Developmental and Tissue-Specific Structural Alterations of the Cell-Wall Polysaccharides of Arabidopsis thaliana Roots

The plant cell wall is a dynamic structure that plays important roles in growth and development and in the interactions of plants with their environment and other organisms. We have used monoclonal antibodies that recognize different carbohydrate epitopes present in plant cell-wall polysaccharides to locate these epitopes in roots of developing Arabidopsis thaliana seedlings. An epitope in the pectic polysaccharide rhamnogalacturonan I is observed in the walls of epidermal and cortical cells in mature parts of the root. This epitope is inserted into the walls in a developmentally regulated manner. Initially, the epitope is observed in atrichoblasts and later appears in trichoblasts and simultaneously in cortical cells. A terminal [alpha]-fucosyl-containing epitope is present in almost all of the cell walls in the root. An arabinosylated (1->6)-[beta]-galactan epitope is also found in all of the cell walls of the root with the exception of lateral root-cap cell walls. It is striking that these three polysaccharide epitopes are not uniformly distributed (or accessible) within the walls of a given cell, nor are these epitopes distributed equally across the two walls laid down by adjacent cells. Our results further suggest that the biosynthesis and differentiation of primary cell walls in plants are precisely regulated in a temporal, spatial, and developmental manner.     

Relative apparent synapomorphy analysis (RASA). I: The statistical measurement of phylogenetic signal

We have developed a new approach to the measurement of phylogenetic signal in character state matrices called relative apparent synapomorphy analysis (RASA). RASA provides a deterministic, statistical measure of natural cladistic hierarchy (phylogenetic signal) in character state matrices. The method works by determining whether a measure of the rate of increase of cladistic similarity among pairs of taxa as a function of phenetic similarity is greater than a null equiprobable rate of increase. Our investigation of the utility and limitations of RASA using simulated and bacteriophage T7 data sets indicates that the method has numerous advantages over existing measures of signal. A first advantage is computational efficiency. A second advantage is that RASA employs known methods of statistical inference, providing measurable sensitivity and power. The performance of RASA is examined under various conditions of branching evolution as the number of characters, character states per character, and mutations per branch length are varied. RASA appears to provide an unbiased and reliable measure of phylogenetic signal, and the general approach promises to be useful in the development of new techniques that should increase the rigor and reliability of phylogenetic estimates.      

Cloning, sequencing, and expression of the structural genes for the cytochrome and flavoprotein subunits of p-cresol methylhydroxylase from two strains of Pseudomonas putida.

The structural genes for the flavoprotein subunit and cytochrome c subunit of p-cresol (4-methylphenol) methylhydroxylase (PCMH) from Pseudomonas putida NCIMB 9869 (National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland) and P. putida NCIMB 9866 were cloned and sequenced. The genes from P.putida NCIMB 9869 were for the plasmid-encoded A form of PCMH, and the genes from P.putida NCIMB 9866 were also plasmid encoded. The nucleotide sequences of the two flavoprotein genes from P.putida NCIMB 9869 and P.putida NCIMB 9866 (pchF69A and pchF66, respectively) were the same except for 5 bases out of 1,584, and the translated amino acid sequences were identical. The nucleotide sequences of the genes for the cytochrome subunits of PCMH from the two bacteria (pchC69A and pchC66) varied by a single nucleotide in their 303-base sequences, and the translated amino acid sequences differed by a single residue at position 41 (Asp in PchC69A and Ala in PchC66). Both cytochromes had 21-residue signal sequences, as expected for periplasmic proteins, and these sequences were identical. On the other hand, no signal sequences were found for the flavoproteins.pchF69A and pchC69A were expressed, separately or together, in Escherichia coli JM109 and P.putida RA4007, with active PCMH produced in both bacteria. The E. coli-expressed flavocytochrome was purified. Our studies indicated that the E.coli-expressed subunits were identical to the subunits expressed in P.putida NCIMB 9869: molecular weights, isoelectric points, UV-visible spectra, and steady-state kinetic parameters were the same for the two sets of proteins. The subunits readily associated upon mixing two crude extracts of E.coli, one extract containing PchC69A and the other containing PchF69A. The courses of association of PchC69A and PchF69A were essentially identical for pure E. coli-expressed subunits and pure P. putida 9869-expressed subunits. E. coli-expressed PchC69A and PchF69A contained covalently bound heme and covalently bound flavin adenine dinucleotide, respectively, as the proteins expressed in nature.     

Characterization of compost biofiltration system degrading dichloromethane

The effects of acclimatization of microbial populations, compound concentration, and media pH on the biodegradation of low concentration dichloromethane emissions in biofiltration systems was evaluated. Greater than 98% removal efficiency was achieved for dichloromethane at superficial velocities from 1 to 1.5 m(3)/m(3). min (reactor residence times of 1 and 0.7 min, respectively) and inlet concentrations of 3 and 50 ppm Although acclimatization of microbial populations to toluene occurred within 2 weeks of operation start-up, initial dichloromethane acclimatization took place over a period of 10 weeks. This period was shortened to 10 days when a laboratory grown consortium of dichloromethane degrading organism, isolated from a previously acclimatized column, was introduced into fresh biofilter media. The mixed culture consisted to 12 members, which together were able to degrade dichloromethane at concentrations up to 500 mg/L. Only one member of the consortium was able to degrade dichloromethane were sustained for more than 4 months in a biofilter column receiving an inlet gas stream with 3 ppm(v) of dichloromethane acidification of the column and resulting decline in performance occurred when a 50-ppm(v) inlet concentration was used. A biofilm model incorporating first order biodegradation kinetics provided a good fit to observed concentration profiles, and may prove to be a useful tool for designing biofiltration systems for low concentration VOC emissions. (c) 1994 John Wiley & Sons, Inc.     

Variation in heat-shock proteins among species of desert fishes (Poeciliidae, Poeciliopsis)

Analysis of the heat-shock proteins (hsps) of six closely related species of Poeciliopsis demonstrated the existence of biochemical diversity in the hsp100, hsp70, hsp60, and hsp30 protein families among species. Each species expressed five to seven hsp70-related isoforms. Constitutive 70-kD isoforms were identical among species, but four different patterns of heat-inducible isoforms were seen in these six species. Members of the hsp70 family of molecular chaperones are included among the most highly conserved proteins known, and the possibility of variation in hsp70 among closely related species has rarely been addressed. The hsp30 family is known to be less conserved than the hsp70 family, and, as expected, the Poeciliopsis hsp30 patterns showed more variation. Most of the hsp30 isoforms characteristic of a particular species were unique to that species. Hsp100 and hsp60 were identical in five of the species, but alternate isoforms were found in P. monacha. The small size and limited geographical distribution of the P. monacha population have probably contributed to the uniqueness of the monacha pattern. Two of the species were shown to acquire thermotolerance, the ability to withstand normally lethal temperatures when subjected to a gradual temperature increase. Rapid-heating protocols commonly used to establish critical thermal maxima of organisms do not include this inducible component of thermoresistance and therefore do not adequately assess an organism's capacity to withstand thermal stress.      

Phosphorylation and internalization of gp130 occur after IL-6 activation of Jak2 kinase in hepatocytes.

Recent evidence has shown that members of the Jak kinase family are activated after IL-6 binds to its receptor complex, leading to a tyrosine phosphorylation of gp130, the IL-6 signal-transducing subunit. The different members of the IL-6 cytokine subfamily induce distinct patterns of Jak-Tyk phosphorylation in different cell types. Using monospecific antibodies to gp130, Jak2 kinase, and phosphotyrosine, we investigated the kinetics of IL-6 stimulation of members of this pathway in primary hepatocytes. Our findings show that Jak 2 is maximally activated within 2 min of exposure to IL-6, followed by gp130 phosphorylation that reaches its peak in another 2 min then declines to basal level by 60 min. In vitro phosphorylation experiments show that activated Jak 2 is able to phosphorylate both native gp130 and a fusion peptide containing its cytoplasmic domain, demonstrating gp130 is a direct substrate of Jak 2 kinase. Experiments designed to explore the cell surface expression of gp130 show that > or = 2 h are required to get a second round of phosphorylation after the addition of more cytokines. This finding suggests that activated gp130 is internalized from the cell surface after IL-6 stimulation. Additional experiments using protein synthesis inhibitors reveal that new protein synthesis is required to get a second cycle of gp130 phosphorylation indicating gp130 must be synthesized de novo and inserted into the membrane. These findings provide strong evidence that down regulation of the IL-6 signal in hepatocytes involves the internalization and cytosol degradation of gp130.     

The effects of gravity on the circadian timing system

The physiological system responsible for the temporal coordination of an organism is the circadian timing system (CTS). This system provides two forms of temporal coordination. First, the CTS provides for synchronization of the organism with the 24 hour period of the external environment. This synchronization of the organism with the environment is termed entrainment. Second, this system also provides for internal coordination of the various physiological, behavioral, and biochemical events within the organism. When either of these two temporal relationships are disturbed, various dysfunctions can be manifest within the organism. Homeostatic capacity of other physiological systems may be reduced. Performance is decreased and sleep disorders, mental health impairment (e.g., depression), jet lag syndrome, and shift work maladaptation frequently occur. Over the last several years, several studies have evaluated the potential influence of gravity on this physiological control system by examining changes in rhythmic characteristics of organisms exposed to altered gravitational environments. The altered gravitational environments have included the microgravity of spaceflight as well as hyperdynamic fields produced via centrifugation.     

Herpes simplex virus type 1 and pseudorabies virus bind to a common saturable receptor on Vero cells that is not heparan sulfate.

Herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) infect different natural hosts but are very similar in structure, replicative cycle, and entry into cultured cells. We determined whether HSV-1 and PRV use the same cellular components during entry into Vero cells, which are highly susceptible to each virus but are not from native hosts for either. UV-inactivated virions of either HSV-1 or PRV could saturate cell surfaces to block infection of challenge HSV-1 or PRV. In the presence of saturating levels for infection of either virus, radiolabeled virus bound well and in a heparin-sensitive manner. This result shows that heparan sulfate proteoglycans on Vero cells are not the limiting cellular component. To identify the virus component required for blocking, we used an HSV-1 null mutant virus lacking gB, gD, or gH as blocking virus. Virions lacking gB were able to block infection of challenge virus to the same level as did virus containing gB. In contrast, virions lacking gD lost all and most of the ability to block infection of HSV-1 and PRV, respectively. HSV-1 lacking gH and PRV lacking gp50 also were less competent in blocking infection of challenge virus. We conclude that HSV-1 and PRV bind to a common receptor for infection of Vero cells. Although both viruses bind a heparin-like cell component on many cells, including Vero cells, they also attach to a different and limited cell surface component that is bound at least by HSV-1 gD and possibly gH and to some degree by PRV gp50 but not gB. These results clearly demonstrate binding of both HSV-1 and PRV to a common cell receptor that is not heparan sulfate and demonstrate that several types of attachment occur for both viruses during infectious entry.