Cyclic nucleotides affect growth and differentiation of cultured rat embryonic shields

The modified organ culture of rat embryonic shields provides favorable conditions during 2 weeks for the differentiation of main tissue types. Since the terminal differentiation in explants is inferior to that obtained in the homografts of the same shields under the kidney capsule, we tried to improve the culture medium by adding some known regulatory molecules: db-cAMP, db-cGMP, ATP, AMP, and butyric acid. These agents were added to the liquid medium in the concentration of 1 mM. In the first part of the study the explants were fixed and weighed after 8 or 14 days in vitro culture, and histological sections were examined. When the explants were treated with db-cAMP during the second week of culture, the skeletal muscle appeared more frequently in the treated series than in controls, and the weight of the treated explants was sometimes increased when compared with the control series. The db-cGMP had no effect on differentiation, but stimulated the growth of the explants when applied during the first week of culture. On the contrary, the db-cAMP when added during the first week, severely impeded the growth of explants. Other agents seem to be ineffective. In the second part, the content of cAMP and cGMP was measured in normal explants. The radioimmunoassay showed the same content of cAMP and cGMP during the entire culture period. In the third part of our study the incorporation of tritiated uridine and tritiated thymidine was measured during the second week of culture after the addition of db-cAMP. During the first days of treatment with db-cAMP the uptake of tritiated uridine and thymidine was inhibited, whereas on the seventh day the uptake was similar to that of the control. We can conclude that both cyclic nucleotides have a visible effect on growth whereas only cAMP has a positive impact on the differentiation of myotubes in cultured rat embryonic shields.     

Phenylketonuria in U.S. blacks: molecular analysis of the phenylalanine hydroxylase gene

We investigated the frequency, origin, and molecular basis of phenylketonuria (PKU) in U.S. blacks. On the basis of 10 years of Maryland newborn-screening data, we found the frequency to be 1/50,000, or one-third that in whites. We performed haplotype analysis of the phenylalanine hydroxylase (PAH) gene of 36 U.S. blacks, 16 from individuals with classical PKU and 20 from controls. In blacks, 20% of wild-type PAH alleles have a common Caucasian haplotype (i.e., haplotype 1), whereas 80% had a variety of haplotypes, all rare in Caucasians and Asians. One of these, haplotype 15, accounted for a large fraction (30%). Among black mutant PAH alleles, 20% have a haplotype (i.e., either haplotype 1 or haplotype 4) common in Caucasians; 40% have a haplotype rare in Caucasians and Asians, and 40% have one of two previously undescribed haplotypes. Both can be derived from known haplotypes by a single event. One of these haplotypes is characterized by a new MspI restriction site, located in intron 8, which was present in five of 16 black mutant alleles but was not present in 60 U.S. black control, 20 U.S. Caucasian control, or 20 Caucasian mutant PAH alleles. Sequence analysis of DNA from a single individual, homozygous for the new MspI associated haplotype, shows homozygosity for a C----T transition at nucleotide 896 in exon 7 of the PAH cDNA, resulting in the conversion of leucine 255 to serine (L255S).     

Adherent cell requirement for PWM-stimulated factor suppression of natural killer cytotoxicity

Natural killer cytotoxicity is enhanced in a 5-day mixed-leukocyte culture (MLC). This augmentation of NK can be suppressed by factor released from pokeweed-mitogen (PWM)-activated peripheral blood cells. To maximize activity, the PWM-soluble factor must be present within the first 6 hr of the MLC, and is required to be in the culture for at least 4 hr. The affect of the PWM factor is not due to the destruction of either effector or stimulator cells. This factor does, however, require the presence of an adherent, non-T cell in the effector population. Directly incubating adherent cells with the PWM-induced soluble factor activates this population to mediate suppression. Thus the adherent cells are required for the PWM-induced suppression of NK cytotoxicity, indicating a possible regulatory mechanism for this cytotoxicity.     

Somatic antigens of Shigella. The structure of the specific polysaccharide of Shigella newcastle (Sh. flexneri type 6) lipopolysaccharide.

The specific polysaccharide was obtained from the lipopolysaccharide of Shigella newcastle by mild acid hydrolysis and further purified by permeation chromatography on Sephadex G-50. It was found to consist of L-rhamnose, 2-acetamido-2-deoxy-D-galactose, D-galacturonic acid residues and O-acetyl groups in the molar ratios of 2:1:1:1. On the basis of 1H and 13C nuclear magnetic resonance spectroscopy, methylation analysis, partial acid hydrolysis, Smith degradation, and chromium trioxide oxidation, the following structure can be assigned to the repeating oligosaccharide unit of the polysaccharide:-4)DGalA(beta 1-3)DGalNAc-(beta 1-2)LAc3Rha(alpha 1-2)LRha(alpha 1-, where GalA = galacturonic acid. GalNAc = N-acetylgalactosamine, Ac3Rha = 3-O-acetylrhamnose. The structural and immunochemical data presented prove that Sh. newcastle lipopolysaccharide belongs to a 'non-classical' type of somatic antigens with acidic O-specific polysaccharide chains.     

Olomoucine II,but Not Purvalanol A,Is Transported by Breast Cancer Resistance Protein (ABCG2) and P-Glycoprotein (ABCB1)

Purine cyclin-dependent kinase inhibitors have been recognized as promising candidates for the treatment of various cancers; nevertheless, data regarding interaction of these substances with drug efflux transporters is still lacking. Recently, we have demonstrated inhibition of breast cancer resistance protein (ABCG2) by olomoucine II and purvalanol A and shown that these compounds are able to synergistically potentiate the antiproliferative effect of mitoxantrone, an ABCG2 substrate. In this follow up study, we investigated whether olomoucine II and purvalanol A are transported by ABCG2 and ABCB1 (P-glycoprotein). Using monolayers of MDCKII cells stably expressing human ABCB1 or ABCG2, we demonstrated that olomoucine II, but not purvalanol A, is a dual substrate of both ABCG2 and ABCB1. We, therefore, assume that pharmacokinetics of olomoucine II will be affected by both ABCB1 and ABCG2 transport proteins, which might potentially result in limited accumulation of the compound in tumor tissues or lead to drug-drug interactions. Pharmacokinetic behavior of purvalanol A, on the other hand, does not seem to be affected by either ABCG2 or ABCB1, theoretically favoring this drug in the potential treatment of efflux transporter-based multidrug resistant tumors. In addition, we observed intensive sulfatation of olomoucine II in MDCKII cell lines with subsequent active efflux of the metabolite out of the cells. Therefore, care should be taken when performing pharmacokinetic studies in MDCKII cells, especially if radiolabeled substrates are used; the generated sulfated conjugate may largely contaminate pharmacokinetic analysis and result in misleading interpretation. With regard to chemical structures of olomoucine II and purvalanol A, our data emphasize that even drugs with remarkable structure similarity may show different pharmacokinetic behavior such as interactions with ABC transporters or biotransformation enzymes.     

Discovery of the oldest known Pterygota in the Lower Carboniferous of the Upper Silesian Basin in the Czech Republic (Insecta: Archaeorthoptera)

The earliest pterygote (winged insect), dated from the Lower Carboniferous (Namurian A/E1, circa 324 millions years ago) is described from the Upper Silesian Basin in the Czech Republic. On the basis of its wing venation, it is attributed to the Archaeorthoptera Béthoux and Nel, 2002, crown group of the “Orthoptera”. Besides Apterygota (Collembola and Archaeognatha) known from the Lower Devonian, extremely rare pterygote insects are known from Lower Carboniferous deposits when they first appeared. The present discovery supports the hypothesis of the presence of the ancestor lineage of the orthopteroid in the Lower Carboniferous ecosystems.     

Plasmonic Properties of Gold Nanostructures on Gold Film

This paper reports on a systematic study of the plasmonic properties of periodic arrays of gold cylindrical nanoparticles in contact with a gold thin film. Depending on the gold film thickness, it observes several plasmon bands. Using a simple analytical model, it is able to assign all these modes and determine that they are due to the coupling of the grating diffraction orders with the propagating surface plasmons travelling along the film. With finite difference time domain (FDTD) simulations, it demonstrates that large field enhancement occurs at the surface of the nanocylinders due to the resonant excitation of these modes. By tilting the sample, it also observes the evolution of the spectral position of these modes and their tuning through nearly the whole visible range is possible. Such plasmonic substrates combining both advantages of the propagative and localised surface plasmons could have large applications in enhanced spectroscopies.

     

Positive selection and convergent evolution shape molecular phenotypic traits of innate immunity receptors in tits (Paridae)

Despite widespread variability and redundancy abounding animal immunity, little is currently known about the rate of evolutionary convergence (functionally analogous traits not inherited from a common ancestor) in host molecular adaptations to parasite selective pressures. Toll‐like receptors (TLRs) provide the molecular interface allowing hosts to recognize pathogenic structures and trigger early danger signals initiating an immune response. Using a novel combination of bioinformatic approaches, here we explore genetic variation in ligand‐binding regions of bacteria‐sensing TLR4 and TLR5 in 29 species belonging to the tit family of passerine birds (Aves: Paridae). Three out of the four consensual positively selected sites in TLR4 and six out of 14 positively selected positions in TLR5 were located on the receptor surface near the functionally important sites, and based on the phylogenetic pattern evolved in a convergent (parallel) manner. This type of evolution was also seen at one N‐glycosylation site and two positively selected phosphorylation sites, providing the first evidence of convergence in post‐translational modifications in evolutionary immunology. Finally, the overall mismatch between phylogeny and the clustering of surface charge distribution demonstrates that convergence is common in overall TLR4 and TLR5 molecular phenotypes involved in ligand binding. Our analysis did not reveal any broad ecological traits explaining the convergence observed in electrostatic potentials, suggesting that information on microbial symbionts may be needed to explain TLR evolution. Adopting state‐of‐the‐art predictive structural bionformatics, we have outlined a new broadly applicable methodological approach to estimate the functional significance of positively selected variation and test for the adaptive molecular convergence in protein‐coding polymorphisms.     

Patterns of host–parasite associations in tropical lice and their passerine hosts in Cameroon

Coevolutionary processes that drive the patterns of host–parasite associations can be deduced through congruence analysis of their phylogenies. Feather lice and their avian hosts have previously been used as typical model systems for congruence analysis; however, such analyses are strongly biased toward nonpasserine hosts in the temperate zone. Further, in the Afrotropical region especially, cospeciation studies of lice and birds are entirely missing. This work supplements knowledge of host–parasite associations in lice using cospeciation analysis of feather lice (genus Myrsidea and the Brueelia complex) and their avian hosts in the tropical rainforests of Cameroon. Our analysis revealed a limited number of cospeciation events in both parasite groups. The parasite–host associations in both louse groups were predominantly shaped by host switching. Despite a general dissimilarity in phylogeny for the parasites and hosts, we found significant congruence in host–parasite distance matrices, mainly driven by associations between Brueelia lice and passerine species of the Waxbill (Estrildidae) family, and Myrsidea lice and their Bulbul (Pycnonotidae) host species. As such, our study supports the importance of complex biotic interactions in tropical environments.     

Absence of MMACHC in peripheral retinal cells does not lead to an ocular phenotype in mice

Combined methylmalonic aciduria with homocystinuria (cblC type) is a rare disease caused by mutations in the MMACHC gene. MMACHC encodes an enzyme crucial for intracellular vitamin B₁₂ metabolism, leading to the accumulation of toxic metabolites e.g. methylmalonic acid (MMA) and homocysteine (Hcy), and secondary disturbances in folate and one-carbon metabolism when not fully functional. Patients with cblC deficiency often present in the neonatal or early childhood period with a severe multisystem pathology, which comprises a broad spectrum of treatment-resistant ophthalmological phenotypes, including retinal degeneration, impaired vision, and vascular changes. To examine the potential function of MMACHC in the retina and how its loss may impact disease, we performed gene expression studies in human and mouse, which showed that local expression of MMACHC in the retina and retinal pigment epithelium is relatively stable over time. To study whether functional MMACHC is required for retinal function and tissue integrity, we generated a transgenic mouse lacking Mmachc expression in cells of the peripheral retina. Characterization of this mouse revealed accumulation of cblC disease related metabolites, including MMA and the folate-dependent purine synthesis intermediates AICA-riboside and SAICA-riboside in the retina. Nevertheless, fundus appearance, morphology, vasculature, and cellular composition of the retina, as well as ocular function, remained normal in mice up to 6 or 12 months of age. Our data indicates that peripheral retinal neurons do not require intrinsic expression of Mmachc for survival and function and questions whether a local MMACHC deficiency is responsible for the retinal phenotypes in patients.