In vitro culture of Aloe Barbadensis Mill.: Micropropagation from vegetative meristems

A method for rapid and highly effective plant micropropagation from vegetative meristems was established for Aloe barbadensis Mill. Plant micropropagation was achieved culturing apices on medium containing 1.1 M 2,4-dichlorophenoxyacetic acid and 2.3 M kinetin for 15–30 days. High morphogenetic ability was maintained by transferring explants (after 60 days) on media containing 0.11 M 2,4-dichlorophenoxyacetic acid and 2.2 M 6-benzylaminopurine.     

Cell cycle arrest by human cytomegalovirus 86-kDa IE2 protein resembles premature senescence

Primary human embryo lung fibroblasts and adult diploid fibroblasts infected by the human cytomegalovirus (HCMV) display beta-galactosidase (beta-Gal) activity at neutral pH (senescence-associated beta-Gal [SA-beta-Gal] activity) and overexpression of the plasminogen activator inhibitor type 1 (PAI-1) gene, two widely recognized markers of the process designated premature cell senescence. This activity is higher when cells are serum starved for 48 h before infection, a process that speeds and facilitates HCMV infection but that is insufficient by itself to induce senescence. Fibroblasts infected by HCMV do not incorporate bromodeoxyuridine, a prerequisite for the formal definition of senescence. At the molecular level, cells infected by HCMV, beside the accumulation of large amounts of the cell cycle regulators p53 and pRb, the latter in its hyperphosphorylated form, display a strong induction of the cyclin-dependent kinase inhibitor (cdki) p16(INK4a), a direct effector of the senescence phenotype in fibroblasts, and a decrease of the cdki p21(CIP1/WAF). Finally, a replicative senescence state in the early phases of infection significantly increased the number of cells permissive to virus infection and enhanced HCMV replication. HCMV infection assays carried out in the presence of phosphonoformic acid, which inhibits the virus DNA polymerase and the expression of downstream genes, indicated that immediate-early and/or early (alpha) genes are sufficient for the induction of SA-beta-Gal activity. When baculovirus vectors expressing HCMV IE1-72 or IE2-86 proteins were inoculated into fibroblasts, the increase of p16(INK4a) (observed predominantly with IE2-86) was similar to that observed with the whole virus, as was the induction of SA-beta-Gal activity, suggesting that the viral IE2 gene leads infected cells into senescence. Altogether our results demonstrate for the first time that HCMV, after arresting the cell cycle and inhibiting apoptosis, triggers the cellular senescence program, probably through the p16(INK4a) and p53 pathways.     

The Gut of the Soil Microarthropod Folsomia candida (Collembola) Is a Frequently Changeable but Selective Habitat and a Vector for Microorganisms

Interaction potentials between soil microarthropods and microorganisms were investigated with Folsomia candida (Insecta, Collembola) in microcosm laboratory experiments. Microscopic analysis revealed that the volumes of the simple, rod-shaped guts of adult specimens varied with their feeding activity, from 0.7 to 11.2 nl. A dense layer of bacterial cells, associated with the peritrophic membrane, was detected in the midgut by scanning electron microscopy. Depending on the molting stage, which occurred at intervals of approximately 4 days, numbers of heterotrophic, aerobic gut bacteria changed from 4.9 × 10² to 2.3 × 10⁶ CFU per specimen. A total of 11 different taxonomic bacterial groups and the filamentous fungus Acremonium charticola were isolated from the guts of five F. candida specimens. The most abundant isolate was related to Erwinia amylovora (96.2% DNA sequence similarity to its 16S rRNA gene). F. candida preferred to feed on Pseudomonas putida and three indigenous gut isolates rather than eight different type culture strains. When luciferase reporter gene-tagged bacterial strains were pulse fed to F. candida, gut isolates were continuously shed for 8 days to several weeks but Escherichia coli HB101 was shed for only 1 day. Ratios of ingested to released bacterial cells demonstrated that populations of nonindigenous gut bacteria like Sinorhizobium meliloti L33 and E. coli HB101 were reduced by more than 4 orders of magnitude but that the population of gut isolate Alcaligenes faecalis HR4 was reduced only 500-fold. This work demonstrates that F. candida represents a frequently changeable but selective habitat for bacteria in terrestrial environments and that microarthropods have to be considered factors that modify soil microbial communities.     

Bayesian-based selection of metabolic objective functions

MOTIVATION: A critical component of in silico analysis of underdetermined metabolic systems is the identification of the appropriate objective function. A common assumption is that the objective of the cell is to maximize growth. This objective function has been shown to be consistent in a few limited experimental cases, but may not be universally appropriate. Here a method is presented to quantitatively determine the most probable objective function. RESULTS: The genome-scale metabolism of Escherichia coli growing on succinate was used as a case-study for analysis. Five different objective functions, including maximization of growth rate, were chosen based on biological plausibility. A combination of flux balance analysis and linear programming was used to simulate cellular metabolism, which was then compared to independent experimental data using a Bayesian objective function discrimination technique. After comparing rates of oxygen uptake and acetate production, minimization of the production rate of redox potential was determined to be the most probable objective function. Given the appropriate reaction network and experimental data, the discrimination technique can be applied to any bacterium to test a variety of different possible objective functions. SUPPLEMENTARY INFORMATION: Additional files, code and a program for carrying out model discrimination are available at http://www.engr.uconn.edu/~srivasta/modisc.html.     

Water use by perennial plants in the transition zone between river oasis and desert in NW China

The study aimed at establishing the role of two possible water sources (inundation, ground water) for the water supply to the perennial plant species Alhagi sparsifolia, Calligonum caput-medusae, Populus euphratica and Tamarix ramosissima growing in the transition zone between a river oasis and the open desert at the southern fringe of the Taklamakan desert (Xinjiang province, NW China). The basic hypothesis was that inundations, which normally occur in summer when rivers from a nearby mountain range carry high water, contribute significantly to the plants’ water supply. When, in the first summer, inundations did not occur, four sites, each of which covered by a relatively dense stand of one species, were artificially flooded. Soil and plant water relations as well as meteorological variables were measured during two growing seasons. Water use efficiency of production (WUE_P) was calculated by relating biomass production, which was determined using allometric regressions, to water use.The effects of artificial flooding on the plant water relations were negligible. Water use was relatively high, especially in the A. sparsifolia and the P. euphratica stands and in a dense stand of T. ramosissima (up to approx. 500 kgH₂O m⁻² year⁻¹). Using the total above-ground biomass in the calculation, WUE_P was highest in C. caput-medusae and P. euphratica, and lowest, in A. sparsifolia. From soil and plant water relations, and against the background of the climate and the productivity of the vegetation, it is concluded that all perennial plants in the transition zone between oases and desert in that region must have sufficient access to ground water to ensure long-term survival. Management of ground water such that it remains continuously accessible to the perennial plants is a prerequisite for the conservation and sustainable use of the vegetation in the transition zone.     

A threonine synthase homolog from a mammalian genome

The genomes of several vertebrates contain two genes encoding proteins highly similar to threonine synthase (TS), even though the biosynthesis of l-threonine (l-Thr) is not known to occur in these animals. We report a bioinformatic analysis of the two TS-like genes, the recombinant expression of one murine TS homolog (mTSH2) and its initial biochemical characterization. Recombinant mTSH2 contained bound pyridoxal-5'-phosphate (PLP), but did not synthesize l-Thr. The enzyme did, however, bind O-phospho-homoserine (PHS; the actual TS substrate) and degraded it to alpha-ketobutyrate, phosphate, and ammonia-a known side reaction of microbial TSs. mTSH2 also degraded O-phospho-threonine (PThr) to alpha-ketobutyrate, showing that it can act as a catabolic phospho-lyase on both gamma- and beta-phosphorylated substrates. These findings suggest an unusual evolutionary origin for mTSH2, whereby an original TS enzyme became 'recycled' into a phospho-lyase upon dismissal, in metazoa, of the l-Thr biosynthetic pathway.     

Role of the guanidine group in the N-terminal fragment of PTH(1–11)

A series of PTH hybrids containing a diamine [NH₂(CH₂) _n NH₂; n = 4, 5, 6] in the C-terminal position was synthesized based on the H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH₂ (Har = homoarginine) template. The compounds were pharmacologically characterized at PTH1R receptors for agonist activity.