Effects of replacement of active site residue glutamine 231 on activity and allosteric properties of aspartate transcarbamoylase.

Since crystallographic studies on Escherichia coli aspartate transcarbamoylase (ATCase) indicate that Gln 231 is in the active site of the enzyme and participates in the binding of the substrate, aspartate, it seemed of interest to examine mutant enzymes in which Gln 231 was replaced by Asn or Ile. The two mutant forms containing amino acid substitutions were characterized by a combination of steady-state kinetics, hydrodynamic measurements, and equilibrium ligand binding techniques. Both mutant forms exhibited a dramatic reduction in the affinity of the protein for substrates and substrate analogues as well as a very large decrease in catalytic activity. Moreover, the amino acid substitutions introduced within the active site of the enzyme led to unusual allosteric properties in the mutant enzymes. Although the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate promotes the characteristic global conformational change in the mutant forms that is observed with the wild-type enzyme, the combination of substrate and substrate analogue does not. Cooperativity with respect to substrate binding is largely reduced compared to wild-type ATCase. Also, the effector molecules ATP and CTP which bind to the regulatory chains have dramatic effects on the activity of the mutant enzymes containing replacements for Gln 231 in the catalytic chains. In stark contrast to the wild-type enzyme, in which effects of nucleotides are manifested primarily by changes in the K0.5 of the enzyme, ATP and CTP have large effects on the Vmax of the mutant enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concentration of aerosolized 99mTc-albumin in the pulmonary lymph of anesthetized sheep

We examined the lymphatic concentration of 99mTc-albumin deposited in the air spaces of anesthetized sheep to determine whether changes in the concentration reflected changes in lung epithelial function. Five control sheep were ventilated with an aerosol of 99mTc-albumin for 6 min, and the lung lymphatic concentration of the tracer was monitored for the next 2 h. During the last 45 min the lymphatic concentration stabilized at a value that was 0.03 +/- 0.01% of the estimated value in the air spaces. Pulmonary vascular hypertension, induced in seven sheep by increasing the left atrial pressure 20 cmH2O for 4 h, increased the lung lymph flow from a base-line value of 3 +/- 2 to 21 +/- 14 ml/h. This caused the concentration of the 99mTc-albumin in the lymph to double to 0.07 +/- 0.03% of the air space concentration (P less than 0.01). Lung injury induced by infusing 0.08-0.10 ml/kg oleic acid intravenously in seven other sheep increased the lymphatic concentration of the 99mTc-albumin 10-fold to 0.31 +/- 0.09% of the air space concentration (P less than 0.01). The increased tracer concentration in the sheep with pulmonary vascular hypertension could be the result of the increased lymph flow causing a diversion of tracer into the lymphatics. However, a mathematical model showed that the 10-fold increase in the lymphatic concentration in the sheep with lung injury was primarily the result of an increase in both permeability and surface area of the epithelium that participated in the transfer of the 99mTc-albumin from the air spaces into the lung tissue drained by the lymphatics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions between the ankyrin repeat-containing protein Akr1p and the pheromone response pathway in Saccharomyces cerevisiae.

Akr1p, which contains six ankyrin repeats, was identified during a screen for mutations that displayed synthetic lethality with a mutant allele of the bud emergence gene BEM1. Cells from which AKR1 had been deleted were alive but misshapen at 30 degrees C and inviable at 37 degrees C. During a screen for mutants that required one or more copies of wild-type AKR1 for survival at 30 degrees C, we isolated mutations in GPA1, which encodes the G alpha subunit of the pheromone receptor-coupled G protein. (The active subunit of this G protein is G beta gamma, and G alpha plays an inhibitory role in G beta gamma-mediated signal transduction.) AKR1 could serve as a multicopy suppressor of the lethality caused by either loss of GPA1 or overexpression of STE4, which encodes the G beta subunit of this G protein, suggesting that pheromone signaling is inhibited by overexpression of Akr1p. Mutations in AKR1 displayed synthetic lethality with a weak allele of GPA1 and led to increased expression of the pheromone-inducible gene FUS1, suggesting that Akr1p normally (and not just when overexpressed) inhibits signaling. In contrast, deletion of BEM1 resulted in decreased expression of FUS1, suggesting that Bem1p normally facilitates pheromone signaling. During a screen for proteins that displayed two-hybrid interactions with Akr1p, we identified Ste4p, raising the possibility that an interaction between Akr1p and Ste4p contributes to proper regulation of the pheromone response pathway.     

Genomic organization of a mouse MHC class II region including theH2-M andLmp2 loci

The region encompassing theMa, Mb1, Mb2, andLmp2 genes of the mouse class II major histocompatibility complex (MHC) was sequenced. Since this region contains clusters of genes required for efficient class I and class II antigen presentation, it was interesting to search for putative additional genes in the 21 kilobase gap between theMb1 andLmp2 genes. Computer predictions of coding regions and CpG islands, exon trapping experiments, and cross-species comparison with the corresponding human sequence indicate that no additional functional gene is present in that stretch. However, computer analysis revealed the possible existence of an alternative 3 exon forMb1. Except for the fact that the mouse MHC contains twoMb genes, the genomic organization of theH2-M loci was found to be almost identical to the organization of the humanHLA-DM genes. The promoter regions of theMa andMb genes also resemble classical class II promoters, containing typical S, X, and Y boxes. Like the human genes, the threeH2-M genes displayed very limited polymorphism when we compared the cDNA sequences from six haplotypes. Finally, comparison ofDMB withMb1 andMb2, both at the genomic level and in their coding regions, suggests that theMb gene was recently duplicated, probably only in certain rodents.     

Functions of passage cells in the endodermis and exodermis of roots

Passage cells frequently occur in the endodermis and exodermis but are not ubiquitous in either layer. Passage cells occur in the form of short cells in the dimorphic type of exodermis. In both layers, Casparian bands are formed in all cells, but the subsequent development of suberin lamellae and thick, cellulosic walls are delayed or absent in the passage cells. Available evidence suggests that passage cells of the endodermis are important for the transfer of calcium and magnesium into the stele and thus into the transpiration stream. They become the only cells which present a plasmalemma surface to the soil solution (and are thus capable of ion uptake) when the epidermis and central cortex die. This occurs naturally in some herbaceous and woody species and is known to be promoted by drought. Most evidence indicates that the development of suberin lamellae in both the endodermis and exodermis increases the resistance of the root to the radial flow of water. Passage cells thus provide areas of low resistance for the movement of water, and the position of these cells in the endodermis (i.e., in close proximity to the xylem) is explained in terms of function. Exodermal passage cells have a cytoplasmic structure suggesting an active role in ion uptake. This may be related to the tendency of the epidermis to die, leaving the passage cells as the only ones with their membranes exposed to the soil solution. Passage cells in the exodermis attract endomycorrhizal fungi while those in the endodermis do not. It is clear that passage cells of the endodermis and exodermis play a variety of roles in the plant root system.     

Microbial Utilization of Estuarine Dissolved Organic Carbon: a Stable Isotope Tracer Approach Tested by Mass Balance

The natural stable isotope values of different plants have been used to trace the fate of organic carbon that enters estuarine ecosystems. Experiments were designed to determine the magnitude of (delta) (sup13)C changes of dissolved organic carbon (DOC) derived from tidal marsh vegetation that occurred during bacterial decomposition. Bacteria were grown on DOC leached from estuarine Spartina alterniflora and Typhus angustifolia plants. In four experiments, 25 to 80% of the initial carbon (2.6 to 9.1 mM organic C) was converted to bacterial biomass and CO(inf2). Mass balance calculations showed good recovery of total C and (sup13)C at the end of these experiments (100% (plusmn) 14% total C; (plusmn) 1(permil) (delta) (sup13)C). The (delta) (sup13)C values of DOC, bacterial biomass, and respired CO(inf2) changed only slightly in the four experiments by average values of -0.6, +1.4, and +0.5(permil), respectively. These changes are small relative to the range of (delta) (sup13)C values represented by different organic carbon sources to estuaries. Thus, microbial (delta) (sup13)C values determined in the field helped to identify the source of the carbon assimilated by bacteria.     

Thidiazuron-induced adventitious shoot regeneration of sweetpotato (Ipomoea batatas)

Adventitious shoots of sweetpotato (Ipomoea batatas L. Lam.) were produced in vitro using a two-stage culture method. Petiole explants were incubated on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxy acetic acid (0.2 mg·liter^−1) for 3 d, and transferred to MS medium with thidiazuron (0 to 0.4 mg·liter^−1). Shoot regeneration was observed in most explants (78.2%) of genotype PI 318846-3 within 28 days when cultured on thidiazuron at 0.2 mg·liter^−1. Histological studies of cultured petiole explants showed meristematic activity within cells of vascular bundles and throughout the ground tissue. Explants isolated from apical leaves exhibited higher shoot regeneration frequency than those isolated from the basal portion of the shoot. Leaf lamina explants exhibited lower frequency of regeneration than petiole explants. In contrast to thidiazuron, the use of zeatin riboside, and kinetin resulted in a lower frequency of shoot regeneration although more sweetpotato genotypes could be regenerated using either of these two cytokinins. The sweetpotato plants regenerated using thidiazuron grew vigorously and rooted easily when transferred to the greenhouse.     

Displacement of housekeeping proteasome subunits by MHC-encoded LMPs: a newly discovered mechanism for modulating the multicatalytic proteinase complex.

The degradation of cytoplasmic antigens to peptides presented by class I MHC molecules is thought to be mediated by the ubiquitin/proteasome pathway. Support for this view came from our observation that the subunit composition of proteasomes can be changed by interferon-gamma (IFN-gamma) treatment. Thereby two subunits, LMP2 and LMP7, which are encoded in the MHC class II region, are incorporated into the proteasomal complex, whereas other subunits disappear. In the experiments reported in this communication we studied the subunit changes occurring in cell lines where the expression of LMP2 or LMP7 can be regulated individually either by IFN-gamma induction or by applying a new system to control the expression of transfected LMPs. In both situations LMP2 induction leads exclusively to the disappearance of housekeeping subunit 2, whereas LMP7 affects only subunit 10. Subunit 2 was found to be 76% homologous to LMP2. Since incorporation of LMP2 into the proteasomal complex prevents processing of the subunit 2 precursor, we conclude that LMP2 displaces subunit 2 during assembly. Subunit displacement is most likely a general mechanism to modulate the catalytic activity of the proteasomal complex without changing its structure. Furthermore, the controlled incorporation of transfected subunits into the complex offers a new approach to study proteasome function in vivo.     

Alliances of Muhlenbergia (Poaceae) within New World Eragrostideae are identified by phylogenetic analysis of mapped restriction sites from plastid DNAs

Restriction sites for six enzymes were mapped for the plastid DNAs of 25 species of Eragrostideae, one species of Cynodonteae (Eustachys distichophylla), and one species of Pooideae. Of the 124 restriction sites observed, 67 were variably present and shared by two or more species. These data were analyzed by the parsimony method using equal and unequal weights and by bootstrap analysis. The cladistic analyses established that members of the Muhlenbergiinae, including the genera Muhlenbergia, Blepharoneuron, Bealia, Chaboissaea, Lycurus, and Pereilema, share seven restriction site mutations and are strongly supported by the data as a monophyletic subtribe. Surprisingly, Redfieldia flexuosa also clustered with the Muhlenbergiinae in the analysis, perhaps indicative of a past interspecific hybridization event. The restriction sites data also weakly support a relationship (six shared mutations) between Erioneuron, Munroa, and Dasyochloa.     

Plant and algal interference in bacterial beta-D-galactosidase and beta-D-glucuronidase assays.

Several commonly occurring freshwater and marine plants and algae were screened for beta-D-galactosidase and beta-D-glucuronidase activities by using a 60-min enzyme assay based on the hydrolysis by these enzymes of 4-methylumbelliferyl-beta-D-galactoside and 4-methylumbelliferyl- beta-glucuronide, respectively. All freshwater plant extracts tested showed beta-D-galactosidase activity several at relatively high levels, and a number also showed beta-D-glucuronidase activity. A number of the macroalgae showed no activity of either enzyme, but those showing beta-D-galactosidase activity also showed beta-D-glucuronidase activity. The majority of microalgae showed some beta-D-galactosidase activity, but few showed beta-D-glucuronidase activity. Further studies, using the commercial Colilert test and the marine water formulation of Colilert, revealed that 2 of 11 of the microalgal species and several of the plant extracts tested caused positive reactions. It was concluded that several plant extracts and algae could significantly interfere with the detection of coliform bacteria and Escherichia coli with the use of rapid assays, on the basis of their production of beta-D-galactosidase and beta-D-glucuronidase, respectively. The significance of the plant and algal interferences in tests such as Colilert is dependent on the levels of enzymes released under natural conditions, the dilution which they may undergo, and the numbers of algal cells present. This also applies to interferences in rapid enzyme assays.(ABSTRACT TRUNCATED AT 250 WORDS)