Microtiter micromass cultures of limb-bud mesenchymal cells

Summary A method is described for growing high-density micromass cultures of chick and mouse limb mesenchyme cells in 96-well microtiter plates (μTμM cultures). Rapid quantitative estimates of chondrogenic expression were obtained by automated spectrophotometric analysis of Alcian-blue-stained cartilage matrix extracts performed in the wells in which the cells had been grown. Quantitative estimates of myogenic expression were obtained similarly using anti-sarcomere myosin monoclonal antibody and modified ELISA techniques. This μTμM-ELISA method may be adapted for use with other antigens for which specific antibodies are available. These methods were used to compare cartilage and muscle differentiation in 1 to 4 d μTμM cultures grown in serum-containing (SCM) and defined (DM) media. The DM contains minimal additives (insulin, hydrocortisone, and in some cases, ascorbate or transferrin) and supports both chondrogenesis and myogenesis. The colorimetric analyses agree well with the morphologic appraisal of chondrogenesis and myogenesis. Similar numbers of cartilage nodules formed in all cultures, but in DM the nodules failed to enlarge; explaining the reduced matrix synthesis in DM as compared with SCM, and suggesting that nodule enlargement is a discrete, serum-dependent step. Studies of selected additives to DM show that transferrin enhances myogenesis, ascorbic acid enhances chondrogenesis, and retinoic acid inhibits chondrogenesis. Together, the μTμM system, in situ colorimetric assays of chondrogenesis and myogenesis, and DM will allow rapid prescreening of teratogens and screening of various bioactive compounds (e.g., hormones, growth factors, vitamins, adhesion factors) for effects on limb mesenchymal cell differentiation. This work was supported by grants RR08006-13 (DFP) and HD05505 and HD18577 (MS) from the National Institutes of Health, Bethesda, MD. MF-20 hybridoma supernatant was obtained from the Developmental Studies Hybridoma Bank, Department of Biology, University of Iowa, Iowa City, Iowa 52242 (maintained by NIH grant NO1-HD62915).     

Enzyme-linked immunosorbent assays for the measurement of blood group A and B glycosyltransferase activities

ELISA assays have been developed for (1–3)N-acetylgalactosaminyltransferase (blood group A transferase) and (1–3)galactosyltransferase (blood group B transferase) activities. In these assays, microtitre plates coated with the bovine serum albumin conjugate of a synthetic Fuc1–2Gal-R acceptor substrate are incubated with the appropriate nucleotide donor (UDP-GalNAc or UDP-Gal) and human serum as the enzyme source. The resulting trisaccharide products Fuc1–2(GalNAc1–3)Gal-R-BSA or Fuc1–2(Gal1–3)Gal-R-BSA are detected and quantified with monoclonal antibodies selected not to cross-react with the substrate structure. With less than a microliter of human serum, product formation is proportional to enzyme concentration and to time of incubation of up to 90 min.     

Hybrid Bacillus (1-3,1-4)-β-glucanases: engineering thermostable enzymes by construction of hybrid genes

Summary Hybrid (1-3,1-4)--glucanase genes were constructed by extension of overlapping segments of the (1-3,1-4)--glucanase genes from Bacillus amyloliquefaciens and B. macerans generated by the polymerase chain reaction (PCR). Four hybrid genes were expressed in Escherichia coli cells. The mature hybrid enzymes contain a 16, 36, 78, or 152 amino acid N-terminal sequence derived from B. amyloliquefaciens (1-3,1-4)--glucanase followed by a C-terminal segment derived from B. macerans (1-3,1-4)--glucanase. Biochemical characterization of parental and hybrid enzymes shows a significant increase in thermostability of three of the hybrid enzymes when exposed to an acidic environment thus combining two important enzyme characteristics within the same molecule. At pH 4.1, 85%-95% of the initial activity was retained after 1 h at 65° C in contrast to 5% and 0% for the parental enzymes from B. amyloliquefaciens and B. macerans. After 60 min incubation at 70° C, pH 6.0, the parental enzymes retained 5% or less of the initial activity whilst one of the hybrids still exhibited 90% of the initial activity. Of the parental enzymes B. macerans (1-3,1-4)--glucanase had the lower specific activity while the hybrid enzymes exhibited specific activities that were 1.5- to 3-fold higher. These experimental results demonstrate that exchange of homologous gene segments from different species may be a useful technique for obtaining new and improved versions of biologically active proteins.Abbreviations AMY mature form of Bacillus amyloliquefaciens (1-3,1-4)--glucanase; - MAC mature form of B. macerans (1-3,1-4)--glucanase - SUB mature form of B. subtilis (1-3,1-4)--glucanase - H(A16-M), H(A36-M), H(A78-M), H(A107-M), H(A152-M) mature forms of hybrid enzymes having 16, 36, 78, 107, 152 N-terminal amino acids, respectively, derived from AMY with the remaining amino acids derived from MAC     

Membrane transport proteins: implications of sequence comparisons.

Analyses of the sequences and structures of many transport proteins that differ in substrate specificity, direction of transport and mechanism of transport suggest that they form a family of related proteins. Their sequence similarities imply a common mechanism of action. This hypothesis provides an objective basis for examining their mechanisms of action and relationships to other transporters.     

Genome analysis ofElymus angulatus andE. patagonicus (Poaceae) and their hybrids with N. and S. AmericanHordeum spp.

The results of genome analysis of five hybrids, viz.Elymus patagonicus ×Hordeum procerum, E. patagonicus ×H. tetraploidum, E. angulatus ×H. jubatum, E. angulatus ×H. lechleri, andE. angulatus ×H. parodii, are reported. The genomic constitution ofHordeum tetraploidum andH. jubatum is best given as H₁H₁H₂H₂, ofH. lechleri andH. parodii as H₁H₁H₂H₂H₄H₄, ofH. procerum as H₁H₁H₂H₂H₃H₃, and ofElymus patagonicus andE. angulatus as SSH₁H₁H₂H₂.     

tRNA topography during translocation: steady-state and kinetic fluorescence energy-transfer studies

The distances between the anticodon loops of fluorescent tRNAPhe bound to the E site and to either the A or the P site of poly(U)-programmed Escherichia coli ribosomes were measured by fluorescence energy transfer. Donor and acceptor molecules were wybutine and proflavin, respectively, both located 3' to the anticodon of tRNAPhe. The anticodon loops were found to be separated by 42 +/- 10 A (A to E site) and 34 +/- 8 A (P to E site). The latter distance is much larger than the one measured between the anticodon loops of A and P site bound tRNAs [24 +/- 4 A; Paulsen, H., Robertson, J. M., & Wintermeyer, W. (1983) J. Mol. Biol. 167, 411-426], rendering unlikely simultaneous codon-anticodon interaction in the P and E sites. In kinetic stopped-flow measurements, the energy transfer between the anticodon loops of the tRNA molecules was followed during translocation. The transfer efficiency decreases in three steps with apparent rate constants on the order of 1, 0.1, and 0.01 s-1. The fast step is ascribed to the simultaneous displacement of the deacylated tRNAPhe out of the P site and of the N-AcPhe-tRNAPhe from the A site to the P site. The distance between the anticodon loops does not change appreciably during this reaction. A significant separation of the two tRNAs occurs during the intermediate and the slow steps. The latter most likely represents a rearrangement of the posttranslocation complex containing both tRNA molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Separate,Ca2+-activated K+ and Cl− transport pathways in Ehrlich ascites tumor cells

Summary The net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K⁺ and Cl^– transport pathways. RVD is accelerated when a parallel K⁺ transport pathway is provided by addition of gramicidin, indicating that the K⁺ conductance is rate limiting. Addition of ionophore A23187 plus Ca²⁺ also activates separate K⁺ and Cl^– transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K⁺ and Cl^– conductance is increased 14-and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl^– conductance is rate limiting. An A23187-induced activation of⁴²K and³⁶Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: (i) inhibited by quinine which blocks the Ca²⁺-activated K⁺ channel. (ii) unaffected by substitution of NO ₃ ^– or SCN^– for Cl^–, and (iii) inhibited by the anti-calmodulin drug pimozide. When the K⁺ channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl^– conductance. The Cl^– conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl^– transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca²⁺-free media (probably by release of Ca²⁺ from internal stores) is also transient, whereas the activation is persistent in Ca²⁺-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl^– transport pathway. These findings suggest that a transient increase in free cytosolic Ca²⁺ may account for the transient activation of the Cl^– transport pathway. The activated anion transport pathway is unselective, carrying both Cl^–, Br^–, NO ₃ ^– , and SCN^–. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl^– transport pathway and also blocks the activation of the K⁺ transport pathway. This is demonstrated directly by⁴²K flux experiments and indirectly in media where the dominating anion (SCN^–) has a high ground permeability. A comparison of the A23187-induced K⁺ conductance estimated from⁴²K flux measurements at high external K⁺, and from net K^– flux measurements suggests single-file behavior of the Ca²⁺-activated K⁺ channel. The number of Ca²⁺-activated K⁺ channels is estimated at about 100 per cell.     

Salicylic acid blocks indomethacin-induced cyclooxygenase inhibition and lesion formation in rat gastric mucosa

Salicylic acid has been shown to decrease gastric mucosal lesions induced by indomethacin in the rat. In vitro, it has also been shown to counteract the inhibitory effect of indomethacin and aspirin on the cyclooxygenase enzyme system in seminal vesicle microsomes and in platelets and vascular tissue. The hypothesis that the mechanism of salicylic acid "protection" against indomethacin-induced gastric lesions involves interference with indomethacin-induced mucosal cyclooxygenase inhibition was tested. Male, fasted rats were treated with intragastric salicylic acid in doses of 50, 100, 200, 300, or 400 mg/kg concomitantly with a sc injection of 20 mg/kg of indomethacin. Gastric mucosal lesions and mucosal cyclooxygenase activity (as measured by ex vivo prostaglandin F2 alpha synthesis) were examined 3 hr later. Intragastric salicylic acid, 200-400 mg/kg, significantly reduced indomethacin-induced lesion formation, while counteracting significantly indomethacin inhibition of prostaglandin synthesis. Salicylic acid alone did not significantly change cyclooxygenase activity. It is concluded that topical salicylic acid can decrease indomethacin-induced gastric mucosal lesion in the rat, in part, by counteracting the inhibitory effect of indomethacin at the cyclooxygenase level.     

An interspecies comparison of normal levels of glycosylated hemoglobin and glycosylated albumin

Aminophenylboronic acid affinity chromatography was used to measure glycosylated hemoglobin and glycosylated albumin levels in a variety of species. The highest glycosylated hemoglobin levels were found in man, the lowest in the chicken and the pig. The highest glycosylated albumin levels were found in avian species, the lowest in the mouse and the rat. A simple kinetic model was used to analyze the rates of formation of glycosylated hemoglobin and albumin in the various species. Rates of glycosylated albumin formation were very similar across the species while rates of glycosylated hemoglobin formation were quite different, presumably reflecting wide differences in erythrocyte permeability to glucose among the species.     

Mode of high temperature injury to wheat during grain development

High temperature stress adversely affects wheat growth in many important production regions, but the mode of injury is unclear. Wheat ( Triticum aestivum L. cv. Newton) was grown under controlled conditions to determine the relative magnitude and sequences of responses of source and sink processes to high temperature stress during grain development. Regimes of 25°C day/15°C night, 30°C day/20°C night, and 35°C day/25°C night from 5 days after anthesis to maturity differentially affected source and sink processes. High temperatures accelerated the normal decline in viable leaf blade area and photosynthetic activities per unit leaf area. Electron transport, as measured by Hill reaction activity, declined earlier and faster than other photosynthetic processes at the optimum temperature of 25/15 °C and at elevated temperatures. Changes in RUBP carboxylase activities were similar in direction but smaller in magnitude than changes in photosynthesic rate. Increased protease activity during senscence was markedly accentuated by high temperature stress. Specific protease activity increased 4-fold at 25/15 °C and 28-fold at 35/25 °C from 0 to 21 days after initiation of temperature treatments. Grain-filling rate decreased from the lowest to the highest temperature, but the change was smaller than the decrease in grain-filling duration at the same temperatures. We concluded that a major effect of high temperature is acceleration of senescence, including cessation of vegetative and reproductive growth, deterioration of photosynthetic activities, and degradation of proteinaceous constituents.