Immunolocalization of alpha-transforming growth factor in the developing rat mammary gland in vivo, rat mammary cells in vitro and in human breast diseases

Summary Immunoreactive alpha-transforming growth factor (-TGF) was shown by immunocytochemistry to be present in the rat mammary gland at various stages of development, the staining being most intense in mature myoepithelial cells. -TGF was also detected in the secretions of the mammary glands of pregnant and lactating rats. -TGF in the extracts of rat mammary glands at each stage of development, and in several rat mammary cell lines and in culture medium in which they had been grown, was shown by Western blotting to consist primarily of a protein of molecular weight 50 kDa. The amount of this protein was greater in the mammary gland of the lactating rat than in resting or involuting glands. -TGF was also found in some, but not all, human breast carcinomas, and in benign hyperplastic breast diseases.     

Influence of nitrate on uptake of ammonium by nitrogen-depleted soybean: is the effect located in roots or shoots?

In non-nodulated soybean [Glycine max (L.) Merrill cv. Ransom]plants that were subjected to 15 d of nitrogen deprivation inflowing hydroponic culture, concentrations of nitrogen declinedto 1.0 and 1.4mmol Ng^–1 dry weight in shoots and roots,respectively, and the concentration of soluble amino acids (determinedas primary amines) declined to 40µmol g^–1 dry weightin both shoots and roots. In one experiment, nitrogen was resuppliedfor 10 d to one set of nitrogen-depleted plants as 1.0 mol m^–3NH₄⁺ to the whole root system, to a second set as 0.5 mol m^–3NH₄⁺ plus 0.5 mol m^–3 NO₃^– to the whole root system,and to a third set as 1.0 mol m^–3 NH₄⁺ to one-half ofa split-root system and 1.0 mol m^–3 NO₃^– to theother half. In a second experiment, 1.0 mol m^–3 of nitrogenwas resupplied for 4 d to whole root systems in NH₄⁺ : NO₃^–ratios of 1:0, 9:1, and 1:1. Nutrient solutions were maintainedat pH 6.0. When NH₄⁺ was resupplied in combination with NO₃^– to thewhole root system in Experiment I, cumulative uptake of NH₄⁺for the 10 d of resupply was about twice as great as when NH₄⁺was resupplied alone. Also, about twice as much NH₄⁺ as NO₃^–was taken up when both ions were resupplied to the whole rootsystem. When NH₄⁺ and NO₃^– were resupplied to separatehalves of a split-root system, however, cumulative uptake ofNH₄⁺ was about half that of NO₃^–. The uptake of NH₄⁺,which is inhibited in nitrogen-depleted plants, thus is facilitatedby the presence of exogenous NO₃^–, and the stimulatingeffect of NO₃^– on uptake of NH₄⁺ appears to be confinedto processes within root tissues. In Experiment II, resupplyof nitrogen as both NH₄⁺ and NO₃^– in a ratio of either1:1 or 9:1 enhanced the uptake of NH₄⁺. The enhancement of NH₄⁺uptake was 1.8-fold greater when the NH₄⁺: NO₃^–-resupplyratio was 1:1 than when it was 9:1; however, only 1.3 timesas much NO₃^– was taken up by plants resupplied with the1 :1 exogenous ratio. The effect of NO₃^– on enhancementof uptake of NH₄⁺ apparently involves more than net uptake ofNO₃^– itself and perhaps entails an effect of NO₃^–uptake on maintenance of K⁺ availability within the plant. Theconcentration of K⁺ in plants declined slightly during nitrogendeprivation and continued to decline following resupply of nitrogen.The greatest decline in K⁺ concentration occurred when nitrogenwas resupplied as NH₄⁺ alone. It is proposed that decreasedavailability of K⁺ within the NH₄⁺-resup-plied plants inhibitedNH₄⁺ uptake through restricted transfer of amino acids fromthe root symplasm into the xylem. Key words: Ammonium, Glycine max, nitrate, nitrogen-nutrition, nitrogen stress, split-root cultures     

Spotlight on phytochrome nomenclature

These recommendations for genes encoding phytochromes were developed independently by Quail et al., but are broadly consistent with the Commission's guidelines. Their original article, kindly provided in advance of publication, appeared as a Letter to the Editor inPlant Cell (6:468–471, 1994) and is published with permission of the American Society of Plant Physiologists.     

Simple electromyographic biofeedback treatment for chronic pediatric constipation/encopresis: Preliminary report

Pediatric constipation/encopresis is thought to be due, in part, to paradoxical constriction of the external anal sphincter (EAS) muscle during attempted defecation. This inappropriate contraction can lead to delayed, impacted, painful, and infrequent bowel movements. Standard Medical Care (SMC) involves disimpaction with enemas, followed by laxative therapy and diet modification, to maintain frequent soft stools. Using the case control method, the efficacy of SMC alone was compared with SMC plus EAS electromyographic biofeedback aimed at eliminating paradoxical contraction. Thirteen consecutive chronically constipated children received SMC plus biofeedback, and were compared with 13 age- and sex-matched children who received only SMC. Biofeedback subjects demonstrated post-treatment elimination of EAS paradoxical constriction. At 16 months follow-up parents of biofeedback children reported significantly greater improvement in constipation, encopresis, laxative use, and painful bowel movements compared to SMC.This research report was supported by the NIH under grant No. RO1 HD 28160.     

Host selection and survival of the parasitoidEncarsia formosa on greenhouse whitefly,Trialeurodes vaporariorum, in the presence of hosts infected with the fungusAschersonia aleyrodis

Successful control of greenhouse whitefly may be achieved by complementary activity of the parasitoidEncarsia formosa and the fungusAschersonia aleyrodis. One way to obtain an additive mortality effect of both entomopathogen and parasitoid would be achieved by the selection of healthy hosts by the parasitoid and rejection of fungus-infected hosts. Third and fourth instar larvae ofTrialeurodes vaporariorum which had been treated with a spore suspension ofA. aleyrodis 0, 4, 7, 10 or 14 days beforehand, were presented to female parasitoids. The parasitoids adopted the oviposition posture on untreated hosts as well as on treated hosts, irrespective of the different stages of infection in the hosts. However, significantly more hosts were parasitized byE. formosa in the control treatment than in the fungal treatment. The parasitoids offered treated hosts, showed rejection behaviour after probing on hosts showing detectable signs of infection (containing hyphal bodies or mycelium in the haemolymph). For instance, when hosts were offered seven days after spore treatment, the parasitoids showed an oviposition posture on a total of 83 (95.4%) out of 87 infected larvae, but laid only 4 eggs (4.6%). In contrast, on 48 (94.1%) out of 51 noninfected (or showing no detectable signs of infection) hosts an oviposition posture was adopted and 40 eggs (78.4%) were found after dissection. When infected hosts were encountered the oviposition posture lasted less than 1′40″ while rejection of non-infected hosts occurred after more than 1′40″. Other experiments were carried out offering treated hosts for 24 h to the parasitoids. The hosts were dissected afterwards. Again, significantly more eggs were laid in the non-infected hosts. When hosts were parasitized shortly after fungal spore treatment they were colonized by the fungus and the parasitoids did not develop. Transmission of the entomopathogen after probing infected hosts was observed to a limited extent. In conclusion,A. aleyrodis andE. formosa can be used together in a glasshouse situation. The parasitoid will be most effective when introduced more than seven days after application ofA. aleyrodis, because from that time onwards it is able to detect and reject fungus-infected hosts.     

Considerations for operational space definition and optimization of a no-salt flowthrough hydrophobic interaction chromatography purification step

No-salt flowthrough hydrophobic interaction chromatography (HIC) has been shown to effectively remove process and product-related impurities from bioprocess streams. In this publication, a panel of six antibodies has been used to demonstrate operating principles for the application of no-salt flowthrough HIC in antibody purification processes. The results indicate that no-salt flowthrough HIC provides robust aggregate clearance across operating conditions including flow rate, and variations in resin ligand density. Additionally, HMW reduction has an optimal pH range relative to the isoelectric point of each molecule and high molecular weight (HMW) reduction can be improved by altering the total protein load and/or HMW concentration to drive binding of high molecular weight species to the resin.     

Biosynthesis of Cephalosporin C

The superiority of d-methionine over l-methionine for stimulation of cephalosporin C synthesis in a crude medium was confirmed. The optimal level of dl-methionine was 0.5%. Methionine stimulates growth slightly but this is not thought to be the cause of the marked stimulation of antibiotic synthesis. Of a large number of sulfur compounds tested, only dl-methionine-dl-sulfoxide and S-methyl-l-cysteine showed considerable methionine-replacing activity. Lysine and α-aminoadipic acid were inactive.     

Chirality differences in amino acid retention and release from the acid-extractable pool of cultured mammalian cells

In previous work, no chiral differences were found between D and L enantiomers of Leu in their ability to displace one another from the acid-extractable pool in mammalian cells. Recent evidence suggested otherwise. Our aim is to examine whether, in physiological range, D-amino acids have an equivalent ability to displace L-amino acids from the acid-extractable pool of HeLa cells, and vice versa. In the millimolar range, D-Leu and L-Leu have similar uptake and displacement properties with regard to the acid-extractable pool in HeLa cells, despite only the latter isomer being incorporated into protein. Below millimolar concentrations however, a distinct difference was found in the displacement of tritium-labelled L-Leu from the pool by unlabelled D-Leu compared with unlabelled L-Leu. Thus, unlabelled L-Leu in the external medium at 10⁻⁴ or 10⁻⁵ M displaced an equivalent amount of label from the pool as D-Leu introduced at a concentration approx. one order of magnitude higher, respectively. Reciprocal experiments, in which the acid-extractable pool was preloaded with ³H-D-Leu, confirmed this finding. The chirality difference was noted whether pool prelabelling was carried out at 37 or 0°C; but in order to avoid the complications of active transport mechanisms, the competition work reported here was done at 0°C. Similar chirality differences were observed with other hydrophobic amino acids, including His, Ile and Phe, such as, preferential displacement by the L-Leu racemer compared with the D-Leu racemer below mM levels. This was also true for the D and L forms of the non-utilisable isomer of Leu, norleucine (nLeu). We conclude that D-forms of hydrophobic amino acids have lower affinity for similar or the same intracellular binding sites involved in the acid-extractable pool than their L-forms. The significance of these chirality findings to amino acid pools in cells, and to the predominance of L-forms of amino acids in the biosphere is considered.     

Characterization of anther-expressed genes encoding a major class of extracellular oleosin-like proteins in the pollen coat of Brassicaceae†

A large, heterogeneous, highly expressed gene family encoding oleosin-like proteins is described in the Brassicaceae. íeven related cDNA sequences were isolated from Brassica napus anther mRNA using RACE-PCR and compared with other recently described anther-specific oleosin-like genes from B. napus. The expression patterns of four representative members of this diverse gene family were analyzed by Northern blotting and in situ hybridization. In all cases, the genes were expressed specifically in the tapetum of 3–5 mm B. napus buds, which contained microspores at the late-vacuolate and bicellular stages of development. The predicted protein products are ordered into subclasses, each of which has a characteristic C-terminal domain, containing different amino acid motifs or repeated residues. Tryphine (pollen coat) fractions from mature B. napus pollen were found to be particularly enriched in polypeptides of apparent molecular weights 32–38 kDa, plus numerous less abundant polypeptides of less than 15 kDa. The N-terminal 15–20 residues of three of these polypeptides (12, 32 and 38 kDa) were found by microsequencing to be identical to parts of the predicted amino acid sequences of three of the tapetal-expressed oleosin-like genes. This indicates the possibility of post-translational modification of these proteins resulting in a cleavage of the primary translation products in order to generate the mature tryphine polypeptides. These data imply that a large and diverse group of oleosin-like proteins is synthesized in the tapeturn of B. napus anthers and that following tapetal degradation, these proteins, possibly in modified form, then relocate to the developing microspores where they eventually constitute some of the major components of the extracellular tryphine of mature pollen grains. These proteins share a conserved 70 amino acid residue hydrophobic domain and are related structurally to the seed-specific intracellular oleosins, although their biological function may be different.