we give an overview of current knowledge with respect to redox-sensitive alterations in Na⁺ and Ca²⁺ handling in the heart. In particular, we focus on redox-activated protein kinases including cAMP-dependent protein kinase A (PKA), protein kinase C (PKC), and Ca/calmodulin-dependent protein kinase II (CaMKII), as well as on redox-regulated downstream targets such as Na⁺ and Ca²⁺ transporters and channels. We highlight the pathological and physiological relevance of reactive oxygen species and some of its sources (such as NADPH oxidases, NOXes) for excitation—contraction coupling (ECC). A short outlook with respect to the clinical relevance of redox-dependent Na⁺ and Ca²⁺ imbalance will be given.     

Detection Probability and Sources of Variation in White-Tailed Deer Spotlight Surveys

Abstract: As a first step in understanding structure and dynamics of white-tailed deer (Odocoileus virginianus) populations, managers require knowledge of population size. Spotlight counts are widely used to index deer abundance; however, detection probabilities using spotlights have not been formally estimated. Using a closed mark—recapture design, we explored the efficiency of spotlights for detecting deer by operating thermal imagers and spotlights simultaneously. Spotlights detected only 50.6% of the deer detected by thermal imagers. Relative to the thermal imager, spotlights failed to detect 44.2% of deer groups (≥1 deer). Detection probabilities for spotlight observers varied between and within observers, ranging from 0.30 (SE = 0.053) to 0.66 (SE = 0.058). Managers commonly assume that although road counts based on convenience sampling designs are imperfect, observers can gather population-trend information from repeated counts along the same survey route. Our results indicate detection rate varied between and within observers and surveyed transects. If detection probabilities are substantially affected by many variables, and if transect selection is not based on appropriate sampling designs, it may be impractical to correct road spotlight counts for detection probabilities to garner unbiased estimates of population size.     

Pertactin-deficient Bordetella pertussis isolates in Poland—a country with whole-cell pertussis primary vaccination

The introduction of pertussis vaccination in the 1950s resulted in a significant decrease in the incidence of disease. However, since the 1990s many highly vaccinated countries have observed the re-emergence of the disease. One of the causes of this phenomenon might be related to the adaptation of Bordetella pertussis to vaccination. The purpose of the presented study was an investigation of the emergence and spread of vaccine antigen-deficient B. pertussis isolates in Poland and genomic characterization of the currently circulating pathogen population using PFGE, MLVA and MAST. The results revealed that all tested isolates expressed Ptx, FHA and ACT antigens but 15.4% (4/26) of isolates from 2010 to 2016 were Prn-deficient. Moreover, one TcfA-deficient isolate was collected in 2015. The genotyping showed a genetic distinction between the isolates circulating in 2010–2016 and isolates from previous periods. The majority of currently circulating isolates belonged to PFGE group IV (96.2%), type MT27 (73.1%), and carried ptxA1-ptxC2-ptxP3-prn2-tcfA2-fim2-1-fim3-1 alleles (61.5%). The unique genetic structure of the B. pertussis population in Poland has changed since 2010 and became similar to that observed in countries with aP vaccination. This could be a result of increasing use of aP vaccines (60% of primary vaccination in 2013) over wP vaccines, which have been broadly used for primary vaccination in Poland for decades.     

Biochemical synthesis of uniformly 13C-labeled diterpene hydrocarbons and their bioconversion to diterpenoid phytoalexins in planta

Phytocassanes and momilactones are the major diterpenoid phytoalexins inductively produced in rice as bioactive substances. Regardless of extensive studies on the biosynthetic pathways of these phytoalexins, bioconversion of diterpene hydrocarbons is not shown in planta. To elucidate the entire biosynthetic pathways of these phytoalexins, uniformly ¹³C-labeled ent-cassadiene and syn-pimaradiene were enzymatically synthesized with structural verification by GC–MS and ¹³C-NMR. Application of the ¹³C-labeled substrates on rice leaves led to the detection of ¹³C-labeled metabolites using LC-MS/MS. Further application of this method in the moss Hypnum plumaeforme and the nearest out-group of Oryza species Leersia perrieri, respectively, resulted in successful bioconversion of these labeled substrates into phytoalexins in these plants. These results demonstrate that genuine biosynthetic pathways from these diterpene hydrocarbons to the end product phytoalexins occur in these plants and that enzymatically synthesized [U-¹³C₂₀] diterpene substrates are a powerful tool for chasing endogenous metabolites without dilution with naturally abundant unlabeled compounds.     

Immunochemical evidence for the presence of 5mC, 6mA and 7mG in human, Drosophila and mealybug DNA

We have reported that production and characterization of antibodies highly specific to 5-methyl-cytosine (5mC) and the development of a sensitive immunochemical method for the detection of 5mC in DNA [FEBS Lett. (1982) 150, 469]. Extension of this method to two other modified bases, 6-methyladenine (6mA) and 7-methylguanine (7mG), is reported here. By use of this immunochemical approach, we are able to detect 5mC, 6mA and 7mG in human and Drosophila DNA and confirm their presence in the DNA of two mealybug species.     

Noninvasive Hair Sampling and Genetic Tagging of Co-Distributed Fishers and American Martens

ABSTRACT Estimation of abundance is important for assessing population responses to management actions. Accurate abundance estimates are particularly critical for monitoring temporal variation following reintroductions when the management goal is to attain population sizes capable of sustaining harvest. Numerous reintroductions have taken place in the Great Lakes region of North America, including efforts to restore extirpated fishers (Martes pennanti) and American martens (M. americana). We used a DNA-based noninvasive hair-snaring method based on one trap design and trapping -grid configuration, and evaluated capture—mark—recapture (CMR) analytical approaches to simultaneously estimate population size for co-distributed fishers and American martens in a 671-km² area of the Ottawa National Forest in the western Upper Peninsula of Michigan, USA. We included harvest as a final recapture period to increase probability of recapture and to evaluate potential violations of geographic closure assumptions. We used microsatellite markers to identify target species, eliminate congener species, and provide individual identity for estimation of abundance. Population estimates for fishers and martens on the study area ranged from 35 to 60 and 8 to 28, respectively. Estimators incorporating harvest data resulted in up to a 40% increase in abundance estimates relative to estimators without harvest. We considered population estimates not including harvest data the most appropriate for the study due to timing of sampling and environmental factors, but inclusion of harvested individuals was shown to be useful as a means to detect violations of the assumption of geographic closure. We suggest improvements on future CMR sampling designs for larger landscape scales of relevance to management through incorporation of habitat or historical harvest data. Noninvasive genetic methods that simultaneously estimate the numerical abundance of co-distributed species can greatly decrease assessment costs relative to traditional methods, and increase resulting demographic and ecological information.     

Ethylation pf DNA and protamine by ethyl methanesulfonate in the germ cells of male mice and the relevancy of these molecular targets to the induction of dominant lethals

The molecular dosimetry of ethyl methanesulfonate (EMS) in the germ cells of male mice has been investigated. The mice were injected i.p. with 200 mg/kg of [³H]EMS and the ethylations per sperm head, per deoxynucleotide, and per unit of protamine were then determined over a 2-week period. The ethylations per sperm head closely paralleled the dominant-lethal frequency curve for EMS, reaching a maximum of 5 to 6.5 million ethylations per vas sperm head at 8 to 10 days after treatment. Ethylation of sperm DNA was greatest at 4 h after treatment, with 5.7 ethylations/10⁵ deoxynucleotides, and gradually decreased to 2.2 ethylations/10⁵ deoxynucleotides at 15 days after treatment. The ethylation of sperm DNA did not increase in the germ-cell stages most sensitive to EMS, ans was not correlated with the dominant-lethal frequency curve for EMS. However, ethylation of sperm protamine did increase in the germ-cell stages most sensitive to EMS, and showed an excellent correlation with the incidence of dominant lethals produced by EMS in the germ cells.A model is presented to explain, at a molecular level, how dominant lethals may be induced in mouse germ cells by EMS. Ethylation by cysteine sulfhydryl groups contained in mouse-sperm protamine could block normal disulfidebond formation, preventing proper chromatin condensation in the sperm nucleus. Stresses in the chromatin structure could then eventually lead to chromosome breakage, with resultant dominant lethality.