Altered Growth and Wood Characteristics in Transgenic Hybrid Aspen Expressing Agrobacterium tumefaciens T-DNA Indoleacetic Acid-Biosynthetic Genes

A key regulator of cambial growth is the plant hormone indoleacetic acid (IAA). Here we report on altered wood characteristics and growth patterns in transgenic hybrid aspen (Populus tremula L. x Populus tremuloides Michx.) expressing Agrobacterium tumefaciens T-DNA IAA-biosynthetic iaaM and iaaH genes. Eighteen lines simultaneously expressing both genes were regenerated. Of these, four lines, verified to be transgenic by northern blot analysis, were selected and raised under controlled growth conditions. All four lines were affected in their growth patterns, including alterations in height and stem diameter growth, internode elongation, leaf enlargement, and degree of apical dominance. Two transgenic lines, showing the most distinct phenotypic deviation from the wild type, were characterized in more detail for free and conjugated IAA levels and for wood characteristics. Both lines showed an altered IAA balance, particularly in mature leaves and roots where IAA levels were elevated. They also exhibited changes in wood anatomy, most notably a reduction in vessel size, an increase in vessel density, and changes in ray development. Thus, the recent development of techniques for gene transfer to forest trees enabled us to investigate the influence of an altered IAA balance on xylem development in an intact experimental system. In addition, the results demonstrate the possibility of manipulating wood properties in a forest tree through controlled changes of IAA concentration and distribution.     

Do Fleas,and/or Old Nest Material,Influence Nest-site preference in Hole-nesting Passerines?

This study examined nest-site choice in a migratory population of pied flycatchers (Ficedula hypoleuca) and sympatric populations of three resident tit species (Parus major, P. caeruleus and P. palustris) in central Sweden. All four species are secondary-cavity nesters which naturally breed in pre-formed tree cavities but readily use artificial nest boxes. We asked whether flycatchers and tits discriminate between nest boxes that: 1. Are ‘empty’; 2. Contain old nests without ectoparasites (fleas Ceratophyllus sp.); or 3. Contain old nests with ectoparasites. We found that pied flycatchers preferred nest boxes containing old nests, regardless of whether these nests held parasites. In contrast, tits did not discriminate between the three types of boxes. Tits may pay a cost for their lack of choosiness: after the breeding season, tit nests contained more fleas than flycatcher nests. Nevertheless, parasites did not affect the choice of a nest site in any of the species studied. We suggest that the migratory flycatchers are in a hurry to start breeding upon arrival and use the presence of an old nest as a shortcut cue to assess nest-site quality. Also, they may save valuable time by copying the choice of previous breeders. Non-migratory tits may have more time to inspect nest sites, but do not seem to use the same cues in nest-site selection as the pied flycatcher.     

NK-lysin, a novel effector peptide of cytotoxic T and NK cells. Structure and cDNA cloning of the porcine form, induction by interleukin 2, antibacterial and antitumour activity.

A 78 residue antimicrobial, basic peptide, NK-lysin, with three intrachain disulfide bonds was purified from pig small intestine and characterized. A corresponding clone was isolated from a porcine bone marrow cDNA library. The 780 bp DNA sequence had a reading frame of 129 amino acids which corresponded to NK-lysin. The clone was used to show that stimulation with human interleukin-2 induced synthesis of NK-lysin-specific mRNA in a lymphocyte fraction enriched for T and NK cells. Lower levels of mRNA were detected in tissues known to contain T and NK cells, such as small intestine, spleen and colon. Interleukin-2 also induced both proliferation of the lymphocyte fraction and cytolytic function in these cells. Immunostaining showed that NK-lysin was present in cells positive for CD8, CD2 and CD4. NK-lysin showed high anti-bacterial activity against Escherichia coli and Bacillus megaterium and moderate activity against Acinetobacter calcoaceticus and Streptococcus pyogenes. The peptide showed a marked lytic activity against an NK-sensitive mouse tumour cell line, YAC-1, but it did not lyse red blood cells. The amino acid sequence of NK-lysin exhibits 33% identity with a putative human preproprotein, NKG5, of unknown function but derived from a cDNA clone of activated NK cells. We suggest that NK-lysin is a new effector molecule of cytotoxic T and NK cells.     

Sensitivity of the ELISA Test to Detect Phytophthora fragariae var. rubi in Raspberry Roots

A commercial serological, multiwell assay kit was used to assess the detection limits of Phytophthora fragariae var. rubi in raspberry roots. Detection limits in time lapse after inoculation, were assessed after inoculation of root systems of raspberry plants by zoospores of P. fragariae var. rubi. In extracts taken 3-9 days after inoculation, the pathogen was detected from the fourth day after inoculation. In a test series of simulated P. fragariae var. rubi infection where 0.25, 0.5, 1.0 and 1.5% of infected root tissue respectively, were mixed with healthy tissue (w/w), it was possible to detect the pathogen at 0.25% of simulated infection level. The results obtained show the possibility of an early detection of small amounts of antigen by the ELISA test procedure used. This enhance possibilities for early diagnosis and thereby more effective prevention of Phytophthora diseases in raspberry.     

Male mating success, reproductive success and multiple paternity in a natural population of sand lizards: behavioural and molecular genetics data

Sand lizard Lacerta agilis females characteristically mate with several males which, in staged mating experiments, results in multiple paternity of the offspring. In order to investigate multiple paternity in a natural population and interpret male reproductive behaviours in terms of sired young, we sampled the blood of females, potential fathers and hatchlings, and determined paternity using multilocus DNA fingerprinting as well as the variation at a single locus detected by the probe (TC) _n . The paternity analyses were preceded by a laboratory experiment in which we established that the parental alleles identified by the single locus probe were inherited in a Mendelian way. Our molecular data demonstrated that 12 out of 13 males (92%) that sired offspring were correctly identified from the 56 sexually mature males in the population. Also smaller males were accepted as sexual partners by the females, but sired fewer young in competition with larger males and were less able to maintain prolonged post-copulatory mate guarding. This may result in that some sexually successful males are only observed inside a female's home range, but never in pair-association with the female.     

Are Elevated Aminotransferases and Decreased Bilirubin Additional Characteristics of the Metabolic Syndrome?

Abnormal liver tests, as well as morphological changes in the liver, are frequent among obese patients. Other frequent disturbances are visceral fat accumulation, insulin resistance, non-insulin-dependent diabetes mellitus (NIDDM), hypertriglyceridemia, and hypertension; these are a set of aberrations known as the metabolic syndrome. In order to investigate a possible relationship between the metabolic syndrome and impaired liver status we examined associations between liver tests, metabolic variables (insulin, glucose, and triglycerids), body composition and nutrition in 1083 men (BMI 28.8–63.8 kg/m²) and 1367 women (BMI 26.7–68.0 kg/m²) in the ongoing intervention study of Swedish Obese Subjects (SOS). Standard biochemical techniques were used to assess liver status and metabolic variables. Lean body mass (LBM) and masses of visceral and subcutaneous adipose tissue (AT) were estimated by means of computed tomography (CT) calibrated anthropometric equations. In both genders aspartate aminotransferase and alanine aminotransferase were, or tended to be, positively correlated to fasting serum insulin, visceral AT (women), and alcohol intake. In women, the aminotransferases were also correlated with fasting blood glucose. In both genders alkaline phosphatase was, or tended to be, positively associated with visceral AT, insulin (women), and glucose. Bilirubin was negatively correlated to insulin and visceral AT in men and women. Additional multivariate analyses indicated that alcohol had less explanatory power than serum insulin for the examined liver tests, especially among women. These results suggest that pathological liver tests in the obese may represent an expression of the metabolic syndrome.     

Homeostatic responses to water deprivation or hemorrhage in lactating and non-lactating Bedouin goats

Three lactating and three non-lactating black Bedouin goats were subjected to four days of water deprivation or to hemorrhage. Four days of water deprivation caused body wt losses of 32 and 23% and plasma volume losses of 30 and 34% in lactating and non-lactating goats respectively. Plasma osmolality increased 17 and 15% in lactating and non-lactating goats. Plasma arginine vasopressin concentration rose from about 5 pg/ml to a mean of 36 pg/ml. Plasma renin activity increased from about 0.7 ng/ml/hr to a mean of 3.45 ng/ml/hr in lactating and to 3.15 ng/ml/hr in non-lactating goats. At 4.5 hr post-rehydration plasma osmolality and plasma vasopressin concentration were back to normal in non-lactating, but still elevated in lactating goats. Plasma renin activity increased after rehydration. Rapid blood volume loss of 21-28% increased plasma vasopressin concentration to 16-35 pg/ml in non-lactating and to 70 or greater than 500 pg/ml in lactating goats. It is concluded that black Bedouin goats are well adapted to endure severe dehydration and rapid rehydration, but that they (especially lactating animals) react strongly to rapid volume depletion.     

Elastases from human and canine granulocytes, II. Interaction with protease inhibitors of animal, plant, and microbial origin.

Inhibitors of animal, plant, and microbial origin were tested against human and canine granulocytic elastases. The trypsin-chymotrypsin inhibitors from dog submandibular glands, from soybeans (Bowman-Birk) and from chickpeas show strong interaction with these proteases (Ki = 10(-8) - 10(-9)M). The trypsin-kallikrein inactivator of bovine organs (Trasylol) is not active against granulocytic elastases or against human granulocytic cathepsin G. Elastatinal, a specific inhibitor of elastases, isolated from actinomycetes (Streptomyces griseoruber), forms stable complexes with elastase from human (Ki = 6.2 X 10(-6)M) and canine granulocytes (Ki = 1.1 X 10(-6)M). A possible therapeutic application of these inhibitors for the inactivation of granulocytic proteases, which are able to degrade connective tissue in different pathological states, is discussed.     

Purification of human granulocyte catalase in chronic myeloid leukemia.

Human granulocyte catalase (hydrogen peroxide:hydrogen peroxide oxidoreductase, EC 1.11.1.6) was purified from chronic myeloid leukemia cells. The purification procedure included heat precipitation, ammonium sulphate fractionation, DEAE-Sephadex chromatography, gel chromatography on Sephadex G-200 and isoelectric focusing with an approximate yield of 30% and a 1000-fold purification. The molecular weight of the subunit obtained by sodium dodecyl sulphate electrophoresis was 65 800. So20,w was 11.6 +/- 0.24. The pH-optimum was 6.6-6.7 and the spectrum showed a major peak at 405 nm and shoulders at 500, 540 and 625 nm typical for catalase. The electrophoretic mobility was towards the anode at pH 8.6 and identical to normal granulocyte and erythrocyte catalase. These three species of catalase gave the reaction of identity on immunodiffusion and crossed immunoelectrophoresis. The content of catalase and its activity of isolated granulocytes were approximately identical in normal and chronic myeloid leukemia granulocytes while the specific activity of leukemic catalase was higher than normal. No difference in catalase content was found between mature and immature leukemic granulocytes.     

Effects of four agents that modify microtubules and microfilaments (vinblastine,colchicine, lidocaine,and cytochalasin B) on macrophage-mediated cytotoxicity to tumor cells

Summary The effects of vinblastine, colchicine, lidocaine, and cytochalasin B on tumor cell killing by BCG-activated macrophages were examined. These four drugs were selected for their action on membrane-associated cytoskeletal components, microtubules, and microfilaments. Colchicine and vinblastine, which block microtubular synthesis, inhibit macrophage-mediated tumor-cell cytotoxicity at a concentration of 10^–6 M. Cytochalasin B, which disrupts microfilaments, enhances tumor cell lysis and stasis due to activated macrophages at a concentration of 10^–7 M. Lidocaine, which may induce the disappearance of both microtubules and microfilaments, has the same inhibiting effect as vinblastine at a concentration of 5×10^–7 M. Whereas vinblastine and lidocaine seem to act on the macrophage itself, cytochalasin B exerts its effect predominantly on the tumor cell. These results suggest that microtubules and microfilaments play a role in the destruction of tumor cells by activated macrophages.