Functional Identification of the Hypoxanthine/Guanine Transporters YjcD and YgfQ and the Adenine Transporters PurP and YicO of Escherichia coli K-12

The evolutionarily broad family nucleobase-cation symporter-2 (NCS2) encompasses transporters that are conserved in binding site architecture but diverse in substrate selectivity. Putative purine transporters of this family fall into one of two homology clusters: COG2233, represented by well studied xanthine and/or uric acid permeases, and COG2252, consisting of transporters for adenine, guanine, and/or hypoxanthine that remain unknown with respect to structure-function relationships. We analyzed the COG2252 genes of Escherichia coli K-12 with homology modeling, functional overexpression, and mutagenesis and showed that they encode high affinity permeases for the uptake of adenine (PurP and YicO) or guanine and hypoxanthine (YjcD and YgfQ). The two pairs of paralogs differ clearly in their substrate and ligand preferences. Of 25 putative inhibitors tested, PurP and YicO recognize with low micromolar affinity N⁶-benzoyladenine, 2,6-diaminopurine, and purine, whereas YjcD and YgfQ recognize 1-methylguanine, 8-azaguanine, 6-thioguanine, and 6-mercaptopurine and do not recognize any of the PurP ligands. Furthermore, the permeases PurP and YjcD were subjected to site-directed mutagenesis at highly conserved sites of transmembrane segments 1, 3, 8, 9, and 10, which have been studied also in COG2233 homologs. Residues irreplaceable for uptake activity or crucial for substrate selectivity were found at positions occupied by similar role amino acids in the Escherichia coli xanthine- and uric acid-transporting homologs (XanQ and UacT, respectively) and predicted to be at or around the binding site. Our results support the contention that the distantly related transporters of COG2233 and COG2252 use topologically similar side chain determinants to dictate their function and the distinct purine selectivity profiles.     

Have the bloody cells gone to our heads?

Recent studies have shown that cells expressing neuronal antigens can be derived from a bone marrow transplant. A new report lends support to and extends these previous results by presenting compelling morphological evidence for the generation and integration of highly differentiated bone marrow-derived neurons.     

Cellulases and xylanase of an anaerobic rumen fungus grown on wheat straw, wheat straw holocellulose, cellulose, and xylan

The activities of cellulolytic and xylanolytic enzymes produced by an anaerobic fungus (R1) which resembled Neocallimastix sp. were investigated. Carboxymethylcellulase (CMCase), cellobiase, and filter paper (FPase) activities had pH optima of 6.0, 5.5, and 6.0, respectively. CMCase and cellobiase activities both had a temperature optimum of 50 degrees C, whereas FPase had an optimum of 45 degrees C. The pH and temperature optima for xylanase activity were pH 6.0 and 50 degrees C, respectively. Growth of the fungus on wheat straw, wheat straw holocellulose, or cellulose resulted in substantial colonization, with at least 43 to 58% losses in substrate dry matter and accumulation of comparable amounts of formate. This end product was correlated to apparent loss of substrate dry weight and could be used as an indicator of fungal growth. Milling of wheat straw did not enhance the rate or extent of substrate degradation. Growth of the R1 isolate on the above substrates or xylan also resulted in accumulation of high levels of xylanase activity and lower cellulase activities. Of the cellulases, CMCase was the most active and was associated with either low or trace amounts of cellobiase and FPase activities. During growth on xylan, reducing sugars, including arabinose and xylose, rapidly accumulated in the medium. Xylose and other reducing sugars, but not arabinose, were subsequently used for growth. Reducing sugars also accumulated, but not as rapidly, when the fungus was grown on wheat straw, wheat straw holocellulose, or cellulose. Xylanase activities detected during growth of R1 on media containing glucose, xylose, or cellobiose suggested that enzyme production was constitutive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of phenolic acids and phenolics from plant cell walls on rumenlike fermentation in consecutive batch culture

Information on the interaction between mixed populations in the rumen and plant phenolics is required to fully elucidate the limitations of phenolic compounds on forage digestibility. The objective of this study was to examine the degradation of Italian ryegrass (Lolium multiflorum L.) hay incubated with mixed ruminal populations in consecutive batch culture (CBC) with or without phenolic acids or phenolic compounds extracted from plant cell walls. Each CBC consisted of a series of 10 cultures (3 replicates per culture) inoculated (10%, vol/vol) in sequence at 48-h intervals with microbial suspension from the previous set of cultures. All cultures were grown on a semidefined medium containing Italian ryegrass hay, and each CBC was initiated with an inoculum from the rumen. Rumenlike fermentation characteristics were maintained in control CBCs by repeated inoculum transfer. Treatment CBCs were transferred as described above, but cultures 5, 6, and 7 were incubated in the presence of trans-p-coumaric, cis-p-coumaric, or trans-ferulic acid or phenolics extracted from the cell walls of maize stem or barley straw. Mean apparent dry matter disappearance in control CBC cultures was 495 mg per g of hay, whereas the presence of phenolics reduced the initial dry matter disappearance by 6.3 to 25.6%. trans-p-Coumaric acid and, to a lesser extent, the phenolics from cell walls of maize stem were the most inhibitory compounds for dry matter disappearance and for the production of volatile fatty acids; trans-p-coumaric acid altered the molar ratio of acetate/propionate/butyrate. The CBC further showed variations in the ability of the rumen microbial population to adapt to phenolic compounds.     

Effect of phenolic acids and phenolics from plant cell walls on rumenlike fermentation in consecutive batch culture.

Information on the interaction between mixed populations in the rumen and plant phenolics is required to fully elucidate the limitations of phenolic compounds on forage digestibility. The objective of this study was to examine the degradation of Italian ryegrass (Lolium multiflorum L.) hay incubated with mixed ruminal populations in consecutive batch culture (CBC) with or without phenolic acids or phenolic compounds extracted from plant cell walls. Each CBC consisted of a series of 10 cultures (3 replicates per culture) inoculated (10%, vol/vol) in sequence at 48-h intervals with microbial suspension from the previous set of cultures. All cultures were grown on a semidefined medium containing Italian ryegrass hay, and each CBC was initiated with an inoculum from the rumen. Rumenlike fermentation characteristics were maintained in control CBCs by repeated inoculum transfer. Treatment CBCs were transferred as described above, but cultures 5, 6, and 7 were incubated in the presence of trans-p-coumaric, cis-p-coumaric, or trans-ferulic acid or phenolics extracted from the cell walls of maize stem or barley straw. Mean apparent dry matter disappearance in control CBC cultures was 495 mg per g of hay, whereas the presence of phenolics reduced the initial dry matter disappearance by 6.3 to 25.6%. trans-p-Coumaric acid and, to a lesser extent, the phenolics from cell walls of maize stem were the most inhibitory compounds for dry matter disappearance and for the production of volatile fatty acids; trans-p-coumaric acid altered the molar ratio of acetate/propionate/butyrate. The CBC further showed variations in the ability of the rumen microbial population to adapt to phenolic compounds.     

Growth and fermentation of an anaerobic rumen fungus on various carbon sources and effect of temperature on development.

An anaerobic fungus (strain R1) resembling Neocallimastix spp. was isolated from sheep rumen. When grown on defined medium, the isolate utilized a wide range of polysaccharides and disaccharides, but of the eight monosaccharides tested only fructose, glucose, and xylose supported growth. The organism had doubling times of 5.56 h on glucose and 6.67 h on xylose, and in each case fermentation resulted in production of formate, acetate, lactate, and ethanol. During active growth, formate was a reliable indicator of fungal biomass. Growth on a medium containing glucose and xylose resulted in a doubling time of 8.70 h, but diauxic growth did not occur since both sugars were utilized simultaneously. The optimum temperature for zoospore and immature plant development was 39 degrees C, and no development occurred below 33 degrees C or above 41 degrees C.     

FINE STRUCTURAL STUDIES ON THE INTERPHASE AND DIVIDING APICAL CELLS OF SPHACELARIA TRIBULOIDES (PHAEOPHYTA)1

The apical cells of Sphacelaria tribuloides Menegh. are larger than other thallus cells, contain more organelles and appear polarized. Their tip portion, where they grow, contains a well developed Golgi apparatus, abundant endoplasmic reticulum (ER) membranes, mitochondria, chloroplasts and a large number of small vacuoles. It seems likely that a continuous flow of membranous material from the ER membranes to the dictyosomes and from the latter to the plasmalemma of the extending tip portion takes place. In contrast, the basal pole possesses fewer organelles and is occupied mainly by large-sized, sometimes central vacuoles. The apical cells undergo two distinct types of highly asymmetrical differential divisions giving rise to cells of the thallus and hair initials. During the early stages of mitosis the nuclear envelope remains intact, except for fenestrated poles. Microtubules pass through the fenestrae into the nucleoplasm. During meta-phase, a typical chromosome plate is organized. The sites of attachment of spindle microtubules to the chromosomes are structurally different from the rest of the chromosomes. At late anaphase, the nuclear envelope breaks down completely. During telophase, a new membrane encloses the chromosomes which are decondensed and the nucleoli are reorganized. Cytokinesis proceeds long after mitosis at a stage in which the nuclei have increased in size and have moved farther apart. A membranous furrow develops centripetally, without the participation of microtubules. However, microtubules traverse the thin cytoplasmic strands which, in both interphase and cytokinetic cells, meander among the vacuoles of the basal pole of the cell and the internuclear space. Dictyosomes appear to be involved in the subsequent wall deposition.     

Kurzperiodische Schwankungen im Chlorophyllgehalt von Kotyledonen verschiedener Entwicklungsstadien

Zusammenfassung Junge Kotyledonen vonPerilla ocymoides zeigen beim tagesperiodischen Licht-Dunkel-Wechsel während des Ergrünungsprozesses eine Zunahme beider Chlorophylle in der Lichtphase, eine geringe Abnahme während der Dunkelphase. In 12 Std langen Dunkelphasen nimmt dabei das Chlorophyll a um 6%, das Chlorophyll b um 20% (das Gesamtchlorophyll um 9,5%) ab.In etwas älteren Kotyledonen zeigt sich bei 12:12stündigem Licht-Dunkel-Wechsel ein Maximum beider Chlorophylle etwa gegen Mitte der Lichtperiode, ein Minimum etwa gegen Ende der Dunkelperiode. Das Maximum des nächsten Tages erreicht zunächst noch dasjenige des vorhergehenden, liegt aber in noch älteren Kotyledonen noch geringer als dieses.Die Schwankungen im Chlorphyllgehalt laufen zeitlich parallel mit den früher festgestellten Schwankungen in der Fähigkeit zur Chlorophyllbildung.Mit 8 TextabbildungenDem Andenken anFrank Eberhardt.     

CD1 stimulation of human T cell lines induces a rapid increase in the intracellular free Ca2+ concentration and the production of IL-2.

The effect of a battery of CD1 mAb on intracellular free Ca2+ concentration and IL-2 production has been examined on different T cell lines in this study. Both 0249F and NU-T2 two CD1b specific mAb tested, induced a rapid increase in the intracellular Ca2+ concentration on HPBALL T cells whereas only one (L161) among three different CD1c mAb (L161, 10C3, and M241) produced a similar effect. In contrast the addition of four different CD1a mAb directed against two different epitopic groups of this molecule were uneffective in modifying the intracellular Ca2+. Both L161 and 0249F also induced a comparable increase in the intracellular Ca2+ concentration on MOLT 4 and JURKAT, two other T cell lines of similar phenotype. The effect of L161 mAb on the IL-2 production of the IL-2 producing T cell line JURKAT was also examined. The association of the latter with PMA strongly induced the production of IL-2 on this cell model while either L161 or PMA alone had no effect. Although the natural ligand and the function of CD1 molecules are still unknown, the accumulation of these data strongly suggest that CD1b and CD1c might represent two activatory pathways for immature T cells operating before the classical CD2 and CD3 activation pathways.     

Ragweed sensitization alters pulmonary vascular responses to bronchoprovocation in beagle dogs

Theodorou, Andreas, Natalie Weger, Kathleen Kunke, KyooRhee, David Bice, Bruce Muggenberg, and Richard Lemen. Ragweed sensitization alters pulmonary vascular responses to bronchoprovocation in beagle dogs. J. Appl. Physiol.83(3): 912-917, 1997.In ragweed (RW)-sensitized beagle dogs, wetested the hypothesis that reactivity of the pulmonary vasculature wasenhanced with aerosolized histamine (Hist) and RW. Seven dogs wereneonatally sensitized with repeated intraperitoneal RW injections, and12 dogs were controls (Con). The dogs were anesthetizedwith intravenous chloralose, mechanically ventilated, and instrumentedwith femoral arterial and pulmonary artery catheters. Specific lungcompliance(CL_sp),specific lung conductance (G_sp),systemic vascular resistance index, and pulmonary vascular resistanceindex (PVRI) were measured before and after bronchoprovocation withHist and RW. After Hist inhalation (5 breaths of 30 mg/ml), both Conand RW dogs had significant (P < 0.05) decreases inCL_sp(51 ± 4 and 53 ± 5%, respectively) andG_sp (65 ± 5 and69 ± 3%, respectively), but only RW-sensitized dogs had asignificant increase in PVRI (38 ± 10%). After RW inhalation (60 breaths of 0.8 mg/ml), only RW-sensitized dogs had significant increases (62 ± 20%) in PVRI and decreases inG_sp (77 ± 4%) and CL_sp(65 ± 7%). We conclude that, compared with Con,RW-sensitized beagle dogs have increased pulmonary vasoconstrictiveresponses with Hist or RW inhalation.