Comparative in vitro cytotoxicity of ethyl acrylate and tripropylene glycol diacrylate to normal human skin and lung cells

Summary The potential for occupational exposure to the esters of acrylic acid (acrylates) is considerable, and, thus, requires a greater understanding of the their toxicity. Confluent (70–90%) cultures of normal human epidermal keratinocytes (NHEK), dermal fibroblasts (NHDF), or bronchial epithelium (NHBE) were exposed to the monofunctional ethyl acrylate (EA), the multifunctional tripropylene glycol diacrylate (TPGDA), or TPGDA monomer in a radiation curable lacquer (Lacquer A) at equimolar dosages in order to determine human in vitro cytotoxicity. Viability of the cells after 2–24-h exposure to the representative monofunctional or multifunctional acrylate or solvent control was used to calculate an index of acute cytotoxicity (50% inhibitory dose; ID₅₀) and to determine the shape of the dose-response curves. TPGDA, Lacquer A, and EA were equally cytotoxic (ID₅₀≈0.1 μmol/cm²) to NHEK at equimolar doses. TPGDA or Lacquer A were more cytotoxic (≈100×) to NHDF or NHBE than EA. Sequential exposure of UV_A and TPGDA to NHEK indicate the potential for a synergistic cytotoxic response. These findings are consistent with observed decreases in free sulfhydryl groups (e.g., glutathione or cysteine) that parallel the dose-response-related decreases in viability. Together, these data suggest possible differences in toxicity between the monofunctional EA and multifunctional TPGDA to NHEK, NHDF, or NHBE, possibly due to the difference in the number of functional acrylate groups and/or physicochemical differences (e.g., vapor pressure) between the acrylates investigated.     

Symbiotic properties of sinorhizobia isolated from Acacia and Prosopis nodules in Sudan and Senegal

The host specificity, infection process and effectiveness of nodules produced by several African sinorhizobial strains on different Acacia and Prosopis species (Leguminosae, Mimosoideae) were studied. Sinorhizobium arboris strain HAMBI 1552^T, S. kostiense strains HAMBI 1489^T and HAMBI 1493, S. saheli strain HAMBI 1496 and S. terangae bv. acaciae strain ORS 1058 induced nitrogen fixing nodules on seedlings of the following African or Latin American species (marked with ^*): A. angustissima ^* , A. mellifera, A. nilotica, A. oerfota (synonym A. nubica), A. senegal, A. seyal, A. sieberiana, A. tortilis subsp. raddiana, P. chilensis ^* , P. cineraria, P. juliflora and P. pallida ^* . All strains increased plant yield significantly compared with uninoculated seedlings watered with nitrogen-free medium, but none appeared to be superior. The sinorhizobial strains were unable to effectively nodulate Sesbania rostrata (Papilionoideae).All roots had hairs, but particularly in the case of Acacia spp. they were often sparse. After inoculation root hairs were deformed and, in general, infection in Acacia spp. occurred through short root hairs and in Prosopis spp. through longer ones. After entry, the rhizobia filled infection pockets in the root hair, which later expanded into sac-like structures. When infection threads occurred, they usually started from sac-like structures. Elongation and ramification of the nodules indicated that Acacia spp. and Prosopis spp. have indeterminate nodules. A persistent apical meristem, which is the characteristic feature of the indeterminate nodule type, was much clearer in Prosopis spp. than in Acacia spp. Sinorhizobial strains formed tumour-like structures with undifferentiated cell tissue on the Australian acacia A. holosericea and ineffective, nodule-like structures on the African P. africana.     

Mutations in CTC1, encoding the CTS telomere maintenance complex component 1, cause cerebroretinal microangiopathy with calcifications and cysts

Cerebroretinal microangiopathy with calcifications and cysts (CRMCC) is a rare multisystem disorder characterized by extensive intracranial calcifications and cysts, leukoencephalopathy, and retinal vascular abnormalities. Additional features include poor growth, skeletal and hematological abnormalities, and recurrent gastrointestinal bleedings. Autosomal-recessive inheritance has been postulated. The pathogenesis of CRMCC is unknown, but its phenotype has key similarities with Revesz syndrome, which is caused by mutations in TINF2, a gene encoding a member of the telomere protecting shelterin complex. After a whole-exome sequencing approach in four unrelated individuals with CRMCC, we observed four recessively inherited compound heterozygous mutations in CTC1, which encodes the CTS telomere maintenance complex component 1. Sanger sequencing revealed seven more compound heterozygous mutations in eight more unrelated affected individuals. Two individuals who displayed late-onset cerebral findings, a normal fundus appearance, and no systemic findings did not have CTC1 mutations, implying that systemic findings are an important indication for CTC1 sequencing. Of the 11 mutations identified, four were missense, one was nonsense, two resulted in in-frame amino acid deletions, and four were short frameshift-creating deletions. All but two affected individuals were compound heterozygous for a missense mutation and a frameshift or nonsense mutation. No individuals with two frameshift or nonsense mutations were identified, which implies that severe disturbance of CTC1 function from both alleles might not be compatible with survival. Our preliminary functional experiments did not show evidence of severely affected telomere integrity in the affected individuals. Therefore, determining the underlying pathomechanisms associated with deficient CTC1 function will require further studies.     

Variation in telomeric repeats of Scots pine (Pinus sylvestris L.)

The shortening of telomeres, the specific structures at the end of the eukaryotic chromosomes, has been associated with ageing and loss of cell replication or regeneration capacity. The aim of this study was to determine the variation in telomeric repeats during the life-span of Scots pine (Pinus sylvestris L.) by studying different tissues and age groups ranging from immature embryos to cambium, buds, and needles of mature, 200-year-old trees. The telomeric repeats in Scots pine including interstitial and centromeric repeats ranged from 0.9 up to 25 kb, and true telomeres were evaluated to have a mean length of 19.3 kb (±SE 0.17). Telomeres were observed to shorten with increasing tissue differentiation, embryonal samples having the longest repeats with an average length of 21.1 (±0.34) to 21.7 kb (±0.42) and the needles having, on average, the shortest repeats of 18.1 kb (±0.24). After germination, ageing per se had no significant effect on the length of telomeric repeats in cambium, bud, or needle tissues. In the older trees (50–200 years of age), the telomeres in stem cambium showed shortening towards the tree top. This is the first observation on such a position-related variation in telomeric repeats. Finally, there was a remarkable genotypic variation in the length of telomeric repeats, and this was consistent over the tissue types. In the small number of tested donor trees, the genotypic differences were not related to regeneration ability in tissue culture.     

Variation in Hsp70 levels after cold shock: signs of evolutionary responses to thermal selection among Leptinotarsa decemlineata populations

Individuals of widely spread species are expected to show local adaption in temperature tolerance as they encounter a range of thermal conditions. We tracked thermal adaptations of the Colorado potato beetle (Leptinotarsa decemlineata) that invaded Europe within the last 100 years. It has occupied various conditions although, like the majority of invasive species, it lost a measurable amount of neutral genetic variation due to bottleneck effect when it invaded Europe. We exposed diapausing beetles originated from three different latitudes (54°N, 59°N, 60°N) to cold shock (-5°C, 1.5 hrs) in order to test if beetles from the northern populations express differential levels of cold-induced and constitutive Hsp70 compared to the beetles from milder temperature regime. The level of cold-induced Hsp70 was lowest in the northernmost beetle populations while the level of constitutive Hsp70 did not differ with the population. Moreover, the southernmost beetles were more plastic in their response to cold shock than the northernmost beetles. These results suggest that physiological adaptation, like the synthesis of Hsp70, can evolve very quickly.     

The epidermal basement membrane is a composite of separate laminin- or collagen IV-containing networks connected by aggregated perlecan, but not by nidogens

The basement membrane between the epidermis and the dermis is indispensable for normal skin functions. It connects, and functionally separates, the epidermis and the dermis. To understand the suprastructural and functional basis of these connections, heterotypic supramolecular aggregates were isolated from the dermal-epidermal junction zone of human skin. Individual suprastructures were separated and purified by immunomagnetic beads, each recognizing a specific, molecular component of the aggregates. The molecular compositions of the suprastructures were determined by immunogold electron microscopy and immunoblotting. A composite of two networks was obtained from fibril-free suspensions by immunobeads recognizing either laminin 332 or collagen IV. After removal of perlecan-containing suprastructures or after enzyme digestion of heparan sulfate chains, a distinct network with a diffuse electron-optical appearance was isolated with magnetic beads coated with antibodies to collagen IV. The second network was more finely grained and comprised laminin 332 and laminins with α5-chains. The core protein of perlecan was an exclusive component of this network whereas its heparan sulfate chains were integrated into the collagen IV-containing network. Nidogens 1 and 2 occurred in both networks but did not form strong molecular cross-bridges. Their incorporation into one network appeared to be masked after their incorporation into the other one. We conclude that the epidermal basement membrane is a composite of two structurally independent networks that are tightly connected in a spot-welding-like manner by perlecan-containing aggregates.     

Inhibitory Activity of the Isoflavone Biochanin A on Intracellular Bacteria of Genus Chlamydia and Initial Development of a Buccal Formulation

Given the established role of Chlamydia spp. as causative agents of both acute and chronic diseases, search for new antimicrobial agents against these intracellular bacteria is required to promote human health. Isoflavones are naturally occurring phytoestrogens, antioxidants and efflux pump inhibitors, but their therapeutic use is limited by poor water-solubility and intense first-pass metabolism. Here, we report on effects of isoflavones against C. pneumoniae and C. trachomatis and describe buccal permeability and initial formulation development for biochanin A. Biochanin A was the most potent Chlamydia growth inhibitor among the studied isoflavones, with an IC₅₀ = 12 µM on C. pneumoniae inclusion counts and 6.5 µM on infectious progeny production, both determined by immunofluorescent staining of infected epithelial cell cultures. Encouraged by the permeation of biochanin A across porcine buccal mucosa without detectable metabolism, oromucosal film formulations were designed and prepared by a solvent casting method. The film formulations showed improved dissolution rate of biochanin A compared to powder or a physical mixture, presumably due to the solubilizing effect of hydrophilic additives and presence of biochanin A in amorphous state. In summary, biochanin A is a potent inhibitor of Chlamydia spp., and the in vitro dissolution results support the use of a buccal formulation to potentially improve its bioavailability in antichlamydial or other pharmaceutical applications.     

Effects of a biocontrol agent Chondrostereum purpureum on sprouting of Sorbus aucuparia and Populus tremula after four growing seasons

The efficacy of a decay fungus Chondrostereum purpureum (Pers. ex Fr.) Pouzar to prevent sprouting of two tree species, rowan (Sorbus aucuparia L.) and European aspen (Populus tremula L.) was investigated in a four-growing season study. The efficacy of C. purpureum compared with the control (cutting only) increased during the whole study period indicating that long-term studies are needed to reveal the real effect of the biocontrol treatment. At the end of the study period the mortality of treated rowan stumps was 50 % and that of European aspen 77 %, i.e. clearly higher than in the control with mortality 14 and 52 % respectively. The height of living sprouts was considerably lower in the C. purpureum treatment than in the control. Furthermore, our results indicated that a high number of surrounding conspecific saplings decreased whereas a high number of red raspberry (Rubus idaeus L.) shoots increased mortality of C. purpureum treated stumps.