The role of nurse plants in the establishment of shrub seedlings in the semi-arid subtropical Andes

In the nurse plant syndrome, or nurse association, seedlings (beneficiaries) are associated with adult shrubs/trees (benefactors). This phenomenon has been documented in several regions of the planet. Abiotic stress amelioration (one mechanism of facilitation) is one of the causes of this association. Most of the studies addressing the nurse syndrome have been conducted on spatial scales of a few hectares and have focused on only one or a few species. Moreover, there is an almost complete lack of studies addressing the incidence and characteristics of the nurse phenomenon in the arid Andes of South America. We undertook a first approximation to the study of facilitation in these ecosystems. The study was conducted at local and regional scales and involved the assessment of the spatial distribution of juveniles (seedlings and saplings) of 51 populations of 16 shrub and 12 cactus species in relation to shrub cover at 20 localities of the Prepuna (subtropical Andes of Bolivia and Argentina, 20–26°S). In terms of spatial distribution, the juveniles of most of the populations of shrubs studied were distributed both under the shrubs and in open spaces, thereby showing an apparent indifference to microhabitat. Globose and opuntioid cacti were preferentially distributed below the canopies of shrubs and were usually more associated with the dominant shrub species, which stood out as better potential nurses. The pattern was consistent throughout the region, including the more mesic and arid localities. The fact that Prepuna woody species are capable of establishing in open spaces would confer this region a greater resilience. Our findings further suggest that community dynamics in arid and semi-arid environments are more variable than previously thought.     

Alternative Nonallelic Deletion Is Constitutive of Ruminant αs1-Casein

Multiple forms of α_s1-casein were identified in the four major ruminant species by structural characterization of the protein fraction. While α_s1-casein phenotypes were constituted by a mixture of at least seven molecular forms in ovine and caprine species, there were only two forms in bovine and water buffalo species. In ovine and caprine forms the main component corresponded to the 199-residue-long form, and the deleted proteins differed from the complete one by the absence of peptides 141–148, 110–117, or Gln78, or a combination of such deletions. The deleted segments corresponded to the sequence regions encoded by exons 13 and 16, and by the first triplet of exon 11 (CAG), suggesting that the occurrence of the short protein forms is due to alternative skipping, as previously demonstrated for some caprine and ovine phenotypes. The alternative deletion of Gln78 in α_s1-casein, the only form common to the milk of all the species examined and located in a sequence region joining the polar phosphorylation cluster and the hydrophobic C-terminal domain of the protein, may play a functional role in the stabilization of the milk micelle structure.     

Copper Ions Stimulate Polyphosphate Degradation and Phosphate Efflux in Acidithiobacillus ferrooxidans

For some bacteria and algae, it has been proposed that inorganic polyphosphates and transport of metal-phosphate complexes could participate in heavy metal tolerance. To test for this possibility in Acidithiobacillus ferrooxidans, a microorganism with a high level of resistance to heavy metals, the polyphosphate levels were determined when the bacterium was grown in or shifted to the presence of a high copper concentration (100 mM). Under these conditions, cells showed a rapid decrease in polyphosphate levels with a concomitant increase in exopolyphosphatase activity and a stimulation of phosphate efflux. Copper in the range of 1 to 2 μM greatly stimulated exopolyphosphatase activity in cell extracts from A. ferrooxidans. The same was seen to a lesser extent with cadmium and zinc. Bioinformatic analysis of the available A. ferrooxidans ATCC 23270 genomic sequence did not show a putative pit gene for phosphate efflux but rather an open reading frame similar in primary and secondary structure to that of the Saccharomyces cerevisiae phosphate transporter that is functional at acidic pH (Pho84). Our results support a model for metal detoxification in which heavy metals stimulate polyphosphate hydrolysis and the metal-phosphate complexes formed are transported out of the cell as part of a possibly functional heavy metal tolerance mechanism in A. ferrooxidans.     

Do anti-TNF agents have equal efficacy in patients with rheumatoid arthritis?

Tumor necrosis factor (TNF) antagonists have dramatically improved the outcomes of rheumatoid arthritis (RA). Three agents currently available in the USA--infliximab, etanercept, and adalimumab--have been designed to modify the biologic effects of TNF. Infliximab and adalimumab are monoclonal antibodies, and etanercept is a soluble protein. The pharmacokinetic and pharmacodynamic properties of each differs significantly from those of the others. All three agents are effective and safe, and can improve the quality of life in patients with RA. Although no direct comparisons are available, clinical trials provide evidence that can be used to evaluate the comparative efficacy of these agents. Infliximab, in combination with methotrexate, has been shown to relieve the signs and symptoms of RA, decrease total joint score progression, prevent joint erosions and joint-space narrowing, and improve physical function for up to 2 years. Etanercept has been shown to relieve the signs and symptoms of RA, decrease total joint score progression, and slow the rate of joint destruction, and might improve physical function. Etanercept is approved with and without methotrexate for patients who have demonstrated an incomplete response to therapy with methotrexate and other disease-modifying anti-rheumatic drugs (DMARDs), as well as for first-line therapy in early RA, psoriatic arthritis, and juvenile RA. Adalimumab relieves the signs and symptoms of RA with and without methotrexate and other DMARDs, decreases total joint score progression, prevents joint erosions and joint-space narrowing in combination with methotrexate, and might improve physical function. When selecting a TNF antagonist, rheumatologists should weigh evidence and experience with specific agents before a decision is made for use in therapy.     

Calcium‐induced calcium release contributes to somatic secretion of serotonin in leech Retzius neurons

We analyzed the contribution of calcium (Ca²⁺)‐induced Ca²⁺ release to somatic secretion in serotonergic Retzius neurons of the leech. Somatic secretion was studied by the incorporation of fluorescent dye FM1‐43 upon electrical stimulation with trains of 10 impulses and by electron microscopy. Quantification of secretion with FM1‐43 was made in cultured neurons to improve optical resolution. Stimulation in the presence of FM1‐43 produced a frequency‐dependent number of fluorescent spots. While a 1‐Hz train produced 19.5 ± 5.0 spots/soma, a 10‐Hz train produced 146.7 ± 20.2 spots/soma. Incubation with caffeine (10 mM) to induce Ca²⁺ release from intracellular stores without electrical stimulation and external Ca²⁺, produced 168 ± 21.7 spots/soma. This staining was reduced by 49% if neurons were preincubated with the Ca²⁺‐ ATPase inhibitor thapsigargin (200 nM). Moreover, in neurons stimulated at 10 Hz in the presence of ryanodine (100 μM) to block Ca²⁺‐induced Ca²⁺ release, FM1‐43 staining was reduced by 42%. In electron micrographs of neurons at rest or stimulated at 1 Hz in the ganglion, endoplasmic reticulum lay between clusters of dense core vesicles and the plasma membrane. In contrast, in neurons stimulated at 20 Hz, the vesicle clusters were apposed to the plasma membrane and flanked by the endoplasmic reticulum. These results suggest that Ca²⁺‐induced Ca²⁺ release produces vesicle mobilization and fusion in the soma of Retzius neurons, and supports the idea that neuronal somatic secretion shares common mechanisms with secretion by excitable endocrine cells. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2004     

Extensive Phylogenetic Analysis of a Soil Bacterial Community Illustrates Extreme Taxon Evenness and the Effects of Amplicon Length,Degree of Coverage,and DNA Fractionation on Classification and Ecological Parameters

To thoroughly investigate the bacterial community diversity present in a single composite sample from an agricultural soil and to examine potential biases resulting from data acquisition and analytical approaches, we examined the effects of percent G+C DNA fractionation, sequence length, and degree of coverage of bacterial diversity on several commonly used ecological parameters (species estimation, diversity indices, and evenness). We also examined variation in phylogenetic placement based on multiple commonly used approaches (ARB alignments and multiple RDP tools). The results demonstrate that this soil bacterial community is highly diverse, with 1,714 operational taxonomic units demonstrated and 3,555 estimated (based on the Chao1 richness estimation) at 97% sequence similarity using the 16S rRNA gene. The results also demonstrate a fundamental lack of dominance (i.e., a high degree of evenness), with 82% of phylotypes being encountered three times or less. The data also indicate that generally accepted cutoff values for phylum-level taxonomic classification might not be as applicable or as general as previously assumed and that such values likely vary between prokaryotic phyla or groups.Efforts to describe bacterial species richness and diversity have long been hampered by the inability to cultivate the vast majority of bacteria from natural environments. New methods to study bacterial diversity have been developed in the last two decades (32), many of which rely on PCR-based procedures and phylogenetic comparison of 16S rRNA gene sequences. However, PCR using complex mixtures of templates (as in the case of total microbial community DNA) is presumed to preferentially amplify certain templates in the mixture (23) based on their primary sequence, percent G+C (hereafter GC) content, or other factors, resulting in so-called PCR bias. Moreover, the amplification of template sequences depends on their initial concentration and tends to skew detection toward the most abundant members of the community (23). To further complicate matters, subsequent random cloning steps on amplicon mixtures are destined to result in the detection of numerically dominant sequences, especially where relative abundance can vary over orders of magnitude. Indeed, any analysis based on random encounter is destined to primarily detect numerically dominant populations. This is especially of concern where limited sampling is performed on highly complex microbial communities exhibiting mostly even distribution of populations with only a few showing any degree of dominance, as typically perceived for soils (17). These artifacts and sampling limitations represent major hurdles in bacterial community diversity analysis, since the vast majority of bacterial diversity probably lies in “underrepresented minority” populations (24, 30). This is important because taxa that are present only in low abundance may still perform important ecosystem functions (e.g., ammonia-oxidizing bacteria). Of special concern is that biases in detection might invalidate hypothesis testing on complex communities where limited sampling is performed (5).Recently, there has been a concerted effort toward addressing problems impeding comprehensive bacterial diversity studies (7, 13, 24, 26, 28). In recent years, studies have increased sequencing efforts, with targeted 16S rRNA gene sequence libraries approaching 2,000 clones (11) and high-throughput DNA-sequencing efforts (e.g., via 454 pyrosequencing and newer-generation high-throughput approaches) of up to 149,000 templates from one or a few samples (25, 30). These technological advances have come as researchers recognize that massive sequencing efforts are required to accurately assess the diversity of populations that comprise complex microbial communities (29, 30). Alternatively, where fully aligned sequence comparisons need to be made, novel experimental strategies that allow more-comprehensive detection of underrepresented bacterial taxa can be applied. One such approach involves the application of prefractionation of total bacterial community genomic DNA based on its GC content (hereafter GC fractionation) prior to subsequent molecular manipulations of total community DNA (14). This strategy has been successfully applied in combination with denaturing gradient gel electrophoresis (13) and 16S rRNA gene cloning (2, 21) to study microbial communities. This approach separates community genomic DNA, prior to any PCR, into fractions of similar percent GC content, effectively reducing the overall complexity of the total community DNA mixture by physical separation into multiple fractions. This facilitates PCR amplification, cloning, and detection of sequences in fractions with relatively low abundance in the community, thereby enhancing the detection of minority populations (13). Collectively, this strategy reduces the biases introduced by PCR amplification and random cloning of the extremely complex mixtures of templates of different GC content, primary sequence, and relative abundance present in total environmental genomic DNA.Any large molecular survey that relies on sequencing further requires the analysis of large amounts of data that must be catalogued into phylogenetically relevant groups. This is usually done using high-throughput methods like RDP Classifier or Sequence Match (6) or a tree-based method like Greengenes (8) or ARB (18). Two major pitfalls that are encountered using these former approaches are the presence of huge numbers of unclassified sequences in databases and the lack of representative sequences from all phyla. This leads to most surveys having large portions of their phylotypes designated as unclassified. The latter tree-based approaches, although better suited for classification schemes, are also dependent on having a comprehensive database with well-classified sequences for reproducible results. This reproducibility becomes especially important when trying to compare data across different studies, especially those that utilize different approaches and study systems.In the current study, we analyzed an extensive (∼5,000 clones) partial 16S rRNA gene library from a single soil sample that was generated using very general primers and GC-fractionated DNA. Total DNA was extracted from soil at a cultivated treatment plot at the National Science Foundation Long Term Ecological Research (NSF-LTER) site at the Kellogg Biological Station (KBS) in mid-Michigan (http://www.kbs.msu.edu/lter). To test the effect of GC fractionation on recovery of 16S rRNA gene sequences, we conducted a direct comparison with a nonfractionated library generated from the same soil sample. Using the GC-fractionated library, we also calculated several measures of bacterial diversity and examined the effects of sampling size and sequence length on Shannon-Weaver diversity index, Simpson''s reciprocal index (1/D, where D is the probability that two randomly selected individuals from a sample belong to the same species), evenness, and Chao1 richness estimation. The results show that GC fractionation is a powerful tool to help mitigate limitations of random PCR- and cloning-based analyses of total microbial community diversity, resulting in the recovery of underrepresented taxa and, in turn, reducing the sampling size needed for accurate estimations of bacterial richness. The results also provided evidence for the need to expand the typical scale of sequence-based survey efforts, particularly in environments where evenness abounds or where minority bacterial populations may have important effects on community function and processes. We suggest that there is a need for the establishment of standardized approaches for the analysis of sequence data from community diversity studies in order to maximize data comparisons across independent studies and show examples of software programs developed to facilitate comparative analysis of large sequence datasets.     

Circadian-related heteromerization of adrenergic and dopamine D₄ receptors modulates melatonin synthesis and release in the pineal gland

The role of the pineal gland is to translate the rhythmic cycles of night and day encoded by the retina into hormonal signals that are transmitted to the rest of the neuronal system in the form of serotonin and melatonin synthesis and release. Here we describe that the production of both melatonin and serotonin by the pineal gland is regulated by a circadian-related heteromerization of adrenergic and dopamine D₄ receptors. Through α_{1 B}-D₄ and β₁-D₄ receptor heteromers dopamine inhibits adrenergic receptor signaling and blocks the synthesis of melatonin induced by adrenergic receptor ligands. This inhibition was not observed at hours of the day when D₄ was not expressed. These data provide a new perspective on dopamine function and constitute the first example of a circadian-controlled receptor heteromer. The unanticipated heteromerization between adrenergic and dopamine D₄ receptors provides a feedback mechanism for the neuronal hormone system in the form of dopamine to control circadian inputs.     

Rapid immunofluorescent determination of cells in the S phase in pea root meristems: An alternative to autoradiography

Photosystem II (PS II) activity and the localization of ribulose-l,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39) were studied in primary leaves of young maize plants ( Zea mays L. cv. Fronica) by tetra-nitro-blue-tetrazoliumchloride reduction and immunolocalization, respectively. In tissue of 3-day-old plants all chloroplasts were structurally identical. From day 4 they developed into their typical appearance of mesophyll and bundle sheath chloroplasts. First PS II-activity was present in both types of chloroplasts. From day 4 it disappeared in bundle sheath chloroplasts concomitant with the loss of grana. RuBP carboxylase on the other hand was only present in bundle sheath chloroplasts at all stages of development. Thus, the control of the development of the photosystems and the Calvin cycle enzymes seem to differ.     

Cliffs Used as Communal Roosts by Andean Condors Protect the Birds from Weather and Predators

The quality and availability of resources influence the geographical distribution of species. Social species need safe places to rest, meet, exchange information and obtain thermoregulatory benefits, but those places may also serve other important functions that have been overlooked in research. We use a large soaring bird that roosts communally in cliffs, the Andean condor (Vultur gryphus), as a model species to elucidate whether roost locations serve as a refuge from adverse weather conditions (climatic refuge hypothesis, CRH), and/or from predators or anthropogenic disturbances (threats refuge hypothesis, TRH). The CRH predicts that communal roosts will face in the opposite direction from where storms originate, and will be located in climatically stable, low precipitation areas. The TRH predicts that communal roosts will be large, poorly accessible cliffs, located far from human-made constructions. We surveyed cliffs used as communal roosts by condors in northwestern Patagonia, and compared them with alternative non-roosting cliffs to test these predictions at local and regional scales. We conclude that communal roosting places provide refuge against climate and disturbances such as, for instance, the threats of predators (including humans). Thus, it is not only the benefits gained from being aggregated per se, but the characteristics of the place selected for roosting that may both be essential for the survival of the species. This should be considered in management and conservation plans given the current scenario of global climate change and the increase in environmental disturbances.     

A new enzymatic route to the synthesis of 12-ketoursodeoxycholic acid

Summary Cholic acid (3,7,12-trihydroxy-5-cholanoic acid) was completely and selectively transformed into 12-ketoursodeoxycholic acid (3,7-dihydroxy-12-oxo-5-cholanoic acid) by means of two consecutive enzymatic steps catalyzed, the first, by 7- and 12-hydroxysteroid dehydrogenase and, the second, by 7-hydroxysteroid dehydrogenase. Coenzyme regeneration was carried out with -ketoglutarate-glutamate dehydrogenase and glucose-glucose dehydrogenase, respectively.