Modulation of the induction of ornithine decarboxylase by some opioid receptor agonists in immune cells and cardiomyocytes

The ability of natural and synthetic opioids to modulate the induction of ornithine decarboxylase (ODC) was investigated in immune cells and cardiomyocytes in culture. In particular, Leu-enkephalin, which shows preference for -receptors, enhanced ODC activity in both thymocytes and cardiomyocytes, whereas the effect of U-50488H, a synthetic -selective agonist, was cell-specific. In thymocytes, U-50488H markedly inhibited the induction of the enzyme elicited by the mitogen concanavalin A (Con A) or by a combined treatment with PMA and A23187, and also reduced basal ODC activity. However the drug did not affect ODC induced by other stimuli. The inhibition of the induction of ODC activity was accompanied by a reduction of ODC mRNA level and an acceleration of ODC turnover. The action of U-50488H in thymocytes does not appear to be mediated by or other classical opioid receptors lacking both stereospecificity and antagonist sensitivity, but may involve a pertussis toxin-sensitive G protein. Splenocytes also showed the ODC inhibiting effect of U-50488H, although they were less sensitive compared to thymocytes. In contrast, U-50488H enhanced ODC activity in cardiomyocytes and this effect was blocked by a specific -antagonist. In conclusion, these results indicate that some opioid agonists can modulate ODC expression in non neural cells. In particular, -opioid receptors may be involved in the U-50488H action in cardiomyocytes, and a distinct site, linked to inhibition of cell proliferation, may operate in immune cells.     

Marfan syndrome: exclusion of genetic linkage to five genes coding for connective tissue components in the long arm of chromosome 2

Summary Marfan syndrome represents a heterogeneous connective tissue disease, the symptoms arising in several tissues and organs. The defective gene(s) behind this autosomal dominant condition has not been found despite considerable research. The main targets of the research have been the genes coding for connective tissue components. Several of the candidate genes suspected to be defective in Marfan syndrome are located on the long arm of chromosome 2. These genes include a cluster of two genes coding for fibrillar collagens COL3A1 and COL5A2, and a third member of the collagen gene family: COL6A3. Furthermore, genes for elastin (ELN) and fibronectin (FN) are also located in this area of chromosome 2. We studied this chromosomal area using restriction fragment length polymorphism (RFLP) linkage analysis in five Finnish Marfan families with affected members in three generations. In two point linkage analyses, Lod scores of –3.192 ( = 0.1) to COL3A1, –1.683 ( = 0) to COL6A3 and –2.664 ( = 0.01) to FN were obtained, whereas the linkage analysis between elastin and the disease was non-informative (Lod score 0.444, = 0). With the multipoint linkage analysis that permits simultaneous examination of several loci and more efficient use of family data, we obtained an exclusion of all these loci as the site of the mutation leading to Marfan syndrome in these families.     

Localization of ornithine decarboxylase in the chick embryo during organogenesis

Summary The localization of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis and thus in cell growth, was determined in the 4.5-day-old chick embryo, using two independent methods of analysis. ODC protein was identified by indirect immunofluorescence with a monospecific ODC antibody, and catalytically active ODC was identified by autoradiography with -(5-³H) difluoromethylornithine. Both methods revealed a basically similar distribution of ODC within the embryo. Among the organs, the brain exhibited the highest ODC levels. ODC levels were also high in spinal cord, mesonephric tubules and heart. Similar levels, but confined to limited areas, were found in liver tissue, head mesenchyme, and the oral and pharyngeal regions. Organs that exhibited high ODC levels are all engaged in rapid growth, as well as in extensive tissue remodeling and differentiation.     

Protein synthesis during cold shock in barley tissues

Summary In barley (Hordeum vulgare L.) seedlings, a temperature step-down from 24 °C to 6°C (cold shock) determined a reduction in the incorporation of labeled aminoacids and modified the electrophoretic pattern of total proteins. At 6 °C some new proteins appeared and others were intensified (cold shock-induced proteins= CSPs); meantime, few proteins disappeared or were curtailed (cold-repressed proteins=CRPs). The majority of the proteins of the seedlings were labeled at about the same rate both at 6 °C and 24 °C, whereas at 0 °C only the cold shock proteins and a few others were detectable. The cold shock-induced variations of the protein profile differed in roots and in seed remnants which showed only some of the CSPs detected in roots. Total protein synthesis of barley genotypes Onice and Georgie, which have respectively a winter and spring growth habit, were similarly inhibited by a temperature drop. The two genotypes, however, showed some differences in the CSPs and CRPs pattern. Because Onice and Georgie have also a different thermotolerance, the hypothesis can be made that in barley specific CSPs are involved in conferring various degrees of cold resistance.     

Sensitivity of the oviduct to oestrogens in broiler and layer chickens: differential response in the induction of ovalbumin gene expression

Summary There are large differences between broiler and layer chickens in reproductive performance. These differences are correlated with differences in the tissue sensitivity of the oviductal magnum to oestradiol stimulation and demonstrated by alterations in relative ovalbumin synthesis. This biochemical correlate of selection for fertility also differs between male-parent and female-parent lines of broilers. Ovalbumin synthesis in female-parent lines, selected for fertility as well as growth, shows a greater response to saturating doses of oestradiol than male-parent lines selected for growth alone. Sub-saturating doses of oestradiol produced an even greater difference between the strains. Because of the substantial amount of information available on the molecular biology of steroid induction of egg white proteins, it should now be possible to identify the level at which these differences in gene expression occur.     

Adsorption equilibrium and dynamics of lactase/CM-Sephadex system

Summary Partitioning behaviour and adsorption isotherms of lactase/CM-Sephadex system at equilibrium were investigated together with the adsorption kinetics in this study. Maximum adsorption was obtained at the pH values between 5.5–6.0. Adsorption isotherm was a close fit to the Langmuir model.Nomenclature a specific mass transfer area - D_m molecular diffusion coefficient (m²/sec) - e₁, e₂ charge of the protein and the gel - k apparent mass transfer coefficient (s^-1) - ka global mass transfer coefficient - f partition coefficient - K_p dissociation constant for adsorbent-adsorbate complex, (mg/mL solvent) - p equilibrium concentration of free enzyme, (mg free enzyme/mL solution) - q equilibrium concentration of adsorbed enzyme, (mg ads./mL gel) - q_m maximum adsorption capacity, (mg ads./ml gel) - Re particle Reynolds number - Sh Sherwood number - V_g/V gel volume (mL)/bulk solvent volume (mL) - Z dimensionless extent of adsorption - K_p/P_o , model parameter - (/) +1 , model parameter - V_g q_m / V P_o , model parameter     

Unambiguous through-bond sugar-to-base correlations for purines in 13C, 15N-labeled nucleic acids: The HsCsNb,HsCs(N)bCb,and HbNbCb experiments

Summary A set of three 3D (¹H, ¹³C, ¹⁵N) triple-resonance correlation experiments has been designed to provide H1-H8 intraresidue sugar-to-base correlations in purines in an unambiguous and efficient manner. Together, the H_sC_sN_b, H_sC_s(N)_bC_b, and H_bN_bC_b experiments correlate the H1 sugar proton to the H8 proton of the attached base by means of the {H1, C1, N9, C8, H8} heteronuclear scalar coupling network. The assignment strategy presented here allows for unambiguous H1-H8 intraresidue correlations, provided that no two purines have both the same H1 and C1 chemical shifts and the same C8 and N9 chemical shifts. These experiments have yielded H1-H8 intraresidue sugar-to-base correlations for all five guanosines in the [¹³C, ¹⁵N] isotopically labeled RNA duplex r(GGCGCUUGCGUC)₂.     

Light dependence of protoplasmic streaming in Nitella flexilis L. as measured by means of laser-velocimetry

Laser-velocimetry was applied in order to study the effect of light on the velocity of protoplasmic streaming (pps) in Characean cells. A change from dark to light (= 6 W · m^–2) leads to an acceleration of streaming by about 15–30% with a time-constant of approx. 300 s. The transition from light to dark causes a transient decrease of velocity below the original dark level. This response occurs with a time constant of about 500 s. It returns to its initial value with a time-constant of about 2000 s. This may indicate that a control loop of cytosolic homeostasis takes a decrease in pCa more seriously than an increase. A possible involvement of temperature effects caused by illumination was excluded by measuring the influence of temperature. Steady-state velocity of streaming changed by 5% per 1° C. Irradiation with infra-red light ( > 780 nm) did not cause a change in velocity. The absence of a light effect on streaming velocity in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) shows that photosynthesis and not phytochrome is involved. The role of light-induced changes of pCa is discussed, especially with respect to the hypothesis of Vanselow and Hansen (1989, J. Membr. Biol. 110, 175–187) that photosynthesis acts on the plasmalemma K⁺-channel via light-induced uptake of Ca²⁺ into the chloroplasts.Abbreviations and Symbols ASF auto structure function - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - pps protoplasmic streaming - _L, _D, _C time-constants of the light and dark responses, and of a putative Ca-control system Financial support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged. The first author was granted a scholarship by the state of Schleswig-Holstein. We are indebted to Prof. Dr. G. Pfister for technical advice and helpful discussions and to Mrs. E. Götting for drawing the figures.     

Plastid Sequence Evolution: A New Pattern of Nucleotide Substitutions in the Cucurbitaceae

Nucleotide substitutions (i.e., point mutations) are the primary driving force in generating DNA variation upon which selection can act. Substitutions called transitions, which entail exchanges between purines (A=adenine, G=guanine) or pyrimidines (C=cytosine, T=thymine), typically outnumber transversions (e.g., exchanges between a purine and a pyrimidine) in a DNA strand. With an increasing number of plant studies revealing a transversion rather than transition bias, we chose to perform a detailed substitution analysis for the plant family Cucurbitaceae using data from several short plastid DNA sequences. We generated a phylogenetic tree for 19 taxa of the tribe Benincaseae and related genera and then scored conservative substitution changes (e.g., those not exhibiting homoplasy or reversals) from the unambiguous branches of the tree. Neither the transition nor (A+T)/(G+C) biases found in previous studies were supported by our overall data. More importantly, we found a novel and symmetrical substitution bias in which Gs had been preferentially replaced by A, As by C, Cs by T, and Ts by G, resulting in the GACTG substitution series. Understanding this pattern will lead to new hypotheses concerning plastid evolution, which in turn will affect the choices of substitution models and other tree-building algorithms for phylogenetic analyses based on nucleotide data.     

Effects of serial subculture in vitro on the endogenous levels of indole-3-acetic acid and abscisic acid and rootability in microcuttings of ‘Jonathan’ apple

Proliferating axillary shoots of the difficult-to-root apple cultivar Jonathan acquired an enhanced ability to form adventitious roots with increasing number of subcultures in vitro. The transition between the difficult-to-root and the easy-to-root condition occurred at the fourth subculture.Endogenous levels of free IAA and ABA in shoot tissues were analysed by gas chromatography/mass spectrometry/single ion monitoring (GC/MS/SIM) using negative ion chemical ionisation. Tissues from the mother plants grown in the glasshouse contained more IAA and ABA than those from tissue-culture material. After establishment in vitro there was no variation in the IAA content throughout the subcultures but a decrease in ABA content was observed after the fourth transfer. The IAA/ABA ratio increased from 0.2 in difficult-to-root shoots from the initial culture up to 0.7 in easy-to-root shoots from the long-term subculture.     

Nutrients and Primary Production in the Estuary of the Razdol’naya River (Amur Bay,Sea of Japan)

The behavior of the basic nutrients (NO₃, PO₄, SiO₂) was studied in the estuary of the Razdolnaya River in low and high water, the flow was 4.3 × 10⁶ m³/day and 10.8 × 10⁶ m³/day, respectively. It was shown that within the limits of the euphotic zone the nutrients were characterized by a pronounced nonconservative behavior caused by their removal by phytoplankton in primary production. It was determined that phytoplankton removal of nutrients occurred with ratios C : NO₃ : P : Si = 105 : 18 : 1 : 37 and C : NO₃ : P : Si = 93 : 11 : 1 : 29 at a respective ratio P : NO₃ : Si = 1 : 22 : 140 in low water and P : NO₃ : Si = 1 : 17 : 120 in high water. It was also determined that the maximum rate of nutrient removal was 4 times higher in the high water than in the low water. The maximum value of primary production of phytoplankton was 2.5–4.0 gC/m² day. The estuary area of the Razdolnaya River was specified by rather high production. Such a rate of estuarine primary production, caused by nutrients carried out by the river, being no less than 250 t of dry weight of phytoplankton a day, can provide daily production up to 800 t of biomass in the secondary chain of the ecosystem.Original Russian Text Copyright © 2005 by Biologiya Morya, Zvalinsky, Nedashkovsky, Sagalayev, Tishchenko, Shvetsova.     

The production of γ-linolenic acid by selected members of the Dikaryomycota grown on different carbon sources

In this study, seven fungal strains, representing different phylogenetic groups within the Dikaryomycota, were tested for the presence of -linolenic acid [18:3(6)], when grown in synthetic liquid media devoid of fatty acids, on a series of 40 different carbon sources. The fungal strains represented the species Dipodascopsis uninucleata, Eurotium rubrum, Galactomyces geotrichum, Neurospora crassa, Saccharomyces cerevisiae, Spongipellis unicolor and Talaromyces flavus. Cultures were periodically harvested during growth and the fatty acids in the total lipids analysed as methyl esters, using gas chromatography and mass spectrometry. It was found that 18:3(6) is present in E. rubrum CBS 350.65, S. unicolor CBS 117.16 and in T. flavus CBS 310.38NT, when these strains were grown on certain carbon sources. No correlation between the growth phase of the organism and the presence of 18:3(6) could be detected. In order to confirm the production of 18:3(6), the lipid metabolism of two unrelated dikaryomycotan fungi (S. unicolor CBS 117.16 and E. rubrum CBS 350.65) grown on two different carbon sources each, was examined. Cultures of E. rubrum CBS 350.65 were grown on glucose and sorbose and cultures of S. unicolor CBS 117.16 on glucose and sucrose in synthetic liquid media with a C:N ratio of 50:1 (w/w). The total lipids of these cultures were fractionated and the fatty acids in the fractions analysed as methyl esters, using gas chromatography and mass spectrometry. The lipid metabolism of both E. rubrum CBS 350.65 and S. unicolor CBS 117.16 differed on the two carbon sources used. The ab initio production of 18:3(6) by E. rubrum CBS 350.65 in synthetic liquid media was confirmed. In contrast, the ab initio production of 18:3(6) by S. unicolor CBS 117.16 in synthetic liquid media could not be confirmed.     

Mechanism of B12-dependent ribonucleotide reductase

Summary Ribonucleotide reductase from L. leichmmannii catalyzes cleavage of the carbon cobalt bond of AdoCbl homolytically in a kinetically competent fashion. This cleavage triggers a chain of events which results in cleavage of the 3C-H bond of the nucleotide substrate followed by cleavage of the 2 carbon hydroxyl bond. Involvement of a radical cation has been suggested as a possible mechanism by which this unusual reduction reaction might occur. Furthermore, cleavage of the 3 carbon hydrogen bond of [3-³H]NTP resulted in no ³H release to solvent and no ³H recovered in AdoCbl. These results were interpreted to mean that in this system AdoCbl does not serve as a H abstractor, but rather as a radical chain initiator. A protein residue on the RTPR is postulated to carry out the actual H abstraction from the substrate.These results and the conclusions drawn from them are further supported by recent experiments using [3-³H]CIUTP. Incubation of RTPR with [3-³H]CIUTP resulted in release of ³H₂O, uracil, PPP_i, formation of Coll and 5 deoxyadenosine. The ³H₂O release confirms the enzyme's ability to cleave the 3C-H bond of a nucleotide analog. Furthermore, little if any ³H was recovered in the 5 deoxyadenosine and the rate of ³H₂O release from [3³H]CIUTP was 12 times faster than the rate of ³H₂O release from [5-³H]AdoCbl. These observations support the conclusions drawn from data with the normal substrate; ie, AdoCbl serves as a radical chain initiator rather than a direct H abstractor from substrate.     

Metabolic and organ mass responses to selection for high growth rates in the domestic chicken (Gallus domesticus)

Evolutionary hypotheses suggest that higher rates of postembryonic development in birds should either lower the resting metabolic rate (RMR) in a trade-off between the costs of growth and maintenance or increase RMR because of a buildup of metabolic machinery. Furthermore, some suggest that higher rates of postembryonic development in birds should reduce peak metabolic rate (PMR) through delayed tissue maturation and/or an increased energy allocation to organ growth. We studied this by comparing metabolic rates and organ sizes of fast-growing meat-type chickens (broilers) with those of birds from a laying strain, which grow much slower. During the first week of life, despite growing six times faster, the RMR of the broiler chickens was lower than that of birds of the laying strain. The difference between strains in RMR disappeared thereafter, even though broilers continued to grow twice as fast as layers. The differences between strains in growth rate during the first week after hatching were not reflected in similar differences in the relative masses of the heart, liver, and small intestine. However, broilers had heavier intestines once they reached a body mass of 80 g. In contrast, broilers had relatively smaller brains than did layers. There was a positive correlation, over both strains, between RMR and the masses of leg muscles, intestine, and liver. Furthermore, despite delayed maturation of muscle tissue, broilers exhibited significantly higher PMR. We hypothesize that a balance between the larger relative muscle mass but lower muscle maturation level explains this high PMR. Another correlation, between leg muscle mass and PMR, partly explained the positive correlation between RMR and PMR.     

Cytological aspects of in vitro androgenesis in wheat (Triticum aestivum L.) using fluorescent microscopy

Summary Two winter wheat genotypes (Diószegi 200 and Mv 15) were compared for their in vitro androgenic capacity. On average, the induction frequency of embryogenic structures was 71.7% in Diószegi 200 and only 4.3% in Mv 15. The haploid induction ability of the two genotypes differed considerably, with Diószegi 200 being much higher. The difference in the in vitro inductability of the microspores may result from genetic differences which are manifested in the survival rate of the microspores during the culture period and their adaptability to in vitro conditions. Special DNA fluorochrornes were suitable for studying the different pathways of in vitro androgenesis. Our data indicate that the repeated equal divisions of the microspore nucleus might lead to pollen embryo formation, and subsequent divisions of the vegetative portion of the pollen grain after the first asymmetric microspore mitosis can result in pollen callus formation.     

Primer extension studies on α-amylase mRNAs in barley aleurone. II. Hormonal regulation of expression

Relative levels of different -amylase mRNAs were assessed by primer extension experiments using RNA prepared from aleurone of barley (Hordeum vulgare L. cv. Himalaya). Three different aleurone systems were studied: protoplasts prepared from aleurone layers, isolated aleurone layers, and aleurone from germinated grain. Oligonucleotide primers specific for the low-pI and high-pI -amylase groups allowed the levels of different -amylase mRNAs to be assessed both within and between the two groups.In all aleurone systems the same set of -amylase mRNAs was produced in response to either applied gibberellic acid (aleurone protoplasts, isolated aleurone layers) or, presumably, native gibberellin(s) (germinated grain). This result indicates that the same set of genes is being expressed in each case. Differences were observed between the different aleurone systems in regulation of levels of -amylase mRNAs. In particular, the regulation of -amylase mRNA levels in aleurone of germinated grain has unique features which are not adequately explained by the response of isolated aleurone layers to gibberellic acid.     

Haemoglobin hasharon

Summary 14 instances of Haemoglobin Hasharon (₂ 47 Asp His ₂) from various parts of the world have been characterised over the last 7 years. The properties, geographical and ethnic distribution of this variant are discussed.     

Temperature conditional cAMP-requiring mutant strains ofChlamydomonas reinhardtii arrest in G1 and are rescued by added cAMP

Summary Three independent isolates ofChlamydomonas, selected for caffeine resistance, were found to arrest in G₁ phase, as determined by quantitative fluorescence measurements of DNA, when grown at a non-permissive temperature. This cell cycle arrest correlated with lowered levels of cAMP and of adenylate cyclase activity. The arrested cells could be rescued by added cAMP but not AMP, hence the defect was not one of general purine metabolism. Back-crosses to wild type revealed that the phenotypes observed result from a combination of three separable mutations. It is clear that the mutations define functions that are more stringently required for cell division than for growth since the mutant strains are able to grow up to fifteen times normal size while blocked at the non-permissive temperature. The possible interaction of cAMP dependent events with division is discussed.Abbreviations AMP adenosine 5-monophosphate - ATP adenosine 5-triphosphate - BSA bovine serum albumin - cAMP adenosine 3,5-cyclicmonophosphate - db-cAMP dibutyryl-cAMP - DNA deoxyribonucleic acid - DTT dithiothreitol - -cAMP 1,N⁶-etheno-cAMP - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid - HPLC high performance liquid chromatography - LSA low sulphur-high salt-acetate medium - LYP LSA media containing yeast extract and proteose peptone - M1, 2, 3 mutants 1, 2, 3 - PDE phosphodiesterase - TAP trisacetate-phosphate medium - TLC thin layer chromatography - TYP TAP medium containing yeast extract and proteose peptone     

Puromycin enhances 20-hydroxyecdysone-induced protein synthesis in wing discs of Drosophila melanogaster

The effect of cycloheximide and puromycin on 20-hydroxyecdysone-induced protein synthesis in wing discs of Drosophila melanogaster has been studied by one-dimensional and two-dimensional SDS polyacrylamide electrophoresis. It is found that puromycin, but not cycloheximide, when applied simultaneously with the hormone enhanced the hormone-induced synthesis of the early and late proteins. However, when puromycin was applied after hormone treatment, only the late proteins were induced. The possible implication of these observations is discussed.     

The relationship between age and genotype and circulating concentrations of triiodothyronine (T3), thyroxine (T4), and growth hormone in commercial meat strain chickens

The presence of the sex-linked dwarf gene (dw) in homozygous male (dw/dw) and female (dw/-) meat strain chickens is associated with a significant reduction in circulating levels of triiodothyronine (T3). Heterozygous (Dw/dw) male broiler strain chickens have T3 concentrations similar to those in homozygous (Dw/Dw) male broilers. Genetically normal (Dw/Dw) but significantly slower growing roaster strain male meat chickens had consistently higher T3 than the faster growing broilers at all ages in one experiment but only at 8 weeks in a second experiment. Age and not growth rate appears to have a greater influence on serum T3 concentrations in the slow- and fast-growing normal strains. Growth hormone levels were significantly higher in the dwarf chickens at all ages and in all three experiments. The heterozygous and homozygous broilers had similar GH levels and the slow-growing, genetically normal roasters had intermediate concentrations between the broiler and dwarf lines. GH was influenced to a greater extent by the rate of body weight gain than by increasing age in the genetically normal fast and slow growing strains.