Subunit structure and higher order assembly of the hemocyanins of the Melongenidae family: Melongena corona (Gmelin), Busycon canaliculatum (Linné), B. carica (Gmelin), B. contrarium (Conrad), and B. spiratum (Lamarck)

1. The hemocyanins of the Melongenidae family of marine gastropods: Melongena corona, Busycon canaliculatum, B. carica, B. contrarium, and B. spiratum exist in solution as multi-decameric aggregates characterized by sedimentation coefficients of approximately 105 S, 130 S, 150 S, 170 S, and higher values, corresponding to di-, tri-, tetra-, penta-, and larger multi-decameric particles. 2. The hemocyanins of B. contrarium and B. carica seem to form the largest decameric aggregates with the tri- to penta-decamers respresenting the major constitutents. Scanning transmission electron microscopy (STEM), both of unstained, freeze-dried and negatively-stained specimens, shows the presence of discrete aggregates consisting of up to ten decameric units. 3. The particle masses as determined by STEM mass measurements for individual molecules gave integral multiples of from 4.2 x 10(6) to 4.4 x 10(6) daltons ranging from about 8.2 x 10(6) daltons for the typical di-decamer of B. canaliculatum hemocyanin to as high as about 39 x 10(6) and 43 x 10(6) for the nano-and deca-decamers of B. contrarium hemocyanin. 4. The appearance of the higher multi-decamers in both negatively-stained and freeze-dried specimens suggest that they are formed by the addition of decameric units to a single di-decameric unit "tail-wise" in both directions. The higher aggregates formed seem to terminate with a closed head or collar at both ends of the assembly.     

IR and Raman study on the interactions of the 5′-GMP and 5′-CMP phosphate groups with Mg(II), Ca(II), Sr(II), Ba(II), Cr(III), Co(II), Cu(II), Zn(II), Cd(II), Al(III) and Ga(III)

The chief motive behind this research is the interest provoked by the presence of metal ions as necessary stabilizers of the negative charges of phosphate groups in nucleic acids. The effect that the presence of different metal ions produces on the band principally assigned to the nu(s) PO(3)(2-) mode has been studied using FT-IR and FT-Raman spectroscopy. The results obtained reveal the diagnostic capacity of these techniques in determining the type of metal ion interaction with respect to the mononucleotides that form DNA and RNA, providing a tool for improving the knowledge of the stabilizing or destabilizing effects of these ions on such macromolecules. The metal complexes of the ribonucleotides 5'-CMP and 5'-GMP with Mg(II), Ca(II), Sr(II), Ba(II), Cr(III), Co(II), Cu(II), Zn(II), Cd(II), Al(III) and Ga(III) were obtained in this study. After studying and analyzing the IR and Raman spectra of all these complexes and comparing them with the spectra of the corresponding disodium salts, it was verified that, independently of the type of nucleotide involved, the presence of the metal in the vicinity of the phosphate group produces an alteration in the aforementioned nu(s) PO(3)(2-) band. This effect is related to the type of interaction that the phosphate group has with the metal. Three components are observed: (1) one near 983-975 cm(-1) (detectable in IR and Raman), associated with phosphate groups in an electrostatic type of interaction with the metal ion, separated by two or more water molecules; (2) another near 989-985 cm(-1) (only in IR), associated with phosphate groups in indirect interaction through the water molecules of the coordination sphere of the metal ions; and (3) the IR and Raman bands near 1014-1001 cm(-1), which represent phosphate groups directly bonded to the metal ion. These results are supported by the behavior of 5'-CMP in aqueous solution in the presence of Mg(II) ions.     

Preparation and characterization of oxovanadium(IV), acetatomanganese(III), chloroiron(III), nickel(II), copper(II), zinc(II) and palladium(II) complexes of 3,3′-(1,2-phenylenediimino)diacrolein

The oxovanadium(IV), acetatomanganese(III), chloroiron(III), nickel(II), copper(II), zinc(II) and palladium(II) of 3,3′-(1,2-phenylenediimino)diacrolein were prepared and investigated by means of mass, electronic, vibrational, NMR and ESR spectroscopy as well as magnetic susceptibility measurements. The acetatomanganese(III) and chloroiron(III) complexes were confirmed to be of high spin type. The absorption bands appearing in the energy range greater than 23 000 cm⁻¹ were attributed to π→π^* transitions within a ligand molecule and charge- transfer transitions from metal to ligand. The metal complexes assume the square-planar configuration type since the ligand-field bands were detected in the 12 700–18 500 cm⁻¹ region. Strong bands appearing at 1601 and 1627 cm⁻¹ were assigned to the CC and CO stretching vibrational modes, respectively, and were shifted to lower frequency upon metal-coordination. A VO stretching band was observed at 982 cm⁻¹ for the oxovanadium(IV) complex and a CO stretching band was observed at 1547 cm⁻¹ for the acetatomanganese(III) complex. Upon complex formation the amine proton signal is found to vanish and the aldehydic methine proton signal in the lowest field is shifted upfield for the nickel(II), zinc(II) and palladium(II) complexes. ¹³C NMR spectra support the coordination structure of the complexes which is revealed by ¹H NMR spectra. As judged by the spin Hamiltonian parameters, the oxovanadium(IV) complex is of a square- planar type with an unpaired electron in the d_xy orbital and the copper(II) complex assumes a distorted square-planar coordination due to the presence of five- and six-membered chelate rings with an unpaired electron in the d_x2−y2 orbital.     

Incidental finding of double minutes (DM), single minutes (SM), homogenously staining regions (HSR), premature chromosome condensation (PCC), and premature centromere division (PCD)?

The incidental finding of DM's, minutes, HSR's, PCC, and PCD in two completely unrelated cases--one is a prenatal diagnosis in a twin pregnancy complicated by hydramnios and feto-fetal exsanguination, the other is an adult Klinefelter patient--raises the question whether such findings are coincidental or whether there is a common denominator in such cases. Possible relationships between these phenomena and the observed cases are discussed.     

Associations between Meteorological Parameters and Influenza Activity in Berlin (Germany), Ljubljana (Slovenia), Castile and León (Spain) and Israeli Districts

Background

Studies in the literature have indicated that the timing of seasonal influenza epidemic varies across latitude, suggesting the involvement of meteorological and environmental conditions in the transmission of influenza. In this study, we investigated the link between meteorological parameters and influenza activity in 9 sub-national areas with temperate and subtropical climates: Berlin (Germany), Ljubljana (Slovenia), Castile and León (Spain) and all 6 districts in Israel.

Methods

We estimated weekly influenza-associated influenza-like-illness (ILI) or Acute Respiratory Infection (ARI) incidence to represent influenza activity using data from each country’s sentinel surveillance during 2000–2011 (Spain) and 2006–2011 (all others). Meteorological data was obtained from ground stations, satellite and assimilated data. Two generalized additive models (GAM) were developed, with one using specific humidity as a covariate and another using minimum temperature. Precipitation and solar radiation were included as additional covariates in both models. The models were adjusted for previous weeks’ influenza activity, and were trained separately for each study location.

Results

Influenza activity was inversely associated (p<0.05) with specific humidity in all locations. Minimum temperature was inversely associated with influenza in all 3 temperate locations, but not in all subtropical locations. Inverse associations between influenza and solar radiation were found in most locations. Associations with precipitation were location-dependent and inconclusive. We used the models to estimate influenza activity a week ahead for the 2010/2011 period which was not used in training the models. With exception of Ljubljana and Israel’s Haifa District, the models could closely follow the observed data especially during the start and the end of epidemic period. In these locations, correlation coefficients between the observed and estimated ranged between 0.55 to 0.91and the model-estimated influenza peaks were within 3 weeks from the observations.

Conclusion

Our study demonstrated the significant link between specific humidity and influenza activity across temperate and subtropical climates, and that inclusion of meteorological parameters in the surveillance system may further our understanding of influenza transmission patterns.     

Synthesis, characterization and antitumor studies of N-aroyl-N′-thioaroylhydrazines and their Co(II), Ni(II), Cu(II) and Zn(II) complexes

N-Salicyloyl-N-p-hydroxythiobenzohydrazide (H₂STPH) and N-benzoyl-N-thiobenzohydrazide (H₂BTBH) and their Co(II), Ni(II), Cu(II) and Zn(II) complexes were prepared and characterized by physicochemical studies. IR and NMR spectral studies imply dibasic tetradentate behaviour of the ligands bonding through `thiolato' sulfur, enolic oxygen and the two hydrazinic nitrogens in a polymeric fashion. The electronic spectra indicate [Ni(STPH)(H₂O)₂], [Co(STPH)(H₂O)₂] to be distorted octahedral while [Cu(BTBH)] has a square-planar geometry. In vitro antitumor results of the ligand and the complexes on P-815 (murine mastocytoma) and L-929 (murine fibroblast) indicate that these compounds show significant inhibition of ³H-thymidine and ³H-uridine incorporation in DNA and RNA, respectively, in these tumor cells at dose levels of 1, 2.5 and 5 g cm^–3. Antitumor studies suggest that [Cu(BTBH)] has significant dose dependent inhibitory effect on DNA synthesis. In vivo administration of [Cu(BTBH)] and [Ni(STPH)(H₂O)₂] resulted into prolongation of life span of Dalton's Lymphoma (DL) bearing mice.     

Complexes of Co(II), Ni(II), Cu(II) and Zn(II) with 3′AMP and 2′AMP

Derivatives of Co(II), Ni(II), Cu(II) and Zn(II) with 3′AMP and 2′AMP were synthesized and characterized by IR UV-Vis and fluorescence spectroscopy. There seems to be bonding of the metal ion to the base in all cases. The activation test, using the complexes as allosteric labels, was carried out with rabbit muscle glycogen phosphorylase b, but the enzyme was not activated, confirming that the phosphate group must necessarily be bonded to position 5′ of the ribose in order to activate this enzyme.     

Free metal ion depletion by “good's” buffers: II. N-(2-acetamido)-2-aminoethanesulfonic acid (ACESH): Complexes with Calcium(II), Magnesium(II), Manganese(II), Cobalt(II), Zinc(II), Nickel(II), and Cooper(II)

Potentiometric, visible, and infrared studies of the complexation of N-(2-acetamido)-2-aminoethanesulfonic acid (ACESH) by Ca(II), Mg(II), Mn(II), Co(II), Zn(II), Ni(II), and Cu(II) are reported. Ca(II), Mg(II), and Mn(II) were found not to complex with ACES^?, while Co(II), Zn(II), Ni(II), and Cu(II) were found to form 2:1, ACES^? to M²⁺, complexes, and [Cu(ACES)₂] was found to undergo stepwise deprotonation of the amide groups to form [Cu(H_?1ACES)₂^2?]. Formation (affinity) constants for the various metal complexes are reported, and the probable structures of the various metal chelates in solution are discussed.     

Synthesis, Characterization and Antitumor Studies of Mn(II), Ni(II), Cu(II) and Zn(II) Complexes of N-Nicotinoyl-N′-o-Hydroxythiobenzhydrazide

A new ligand N-Nicotinoyl-N-o-hydroxythiobenzhydrazide (H₂Notbh) forms complexes [Mn(Notbh)(H₂O)], [M(Notbh)] [M=Ni(II) Cu(II) and Zn(II)] which were characterized by various physico-chemical techniques. All the metal complexes were observed to inhibit the growth of tumor in vitro, whereas, ligand did not. In vivo administration of these complexes resulted in prolongation of survival of tumor bearing mice. Tumor bearing mice administered with metal complexes showed reversal of tumor growth associated induction of apoptosis in lymphocytes. The paper discusses the possible mechanisms and therapeutic implication of the H₂Notbh and its metal complexes in tumor regression and tumor growth associated immunosuppression.     

Interaction of Adenosine 5′-Triphosphate with Metal Ions X-ray Structure of Ternary Complexes Containing Mg(II), Ca(II), Mn(II), Co(II), ATP and 2,2′ -Dipyridylamine

Abstract

The X-ray structures of the isomorphous Mg²⁺, Ca²⁺, Mn²⁺ and Co²⁺ complexes of ATP have been determined. The metal ions are wrapped in hexa-coordination by the α, β and γ phosphate groups of two ATP molecules thus blocking the interaction of the metal ions with the adenine base. A second metal ion which is fully hydrated, M(H₂O)²⁺ ₆, is engaged in a strong hydrogen bond with the γ phosphate group of ATP and suggests a possible step in facilitating the cleavage between the β and γ phosphates in phosphoryl transfer reactions.     

Comparative genomics of duplicate γ-glutamyl transferase genes in teleosts: medaka (Oryzias latipes), stickleback (Gasterosteus aculeatus), green spotted pufferfish (Tetraodon nigroviridis), fugu (Takifugu rubripes), and zebrafish (Danio rerio)

The availability of multiple teleost (bony fish) genomes is providing unprecedented opportunities to understand the diversity and function of gene duplication events using comparative genomics. Here we examine multiple paralogous genes of γ-glutamyl transferase (GGT) in several distantly related teleost species including medaka, stickleback, green spotted pufferfish, fugu, and zebrafish. Through mining genome databases, we have identified multiple GGT orthologs. Duplicate (paralogous) GGT sequences for GGT1 (GGT1 a and b), GGTL1 (GGTL1 a and b), and GGTL3 (GGTL3 a and b) were identified for each species. Phylogenetic analysis suggests that GGTs are ancient proteins conserved across most metazoan phyla and those paralogous GGTs in teleosts likely arose from the serial 3R genome duplication events. A third GGTL1 gene (GGTL1c) was found in green spotted pufferfish; however, this gene is not present in medaka, stickleback, or fugu. Similarly, one or both paralogs of GGTL3 appear to have been lost in green spotted pufferfish, fugu, and zebrafish. Syntenic relationships were highly maintained between duplicated teleost chromosomes, among teleosts and across ray-finned (Actinopterygii) and lobe-finned (Sarcopterygii) species. To assess subfunction partitioning, six medaka GGT genes were cloned and assessed for developmental and tissue-specific expression. On the basis of these data, we propose a modification of the "duplication-degeneration-complementation" model of subfunction partitioning where quantitative differences rather than absolute differences in gene expression are observed between gene paralogs. Our results demonstrate that multiple GGT genes have been retained within teleost genomes. Questions remain, however, regarding the functional roles of multiple GGTs in these species.     

RAPD and AFLP tagging and mapping of Beta (B) and Beta modifier (MoB), two genes which influence β-carotene accumulation in fruit of tomato (Lycopersicon esculentum Mill.)

The Beta (B) locus in tomato (Lycopersicon esculentum) increases fruit β-carotene content at the expense of lycopene, resulting in orange-pigmented fruit. Expression of B is influenced by the beta-modifier (Mo _B ) gene which segregates independently of B. RAPD and AFLP analyses were performed using near isogenic lines (NILs) unique for B and bulked segregant analysis (BSA) of a L. esculentum×L. cheesmanii-derived F₂ population segregating for B. Using 1018 random primers for RAPD analysis and 64 primer pairs for AFLP analysis, we identified polymorphic products which distinguished the NILs and the two bulked DNA samples constructed for BSA. A single 100 bp AFLP amplification product (E-ACA/M-CTG₁₀₀) which distinguished the NILs cosegregated with Mo _B and was demonstrated to be tightly linked to the locus. E-ACA/M-CTG₁₀₀ exhibited a recombination frequency of 1.7% in the F₂ progeny derived from an initial cross between the isolines. The Mo _B locus was mapped to the long arm of chromosome 6. Two RAPD products (OPAR18₁₁₀₀ and UBC792₈₃₀) of 1100 bp and 830 bp, respectively, were polymorphic between orange- and red-fruited bulks constructed from F₂ individuals in the L. esculentum and L. cheesmanii mating series. OPAR18₁₁₀₀ and UBC792₈₃₀ displayed recombination frequencies of 4.2% and 7.6%, respectively, in F₂ progeny. The B-linked OPAR18₁₁₀₀ marker was also mapped to the long arm of chromosome 6, proximal to Mo _B , and revealed linkage between B and Mo _B . Received: 9 April 1999 / Accepted: 27 April 1999     

INTERACTION OF MELANIN-CONCENTRATING HORMONE (MCH), NEUROPEPTIDE E-I (NEI), NEUROPEPTIDE G-E (NGE), AND α-MSH WITH MELANOCORTIN AND MCH RECEPTORS ON MOUSE B16 MELANOMA CELLS

Melanin-concentrating hormone (MCH) and α-melanocyte-stimulating hormone (α-MSH) are known to exhibit mostly functionally antagonistic, but in some cases agonistic activities, e.g., in pigment cells and in the brain. Neuropeptide E-I (NEI) displays functional MCH-antagonist and MSH-agonist activity in different behavioral paradigms; the role of neuropeptide G-E (NGE) is not known. This study addressed the question of possible molecular interactions between α-MSH, MCH and the MCH-precursor-derived peptides NEI and NGE at the level of the pigment cell MCH receptor subtype (MCH-R_pc) and the different melanocortin (MC) receptors. Radioreceptor assays using [¹²⁵I]MCH, [¹²⁵I]α-MSH and [¹²⁵I]NEI as radioligands and bioassays were performed with MC1-R-positive and MC1-R-negative mouse B16 melanoma cells and with COS cells expressing the different MC receptors. The IC₅₀s of α-MSH and NEI or NGE for [¹²⁵I]MCH displacement from mouse MCH-R_pc were 80-fold and, respectively, > 300-fold higher than that of MCH, and the IC₅₀s for MCH and NEI or NGE for [¹²⁵I]α-MSH displacement from mouse MC1-R were 50,000-fold and > 200,000-fold higher than that of α-MSH. No high-affinity binding sites for NEI were detected on B16 melanoma cells and there was no significant displacement of [¹²⁵I]α-MSH by MCH, NEI or NGE with MC3-R, MC4-R and MC5-R expressed in COS cells. At concentrations of 100 nM to 10 μM, however, MCH, NEI and NGE induced cAMP formation and melanin synthesis which could be blocked by agouti protein or inhibitors of adenylate cyclase or protein kinase A. This shows that mammalian MCH-precursor-derived peptides may mimic MSH signalling via MC1-R activation at relatively high, but physiologically still relevant concentrations, as e.g. found in autocrine/paracrine signalling mechanisms.     

Assignment of the rat genes coding for α1-antitrypsin (PI), phosphoenolpyruvate carboxykinase (PEPCK), alcohol dehydrogenase (ADH), and fructose-1, 6-bisphosphatase (FDP)

The chromosomal localization of four genes which are expressed mainly in the liver has been undertaken for the rat. Using a panel of hybrid clones segregating rat chromosomes, and Southern blot analysis, 1-AT (PI), PEPCK, ADH and FDP are assigned to rat Chromosomes (Chr) 6, 3, 2 and 17, respectively. Groups of synteny among rat, mouse and human species are discussed in relationship to the new assignments.     

Human-Specific SNP in Obesity Genes, Adrenergic Receptor Beta2 (ADRB2), Beta3 (ADRB3), and PPAR γ2 (PPARG), during Primate Evolution

Adrenergic-receptor beta2 (ADRB2) and beta3 (ADRB3) are obesity genes that play a key role in the regulation of energy balance by increasing lipolysis and thermogenesis. The Glu27 allele in ADRB2 and the Arg64 allele in ADRB3 are associated with abdominal obesity and early onset of non-insulin-dependent diabetes mellitus (NIDDM) in many ethnic groups. Peroxisome proliferator-activated receptor γ (PPARG) is required for adipocyte differentiation. Pro12Ala mutation decreases PPARG activity and resistance to NIDDM. In humans, energy-expense alleles, Gln27 in ADRB2 and Trp64 in ADRB3, are at higher frequencies than Glu27 and Arg64, respectively, but Ala12 in PPARG is at lower frequency than Pro12. Adaptation of humans for lipolysis, thermogenesis, and reduction of fat accumulation could be considered by examining which alleles in these genes are dominant in non-human primates (NHP). All NHP (P. troglodytes, G. gorilla, P. pygmaeus, H. agilis and macaques) had energy-thrifty alleles, Gly16 and Glu27 in ADRB2, and Arg64 in ADRB3, but did not have energy-expense alleles, Arg16, Gln27 and Trp64 alleles. In PPARG gene, all NHP had large adipocyte accumulating type, the Pro12 allele. CONCLUSIONS: These results indicate that a tendency to produce much more heat through the energy-expense alleles developed only in humans, who left tropical rainforests for savanna and developed new features in their heat-regulation systems, such as reduction of body hair and increased evaporation of water, and might have helped the protection of entrails from cold at night, especially in glacial periods.     

Expression of Inhibin/activin Subunits alpha (-α), beta A (-β A) and beta B (-β B) in Placental Tissue of Normal and Intrauterine Growth Restricted (IUGR) Pregnancies

During human pregnancy the placenta produces a variety of proteins like steroid hormones and their receptors that are responsible for the establishment and ongoing of the feto-placental unit. Inhibins are dimeric glycoproteins, composed of an α-subunit and one of two possible β-subunits (β _A or β _B). Aims of the present study were the determination of the frequency and tissue distribution patterns of the inhibin/activin subunits in human placental tissue of normal pregnancies and pregnancies complicated with fetal growth restriction (IUGR). Slides of paraffin embedded placental tissue were obtained after delivery from patients diagnosed with IUGR (n = 6) and normal term placentas (n = 8). Tissue samples were fixed and incubated with monoclonal antibodies inhibin/activin-subunits -α, -β _A, -β _B. Intensity of immunohistochemical reaction on the slides was analysed using a semi-quantitative score and statistical analysis was performed (P<0.05). A significant lower expression of the inhibin-α subunit in IUGR extravillous trophoblast compared to normal pregnancies was observed, while the inhibin-α immunostaining was significantly upregulated in syncytiotrophoblast. Additionally, a significant down-regulation of inhibin-β _B subunit in extravillous trophoblast cells in IUGR syncytiotrophoblast cells was demonstrated. A co-localisation of inhibin-α and the β-subunits was also observed, suggesting a production and secretion of intact inhibin A and inhibin B. Although the precise role of these inhibin/activin subunits in human placenta and IUGR pregnancies is still unclear, they could be involved in autocrine/paracrine signalling, contributing to several aspects like angiogenesis and tissue remodelling.     

Dagmar Dykyjová (1914–2011), her life and work

The late Dagmar Dykyjová (1914–2011), who is being honoured by this issue of Folia Geobotanica, was an excellent scientist as well as university teacher and research coordinator. Her research work stretched from both fundamental and applied plant physiology to the ecology of wetlands and wetland plants in particular, and to the ecology of Central European orchids. Apart from having authored, co-authored or edited numerous scientific publications, also including several books, she had great merits in popularizing a sensitive approach to environmental management, nature conservation and management of natural resources, based on up-to-date scientific knowledge. She played an important role in the Czechoslovak participation in the International Biological Programme (IBP, 1965–74), especially in the fields of plant production ecology and wetlands ecology. The history of her life and work is briefly outlined and evaluated (Fig. 1 ).     

Novel Radiolabeled Peptides for Breast and Prostate Tumor PET Imaging: (64)Cu/and (68)Ga/NOTA-PEG-[d-Tyr(6),βAla(11),Thi(13),Nle(14)]BBN(6-14)

Bombesin (BBN)-based radiolabeled peptides exhibit promising properties for targeted imaging of gastrin-releasing peptide receptors (GRPR)-positive tumors. The aim of this study was to evaluate with positron emission tomography (PET) the pharmacokinetic and imaging properties of two novel BBN-based radiolabeled peptides, (64)Cu/and (68)Ga/NOTA-PEG-BBN(6-14), for diagnosis of breast and prostate cancers using small animal models. Competitive binding assays on T47D breast and PC3 prostate cancer cells showed that the affinity for GRPR depends on the complexed metal and can vary up to a factor of about 3; (64)Cu/NOTA-PEG-BBN(6-14) was found to have the lowest inhibition constant (1.60 ± 0.59 nM). (64)Cu/and (68)Ga/NOTA-PEG-BBN(6-14) presented similar cell uptake on T47D and PC3 cells and were stable in vivo. Biodistribution studies of radiolabeled peptides carried out in Balb/c and tumor-bearing Balb/c nude mice showed that (64)Cu/NOTA-PEG-BBN(6-14) presented higher GRPR-mediated uptake in pancreas and adrenal glands, but comparable PC3 tumor uptake as (68)Ga/NOTA-PEG-BBN(6-14). Finally, receptor-dependent responses were observed during blocking studies with unlabeled peptide in both biodistribution and small-animal PET imaging studies. Our results confirmed the dependence of the affinity and pharmacokinetics of BBN-based radiopeptides on the complexed radiometal. Interspecies differences between mouse and human GRPR binding properties were also noted in these preclinical studies. Considering their good imaging characteristics, both (64)Cu/NOTA-PEG-BBN(6-14) and (68)Ga/NOTA-PEG-BBN(6-14) are promising candidates for GRPR-targeted PET imaging of breast and prostate cancers.