Isolation and cryopreservation of O-methylthreonine-resistant Rosa cell lines altered in the feedback sensitivity of l-threonine deaminase

O-Methylthreonine (OMT) inhibits the growth of plated Rosa cells (ID₅₀6·10^-6M). Isoleucine is able to reverse efficiently and specifically this OMT toxicity. From OMT-resistant colonies occurring at a frequency of 1.58·10^-7 variants per cell plated at 10^-4M OMT, the variant strains OMT^R-1 and OMT^R-2 were isolated, cloned via protoplasts and characterized. Both variants were ten times more resistant to OMT than the wildtype and were cross-resistant to another isoleucine analog, dl-4-thiaisoleucine. The resistant variants retained their resistance after storage for three years in liquid nitrogen. Both resistant strains were stable for several months when subcultured in the absence of OMT although it was shown in a reconstitution experiment that wildtype cells overgrow OMT^R-2 variant cells if co-cultivated for many passages in drug-free medium. One case of instability was observed upon long-term subculturing in drug-free medium: the strain OMT^R-1D^* partially lost phenotypic properties. Resistance to OMT was followed qualitatively by a new method based on inhibition-zone formation in cell suspensions plated in agar medium. The OMT-resistant variants showed a reduction in sensitivity of the enzyme l-threonine deaminase to feedback inhibition by isoleucine, a decreased stability of l-threonine deaminase when stored at-18°C or incubated at +55°C and a two- to threefold increase of the free isoleucine pool within the cells. The genetical events and the biochemical mechanisms which might lead to the observed stable and biochemically defined character are discussed with particular reference to the high ploidy level of the Rosa cell line.Abbreviations OMT l-O-methylthreonine - TD l-threonine deaminase     

Analysis of the sheep MHC using HLA class I,II, and C4 cDNA probes

Four cDNA probes for the human major histocompatibility complex (MHC) were used to investigate the sheep MHC, in conjunction with serological typing for ovine lymphocyte antigen (OLA). Lymphocytes from a family (two parents and five offspring) of Romanov sheep were subjected to genomic DNA digestion by the restriction endonuclease Eco RI, followed by gel electrophoresis. A single Southern blot representing all seven individuals was then consecutively hybridized with the class I, alpha-DC, beta-DR, and C4 probes, which were originally designed to identify HLA class I, class II (DC and DR), and C4 products, respectively. Using each of the three class I/class II probes, several bands showing DNA polymorphism were detected. The segregation of these bands in the five offspring exactly paralleled the OLA haplotype segregation established by serological typing. A further eight individuals carrying haplotypes which were phenotypically identical to those in the above-mentioned family showed bands in the corresponding positions when tested with the same three probes. Using the C4 probe, no polymorphism was detected in these fifteen individuals.Abbreviations used in this paper MHC major histocompatibility complex - OLA ovine lymphocyte antigen - kbp kilobase pair(s) - MLR mixed lymphocyte reaction - RFLP restriction fragment length polymorphism     

Risk of predation,hydrodynamic efficiency and their influence on school structure

Synopsis Laboratory studies were conducted on 15 schools of blackchin shiners, Notropis heterodon, to determine if they altered their structure in response to changing environmental demands. The hypothesis tested was that fish schools should sacrifice a flat, hydrodynamically efficient structure in favour of an unobstructed visual field in the presence of a predator by staggering in the vertical plane. Ten schools were exposed for two weeks to a simple environment with only a current. For the next two-week period a predator was added. Five control schools were exposed to the simple environment for both two-week periods. Six of the ten treated schools increased their staggering in the vertical dimension as predicted while none of the control schools changed. This result was suggestive that hydrodynamic advantages were sacrificed. Respirometer experiments indicated these fish were capable of achieving some hydrodynamic benefits from schooling but these benefits may be a function of fish size.     

Localization of alpha 1-microglobulin (HC protein) in normal human tissues: an immunohistochemical study using monoclonal antibodies

Summary Alpha-1-microglobulin is a low molecular weight (approximately 30 000 d) glycoprotein present in biological fluids. It is heterogeneous in charge. A monoclonal antibody was used to investigate the tissue distribution of the protein in normal human tissues and cell lines by indirect immunofluorescence and immunoperoxidase techniques. The protein was demonstrable in cells of the monocyte-macrophage lineage, in thymus and T cell dependent areas of spleen, lymph node and tonsils. It was detected in several lymphoid or nonlymphoid cell lines but not in peripheral blood lymphocytes. The microglobulin was also detectable in the cytoplasm of hepatocytes. Finally, it was observed in glandular secretions (sudoral glands and mucosal glands of the digestive tract) where it may be associated with IgA. Possible explanations for the highly divergent results previously reported with polyclonal antisera to ₁ microglobulin are discussed.     

Prevention of stress-induced gastric ulcers by dopamine agonists in the rat

Dopamine (DA) and DA agonists have been shown to exert a protective role against the formation of duodenal ulcers. The effect of stimulation of DA receptors on the development of stress-induced gastric ulcers is currently unknown. Accordingly, we evaluated the effect of several DA agonists on the development of gastric ulcers induced by 3 h of cold + restraint stress (CRS) in rats. Apomorphine, d-amphetamine, methylphenidate, and threo-dl-p-hydroxymethylphenidate (an hydroxylated analog of methylphenidate), significantly reduced both the incidence and severity of CRS-induced gastric ulcers. The gastric cytoprotection afforded by these agents was dose-related, and completely antagonized by pretreatment with the peripheally acting DA antagonist domperidone. Because domperidone blocks peripheral, but not central, DA receptors, and since the entry of threo-dl-p-hydroxymethylphenidate across the blood-brain barrier into the brain is restricted to a great extent, we conclude that stimulation of peripheral DA receptors is primarily involved in the gastric cytoprotection induced by dopamimetics.The pathogenesis of stress-induced gastric ulcers remains largely unknown, and significant efforts have been made over the last decade to functionally characterize some of the factors involved in the etiology of this disease. Considerable attention has been focused on gastric acid secretion, but its primary role in stress-induced gastric ulcer disease remains uncertain. In fact, agents which effectively inhibit or neutralize gastric acid secretion such as cimetidine or antacids do not necessarily exert protection against stress-induced gastric ulcers (1,2). Moreover, in our original studies with neurotensin, a brain and gastrointestinal peptide, we have found that central administration of this neuropeptide, which completely prevents the development of cold + restraint stress (CRS)-induced gastric ulcers, does not appreciably alter gastric acid secretion (2). These findings support the contention that gastric acid secretion may not be an important factor in the development of this type of gastric ulcer.There is, however, considerable evidence that the automatic nervous system plays an intermediary role in the development of these ulcers (3,4). In this regard, surgical or pharmacological blockade of the vagal (cholinergic) division of the autonomic nervous system prevents the appearance of stress-associated gastric ulcers (5,6). Direct stimulation of catecholamine receptors, or indirect activation via increased sympathetic outflow to the periphery (7,4,8–11) appears to produce a salutary effect of stress-induced gastric ulcers.Szabo and his associates (12, 13, 14) have extensively studied the anti ulcer effects of dopamine (DA) in duodenal ulcer formation. Whether DA also modifies the development of stress-induced gastric ulcers is currently unknown.We have therefore evaluated the effect of selected DA receptor agonists and antagonists on CRS-induced gastric ulcer formation in rats.     

Effects of Polyamines on the Uptake of Neurotransmitters by Rat Brain Synaptosomes

Abstract: The influence of putrescine, spermidine, spermine, and some aliphatic α,ω-diamines on the uptake of neurotransmitters by rat forebrain synaptosomes was investigated. Choline uptake was most effectively inhibited by spermine (IC₅₀= 0.22 m M ), less so by spermidine (IC₅₀= 4.0 m M ), but not by putrescine (IC₅₀ > 100 m M ). At 10 m M, 1,3-diaminopropane, cadaverine, and 1,8-diaminooctane all inhibited choline uptake by 50% or more. Spermine and spermidine inhibited the uptake of dopamine with IC₅₀ values of 2.7 and 2.2 m M , respectively. Putrescine was only slightly inhibitory (IC₅₀= 17.3 m M ) and the other diamines were inactive. The uptake of γ-aminobutyrate (GABA) was only slightly inhibited (15–40%) by the polyamines at 10 m M . With the exception of inhibition of glycine uptake by 1,8-diaminooctane (60%) and of glutamate uptake by cadaverine (35%) none of the polyamines, tested at 10 m M , affected the uptake of adenosine, glutamate, and glycine significantly. A possible modulatory role for polyamines in synaptic transmission through interaction by negatively charged groups of the synaptic membrane with the polycationic compounds is discussed.     

Seasonal and size-related changes in the food of the short-finned eel,Anguilla australis in Lake Ellesmere,Canterbury, New Zealand

Synopsis An approximately monthly sampling programme in Lake Ellesmere, Canterbury, New Zealand, from January 1974 to April 1976 yielded 487 eels. The stomachs were fixed in 10% neutralised formalin and the contents examined. Preliminary analysis indicated that the mollusc Potamopyrgus antipodarum, the isopod Austridotea annectens, the mysid Tenagomysis chiltoni, the amphipod Paracalliope fluviatilis, the midge larva Chironomus zealandicus and the teleosts Retropinna retropinna, Galaxias maculatus and Gobiomorphus cotidianus together made up the bulk of the diet. The pre-ingested dry weight (i.e. the reconstructed weight) of the most important of these prey species was obtained by relating the length of a digestion resistant part to actual dry weight in field collected specimens. Regression equations for this relationship in each season enabled the reconstructed dry weight of each stomach item to be calculated. In some instances reconstructed weight was less than the actual digested dry weight of the prey specimen. In every case the larger value was used. This method is referred to as Combination Dry Weight (CDW) and is believed to be new. These data, used in conjunction with the energy content of the species concerned, enabled the caloric dietary contribution of each prey species to be determined. Comparison of relative contribution to eel diet between CDW and energy values calculated from CDW and bomb calorimetry revealed large differences. Marked variations in diet between ⩽40 cm, 40.1–50 cm, and>50.1 cm size classes were also shown. Eels ≤40 cm feed primarily on invertebrates and become progressively more piscivorous as they grow. Eels >50.1 cm are almost entirely piscivorous. Seasonal differences in diet also exist within each size class examined.     

Confirmation of Occurrence of Hydroxamate Siderophores in Soil by a Novel Escherichia coli Bioassay

The occurrence of ferrichrome-type hydroxamate siderophores in soil was confirmed. In the presence of the iron-scavenging chelator ethylenediamine[di(o-hydroxyphenylacetic)acid], soil extract stimulated the growth of an Escherichia coli strain possessing the ferrichrome transport protein (TonA) but did not stimulate growth of a strain lacking this protein (TonA⁻). The siderophore concentration in a 1:1 (soil-water) extract was estimated to be approximately 78 nM. Specificity of the assay was supported by the absence of significant differential strain responses to ferric citrate, ferric 2,3-dihydroxybenzoate, enterochelin, ferrioxamine B, coprogen, and triacetylfusigen.     

The effect of grazing intensity on phosphorus spiralling in autotropic streams

The effect of grazing on primary productivity and phosphorus cycling in autotrophic streams was studied using the snail Goniobasis clavaeformes. Snails were added to each of three replicate laboratory stream channels, receiving once-through flow of groundwater, in densities of 2.1, 3.0, and 4.2 g ash free dry mass (AFDM)/m². A fourth channel received no snails and served as an ungrazed control. Presence of snail grazers resulted in a large reduction in aufwuchs biomass, primary productivity, and biotic phosphorus uptake; a modest reduction in fine particulate organic matter (FPOM); and an increase in the fraction of stream particulate organic matter (POM) exported as seston. Although primary production and aufwuchs biomass continued to decline with increasing snail density, phosphorus uptake increased. This increased phosphorus uptake is attributed to abiotic sorption to inorganic surfaces exposed as a result of efficient removal of aufwuchs at high snail densities. Although snail densities were chosen to bracket the density measured in a natural stream, the experimental densities may result in considerably higher grazing pressure on aufwuchs due to the absence of alternate food sources (e.g., coarse particulate organic matter) usually found in natural streams. Presence of snail grazers increased the spiralling length of phosphorus, primarily by reducing aufwuchs biomass and consequently reducing uptake of phosphorus from the water. Presence of snails also increased downstream transport velocity of phosphorus bound to organic particles. These results follow the patterns predicted in a previous theoretical analysis for mildly phosphorus-limited streams.