Effects of continuous nitrogen application and nitrogen preconditioning on nodulation and growth ofCeanothus griseus varhorizontalis

Rooted cuttings ofCeanothus griseus varhorizontalis were irrigated with 0, 10, 20, 50, 75 or 100ppm nitrogen as NH₄NO₃ for eight weeks prior to inoculation with infectiveFrankia. After inoculation, half of the plants for each treatment nitrogen level continued to be irrigated with the preconditioning nitrogen level and half were given no more supplemental nitrogen. For plants continuously receiving nitrogen, nodule initiation (nodule number) was inversely correlated with increasing supplemental nitrogen levels, and suppressed above 50 ppm N. Leaf nitrogen above 2% in continuous-N plants correlated with greatly reduced or suppressed nodulation. Plants maintained after inoculation without supplemental nitrogen showed influence of the prior nitrogen treatment on nodulation. Preconditioning at 50 ppm and above greatly reduced the number of nodules formed. The evidence suggests that stored internal nitrogen can regulate nodulation.Plant biomass accumulated maximally when nodulation was suppressed, at 75 and 100 ppm supplemental N applied continuously. Internode elongation during the nodulation period occurred only on nodulated plants, or in the presence of supplemental N (10 ppm and above).     

The effect of cortisol on thyroid hormone kinetics in the ovine fetus

The mechanism underlying the association of rising concentrations of circulating triiodothyronine (T3) with the prepartum surge in the concentration of cortisol was investigated in 11 fetal sheep. The concentrations and metabolic clearance rates of T3 and thyroxine (T4) were measured prior to and following a continuous intravascular infusion of cortisol (1 mg/h for 84 h). Mean plasma T3 concentrations increased 10-fold following cortisol infusion whereas the concentrations of T4 either remained stable or exhibited a variable decline. Cortisol induced a 5-fold decrease in the metabolic clearance rate of T3 and a 6-fold increase in that of T4. The corresponding mean production rates of T3 and T4 increased significantly although the magnitude of the change varied between fetuses. We conclude that the prepartum rise in plasma T3 concentrations is likely to be a consequence of both a decreased metabolic clearance of T3 and increased peripheral conversion of T4 to T3 caused by rising concentrations of cortisol in fetal plasma.     

The hypogonadotropic state of the prepubertal male rhesus monkey (Macaca mulatta) is not associated with a decrease in hypothalamic gonadotropin-releasing hormone content

Hypothalamic contents of gonadotropin-releasing hormone (GnRH) in neonatally orchidectomized infant, juvenile, and adult monkeys were measured by a radioimmunoassay (RIA) and by an in vivo bioassay that utilized luteinizing hormone (LH) secretion in estrogen- and progesterone-treated ovariectomized rats. The results of the bioassay provided no evidence to suggest that hypothalamic GnRH content in juvenile monkeys (mean = 83 ng/hypothalamus; n = 3) was less than that in infants (mean = 54 ng/hypothalamus; n = 4) and adults (mean = 36 ng/hypothalamus; n = 3). A similar developmental pattern in hypothalamic GnRH content was also observed when the decapeptide was measured by RIA. In striking contrast to the maintenance of hypothalamic GnRH content throughout postnatal development, pituitary gonadotropin contents and serum gonadotropin concentrations were markedly reduced in juvenile monkeys.     

Expression of human plasminogen cDNA in a baculovirus vector-infected insect cell system

A cDNA that encodes the human plasminogen (HPg) amino acid sequence has been inserted adjacent to the polyhedrin promoter in the genome of the baculovirus, Autographa californica nuclear polyhedrosis virus, which was then used to infect cultured cells of the farm armyworm, Spodoptera frugiperda. Under the conditions of cell growth employed, recombinant (rec)-HPg was secreted into the medium after 24 h postinfection (p.i.), at which point virtually no rec-HPg antigen remained inside the cells. At 48 h p.i., a maximal level of intact rec-HPg was present in the medium, which underwent substantial proteolytic digestion after that time. The rec-HPg produced by this expression system possessed a molecular weight equivalent to that of plasma [Glu1]-plasminogen. In addition, the rec-HPg adsorbed to Sepharose-lysine, and was eluted with epsilon-aminocaproic acid (EACA). The recombinant protein also interacted with polyclonal antibodies generated to plasma HPg, as well as with a monoclonal antibody directed against a distinct region (kringle 1-3) of the plasma HPg molecule. Finally, the insect-expressed rec-HPg was activatable to plasmin (HPm) by urokinase. The results demonstrate that this expression system produces a full-length functional single-chain rec-HPg, which can be isolated intact from the culture medium, with some consideration for the temporal events that occur in secretion and longer-term degradation of the protein. The fact that this rec-HPg was converted to HPm with a plasminogen activator, and that it interacted with anti-plasma HPg polyclonal and monoclonal antibodies, as well as with the ligand, EACA, indicates that the molecule retains many of its important functional properties and is folded in an integral manner.     

Patterns of [13N]ammonium uptake and assimilation by Frankia HFPArl3

Nitrogen-starved cells of Frankia strain HFPArl3 incorporated [¹³N]-labeled ammonium into glutamine serine (glutamate, alanine, aspartate), after five-minute radioisotope exposures. High initial endogenous pools of glutamate were reduced, while total glutamine increased, during short term NH _inf4 ^sup+ incubation. Preincubation of cells in methionine sulfoximine (MSX) resulted in [¹³N]glutamine reduced by more than 80%, while [¹³N]glutamate and [¹³N]alanine levels increased. The results suggest that glutamine synthetase is the primary enzyme of ammonium assimilation, and that glutamate dehydrogenase and alanine dehydrogenase may also function in ammonium assimilation at low levels. Efflux of [¹³N]serine and lesser amounts of [¹³N]glutamine was detected from the Frankia cells. The identity of both Ser and Gln in the extracellular compartment was confirmed with gas chromatography/mass spectrometry. Serine efflux may be of significance in nitrogen transfer in Frankia.Abbreviations Pthr phosphothreonine - Aad -amino-adipate - MSX methionine sulfoximine     

Phototoxic liposomes coupled to an antibody that alone cannot modulate its cell-surface antigen kill selected target cells

Summary Molecules such as antibodies that bind to cell surfaces can be used to deliver cytotoxic drugs to selected cells. To be effective the drug must usually be taken into the cells by endocytosis. In this study a T-cell line (CCRFCEM) was effectively killed by liposomes carrying a photosensitizer and bearing the antibody OKT4 (anti-CD4). The unconjugated antibody does not induce antigenic modulation in the target cells, an indication of the absence of endocytosis, and would therefore not normally have been selected as an agent for drug delivery. It cannot, however, be concluded with certainty that the conjugates act at the cell surface and several alternative explanations of their efficacy are offered.     

Endo-exonuclease of Aspergillus nidulans

Endo-exonuclease (EE) has been found in both active and inactive, but trypsin-activatable, forms in Aspergillus nidulans. Active EE was present mainly in nuclei, mitochondria, and vacuoles, while trypsin-activatable EE was mainly in the cytosol. The active form accounts for over 90% of the neutral deoxyribonuclease activity extracted from mycelia. A single strand (ss) DNA-binding EE associated with a 28 kilodalton (kDa) polypeptide was partially purified and characterized. It was found to closely resemble, in size and enzymological properties, the ss-DNA-binding EE previously purified from Neurospora crassa. Aspergillus nidulans EE was also found to be immunochemically related to the N. crassa EE and, like that enzyme, was probably derived from a polypeptide of 90 kDa or larger through proteolysis during extraction and purification. It had divalent metal ion-dependent (Mg2+, Mn2+, or Zn2+) activity on both DNA and RNA, which ultimately produced small 5'-P-terminated oligonucleotides. The nuclease activity was mixed endo- and exo-nucleolytic with ss-DNA as substrate, but largely exonucleolytic with double strand (ds) DNA. Superhelical phi X-174 DNA was nicked by EE to form relaxed circular and then linear ds-DNA, which was rapidly degraded to shorter fragments. Linearized pBR322 DNA was extensively nicked internally under conditions where there was relatively low exonuclease activity, but this nicking required that 5'-P-termini be present on the linear ds-DNA. The levels of active EE found in extracts of two recombination-deficient mutants of A. nidulans, uvsC and uvsE, dit not differ significantly from those in extracts of the wild type.     

Differential methylation of the hypervariable locus DXS255 on active and inactive X chromosomes correlates with the expression of a human X-linked gene

Consistent differences in methylation of particular cytosine residues in the DNA of active and inactive X chromosomes can be used for rapid, direct analysis of X-inactivation patterns in different female tissues. We have studied methylation of the highly polymorphic DXS255 locus in tissues from patients with deficiency of the E1 alpha subunit of the pyruvate dehydrogenase complex in whom the results can be correlated directly with total enzyme activity, levels of immunoreactive protein, and patterns of cell mosaicism. The results confirm that methylation of the DXS255 locus correlates with X-chromosome expression. In patients and normal controls, the pattern of X inactivation varied widely from tissue to tissue and often deviated markedly from a 50:50 proportion. These deviations are likely to reflect small numbers of tissue-specific stem cells at the time of random X inactivation and cannot be taken alone as evidence for selection or "nonrandom" inactivation.     

Dynamic changes in optic fiber terminal arbors lead to retinotopic map formation: an in vivo confocal microscopic study

Dynamic remodeling of retinal ganglion cell terminal arbors has been proposed to contribute to formation of the topographically ordered retinotectal projection. To test this directly, the growth of individual terminal arbors was observed in live X. laevis tadpoles using a confocal microscope to visualize their complex three-dimensional structure. During initial development, nasal and temporal retinal arbors covered overlapping tectal areas. Despite subsequent remodeling, the dimensions and positions of the temporal arbors remained relatively stable. In contrast, the nasal arbors grew caudally, as they extended caudal branches and retracted rostral branches. These results suggest that differences in the remodeling of the nasal and temporal arbors lead to the emergence of retino-topography along the rostrocaudal axis of the tectum. All the terminal arbors were dynamic, including those with stable dimensions, suggesting that continual remodeling of arbors may be a universal feature of neuronal projections.     

Uptake and degradation of hyaluronan in lymphatic tissue.

Afferent lymph vessels entering popliteal lymph nodes of sheep were infused with [3H]acetyl-labelled hyaluronan of high Mr (4.3 x 10(6)-5.5 x 10(6)) and low Mr (1.5 x 10(5)). Analysis of efferent lymph and of residues in the nodes showed that hyaluronan presented by this route is taken up and degraded by lymphatic tissue. Labelled residues isolated in node extracts by gel chromatography and h.p.l.c. included N-acetylglucosamine, acetate, water and a fraction provisionally identified as N-acetylglucosamine 6-phosphate. Between 48 and 75% of the infused material was unrecovered, and had been presumably eliminated through the bloodstream as diffusible residues. Rates of degradation reached as high as 43 micrograms/h in a node of 2 g wt. infused with 56 micrograms/h. Some HA passed into efferent lymph and some was detected in the nodes, but fractions of Mr greater than 1 x 10(6) were not found in either. It is concluded that the amounts and Mr values of hyaluronan released from the tissues into peripheral lymph can be significantly underestimated by analysis of efferent lymph, i.e. lymph that has passed through lymph nodes. A substantial role in the normal metabolic turnover of at least one major constituent of intercellular matrix and connective tissue may now be added to the established functions of the lymphatic system.