A Deletion in the α2B‐Adrenergic Receptor Gene and Autonomic Nervous Function in Central Obesity

Objective: We investigated the impact of a three‐amino acid deletion (12Glu9) polymorphism in the α_2B‐adrenergic receptor gene on autonomic nervous function. The short form (Glu⁹/Glu⁹) of the polymorphism has previously been associated with a reduced basal metabolic rate in obese subjects. Because autonomic nervous function participates in the regulation of energy metabolism, there could be a link between this polymorphism and autonomic nervous function. Research Methods and Procedures: Data of a 10‐year follow‐up study with 126 nondiabetic control subjects and 84 type 2 diabetic patients were used to determine the effects of the 12Glu9 polymorphism on autonomic nervous function. A deep breathing test and an orthostatic test were used to investigate parasympathetic and sympathetic autonomic nervous function. In addition, cardiovascular autonomic function was studied using power spectral analysis of heart rate variability. Results: No significant differences were found in the frequency of the 12Glu9 deletion polymorphism between nondiabetic and diabetic subjects. The nondiabetic men with the Glu⁹/Glu⁹ genotype, especially those with abdominal obesity, had significantly lower total and low‐frequency power values in the power spectral analysis when compared with other men. Furthermore, in a longitudinal analysis of 10 years, the decrease in parasympathetic function was greater in nondiabetic men with the Glu⁹/Glu⁹ genotype than in the men with the Glu⁹/Glu¹² or Glu¹²/Glu¹² genotypes. Discussion: The results of the present study suggest that the 12Glu9 polymorphism of the α_2B‐adrenergic receptor gene modulates autonomic nervous function in Finnish nondiabetic men. In the nondiabetic men with the Glu⁹/Glu⁹ genotype, the general autonomic tone is depressed, and vagal activity especially becomes impaired with time. Furthermore, this association is accentuated by central obesity.     

Functional polymorphism in porcine CYP2E1 gene: Its association with skatole levels

Raising intact male pigs would have a significant economic impact on the pork industry. However, the presence of skatole (a major cause of boar taint) in meat from intact male pigs could be highly objectionable to consumer. The excessive accumulation of skatole in fat is a major cause of boar taint, and is associated with defective expression of cytochrome P4502E1 (CYP2E1). In pigs, it has been found that CYP2E1 is negatively correlated with accumulation of skatole. The searching for polymorphism of CYP2E1 and the relevant functional analysis would help develop a genetic marker for the selection of pigs with low skatole levels in fat. The aim of this study was to measure the expression pattern of CYP2E1 mRNA in various tissues of the pig, to identify genetic polymorphisms, and to evaluate the functional relevance of polymorphic sites with respect to the skatole level in fat. We show herein that a substitution of G → A at base 1423 of the CYP2E1 gene in the liver causes a significant decrease in the expressed CYP2E1 level. Our data suggest that the G → A substitute might be at least partially responsible for a high level of skatole in pigs. We believe that this is an important step toward the selection of genetic markers for boar taint by lowering fat levels of skatole in fat.     

Cloning and characterization of novel tumor-targeting immunocytokines based on murine IL7

We generated and characterized novel antibody-cytokine fusion proteins (“immunocytokines”) based on murine interleukin-7 (IL7), an immunomodulatory protein which has previously shown anti-cancer activity in preclinical models and whose human counterpart is currently being investigated in clinical trials. The sequential fusion of the clinical-stage antibody fragment scFv(F8), specific to a tumor-associated splice isoform of fibronectin, yielded an immunocytokine (termed “F8-mIL7”) of insufficient pharmaceutical quality and in vivo tumor targeting performance, with a striking dose dependence on tumor targeting selectivity. By contrast, a novel immunocytokine design (termed “F8-mIL7-F8”), in which two scFv moieties were fused at the N- and C-terminus of murine IL7, yielded a protein of excellent pharmaceutical quality and with improved tumor-targeting performance [tumor: blood ratio = 16:1, 24 h after injection]. Both F8-mIL7 and F8-mIL7-F8 could induce tumor growth retardation in immunocompetent mice, but were not able to eradicate F9 tumors. The combination of F8-mIL7-F8 with paclitaxel led to improved therapeutic results, which were significantly better compared to those obtained with saline treatment. The study indicates how the engineering of novel immunocytokine formats may help generate fusion proteins of acceptable pharmaceutical quality, for those immunomodulatory proteins which do not lend themselves to a direct fusion with antibody fragments.     

DNA polymorphism in Allium cepa cytoplasms and its implications concerning the origin of onions

Summary Mitochondrial and chloroplast DNA was isolated from fertile and cytoplasmic male sterile cultivars of cultivated onions. Restriction fragment length polymorphism led to the distinction between cytoplasms S and M. Mitochondrial DNA patterns from S cytoplasms appeared dentical and characterized mostly male sterile lines. An open-pollinated variety was found to bear this cytoplasm and thought to be the origin of S types. Mitochondrial DNA patterns from M cytoplasms were subdivided into four types, M₁ and M₂ corresponding to normal N cytoplasm, M₃ and M₄ probably corresponding to T cytoplasms. S and M cytoplasms were also distinguished by chloroplast DNA restriction patterns. Our results confirm previous genetic distinction between S, N and T cytoplasms.     

Geographic and breed distribution of an Msp I PCR-RFLP in the bovine growth hormone (bGH) gene

Information is presented on the frequency of the Msp I (-) allele in the third intron of the bovine growth hormone gene in a large number of cattle breeds. Consideration of the breed frequencies in relation to their geographic origin shows a low frequency for breeds originating in Northern Europe, moderate frequencies for breeds originating in Eastern Europe or the countries surrounding the Mediterranean basin, and very high frequencies for breeds originating in the Indian subcontinent. Consideration of breed frequencies in relation to breed type, shows low to moderate frequencies for the humpless breeds, high frequencies for the humped breeds. Various explanations for this distribution are discussed, among them the possibility that the Msp I (-) allele originated in the Bos indicus breeds of the Indian subcontinent, from which it diffused through the humpless Bos taurus breeds of Eastern Europe, the Mediterranean basin, eventually reaching Western, Northern Europe, Western Africa in low frequencies.     

High interleukin-4 expression and interleukin-4 gene polymorphisms are associated with susceptibility to human paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is caused by dimorphic fungi fromtheParacoccidioides brasiliensis complex. Previous studies havedemonstrated that the severity of disease is associated with a T-helper 2 immuneresponse characterised by high interleukin (IL)-4 production. In the present study weanalysed two polymorphisms in the IL-4 gene (-590 C/T and intron-3microsatellite) in 76 patients with PCM and 73 control subjects from an endemic area.The production of IL-4 by peripheral blood mononuclear cells after antigen orphytohaemagglutinin stimulation was determined by ELISA. A significant correlationwas observed between the RP2/RP2 intron-3 genotype and infection withParacoccidioides sp. (p = 0.011), whereas the RP1/RP1 genotypewas correlated with resistance. No significant correlation was observed forthe IL-4 promoter polymorphism. Furthermore, the low IL-4expression observed in the control group compared with patients was associated withthe RP1/RP1 genotype. These results suggest that IL-4polymorphismsmight be associated with the ability of the host to control Paracoccidioidessp. infection. The relevance of this polymorphism is supported by theobservation that patients with disease produce high levels of IL-4 following mitogenor antigen stimulation. The IL-4 gene is located in the cytokinecluster region of chromosome 5 where other polymorphisms have also beendescribed.     

Hydraulic architecture and water relations of Spartium junceum branches affected by a mycoplasm disease

Plant water relations, xylem anatomy and the hydraulic architecture of 1‐year‐old twigs of Spartium junceum, both healthy and affected by a phytoplasm disease, were studied. The disease causes twigs to be either shortened (witches broom disease, WBD) or flat (fasciate disease, FD). WBD twigs show a sevenfold increase in total leaf area, smaller and shorter xylem conduits, a higher stomatal conductance (g_l) and a decline of minimum leaf water potentials ( Ψ _l) below the turgor loss point. FD twigs had nearly twice the leaf area of the healthy controls as well as high g_l values and Ψ _l values below the turgor loss point. Moreover, significant differences between healthy and affected twigs in stem stomatal conductance (g_s) and in the total stem area were recorded. Affected twigs die back under drought stress, which is explained by a pronounced loss of hydraulic conductivity of the infected stems (40 and 60%) in FD and WBD as well as by the unfavourable ratio of weighted conduit radius ( Σ r⁴) to total surface area (A_t), so that the efficiency of the stem in supplying the whole transpiring area with water is strongly reduced.     

Multi‐Length‐Scale Morphologies in PCPDTBT/PCBM Bulk‐Heterojunction Solar Cells

Additives are known to improve the performance of organic photovoltaic devices based on mixtures of a low bandgap polymer, poly[2,6‐(4,4‐bis(2‐ethylhexyl)‐4H‐cyclopenta[2,1‐b;3,4‐b′]‐dithiophene)‐alt‐4,7‐(2,1,3‐benzothiadiazole)] (PCPDTBT) and [6,6]‐phenyl C61‐butyric acid methyl ester (PCBM). The evolution of the morphology during the evaporation of the mixed solvent, which comprises additive and chlorobenzene (CB), is investigated by in‐situ grazing incidence X‐ray scattering, providing insight into the key role the additive plays in developing a multi‐length‐scale morphology. Provided the additive has a higher vapor pressure and a selective solubility for PCBM, as the host solvent (CB) evaporates, the mixture of the primary solvent and additive becomes less favorable for the PCPDTBT, while completely solubilizing the PCBM. During this process, the PCPDTBT first crystallizes into fibrils and then the PCBM, along with the remaining PCPDTBT, is deposited, forming a phase‐separated morphology comprising domains of pure, crystalline PCPDTBT fibrils and another domain that is a PCBM‐rich mixture with amorphous PCPDTBT. X‐ray/neutron scattering and diffraction methods, in combination with UV–vis absorption spectroscopy and transmission electron microscopy, are used to determine the crystallinity and phase separation of the resultant PCPDTBT/PCBM thin films processed with or without additives. Additional thermal annealing is carried out and found to change the packing of the PCPDTBT. The two factors, degree of crystallinity and degree of phase separation, control the multi‐length‐scale morphology of the thin films and significantly influence device performance.     

Integrin binding human antibody constant domains--probing the C-terminal structural loops for grafting the RGD motif

Recently, it has been demonstrated that loops of the crystallizable fragment of IgG1 (IgG1-Fc) can be engineered to form antigen-binding sites. In this work C-terminal structural loops in the CH3 domains of homodimeric IgG1-Fc have been functionalized to form integrin-binding sites in order to probe the effect of engineering on structural integrity and thermal stability of IgG1-Fc as well as on binding to the ligands Protein A, CD16 and FcRn, respectively. The peptide sequence GCRGDCL - a disulfide-bridged cyclic heptapeptide that confers binding to human αvβ3 integrin was introduced into AB, CD and/or EF loops and single and double mutants were heterologously expressed in Pichia pastoris. Integrin binding of engineered IgG-Fc was tested using both binding to coated αvβ3 integrin in ELISA or to αvβ3-expressing K562 cells in FACS analysis. Additionally, blocking of αvβ3-mediated cell adhesion to vitronectin was investigated. The data presented in this report demonstrate that bioactive integrin-binding peptide(s) can be grafted on the C-terminal loops of IgG-Fc without impairing binding to effector molecules. Observed differences between the investigated variants in structural stability and integrin binding are discussed with respect to the known structure of IgG-Fc and its structural loops.     

In vitro chromosome doubling of nineZantedeschia cultivars

Tetraploid plants have been produced from nineZantedeschia cultivars usingin vitro colchicine treatment. Rapidly-multiplying shoot cultures were treated on a medium containing 0.05% colchicine for 1, 2 or 4 days to induce chromosome doubling. Following the treatment, most shoots were killed but the surviving shoots were multiplied for several subcultures. These shoots were then rootedin vitro and transferred to a greenhouse. Plants were screened 2 months later by measuring stomatal length, and 110 out of 565 plants were selected as putative tetraploids with a stomatal length significantly greater than in diploid control plants. Chromosome counts were carried out on root tips from 44 plants and confirmed that 38 were tetraploids, 2 were chimeras (predominantly tetraploid with a few octoploid cells), and 4 were diploids. Stomatal length has been rechecked in mature tetraploid plants of the cultivar Black Magic, demonstrating that stomatal length is a good indicator of ploidy level inZantedeschia. This study has shown that multiplying colchicine-treated shootsin vitro for several subcultures prior to transfer to soil produced very few chimeras. The stomatal length measurements are non-destructive and allow the rapid screening of a population for tetraploids.