Legacies of precipitation fluctuations on primary production: theory and data synthesis

Variability of above-ground net primary production (ANPP) of arid to sub-humid ecosystems displays a closer association with precipitation when considered across space (based on multiyear averages for different locations) than through time (based on year-to-year change at single locations). Here, we propose a theory of controls of ANPP based on four hypotheses about legacies of wet and dry years that explains space versus time differences in ANPP–precipitation relationships. We tested the hypotheses using 16 long-term series of ANPP. We found that legacies revealed by the association of current- versus previous-year conditions through the temporal series occur across all ecosystem types from deserts to mesic grasslands. Therefore, previous-year precipitation and ANPP control a significant fraction of current-year production. We developed unified models for the controls of ANPP through space and time. The relative importance of current-versus previous-year precipitation changes along a gradient of mean annual precipitation with the importance of current-year PPT decreasing, whereas the importance of previous-year PPT remains constant as mean annual precipitation increases. Finally, our results suggest that ANPP will respond to climate-change-driven alterations in water availability and, more importantly, that the magnitude of the response will increase with time.     

Soil animal responses to moisture availability are largely scale,not ecosystem dependent: insight from a cross‐site study

Climate change will result in reduced soil water availability in much of the world either due to changes in precipitation or increased temperature and evapotranspiration. How communities of mites and nematodes may respond to changes in moisture availability is not well known, yet these organisms play important roles in decomposition and nutrient cycling processes. We determined how communities of these organisms respond to changes in moisture availability and whether common patterns occur along fine‐scale gradients of soil moisture within four individual ecosystem types (mesic, xeric and arid grasslands and a polar desert) located in the western United States and Antarctica, as well as across a cross‐ecosystem moisture gradient (CEMG) of all four ecosystems considered together. An elevation transect of three sampling plots was monitored within each ecosystem and soil samples were collected from these plots and from existing experimental precipitation manipulations within each ecosystem once in fall of 2009 and three times each in 2010 and 2011. Mites and nematodes were sorted to trophic groups and analyzed to determine community responses to changes in soil moisture availability. We found that while both mites and nematodes increased with available soil moisture across the CEMG, within individual ecosystems, increases in soil moisture resulted in decreases to nematode communities at all but the arid grassland ecosystem; mites showed no responses at any ecosystem. In addition, we found changes in proportional abundances of mite and nematode trophic groups as soil moisture increased within individual ecosystems, which may result in shifts within soil food webs with important consequences for ecosystem functioning. We suggest that communities of soil animals at local scales may respond predictably to changes in moisture availability regardless of ecosystem type but that additional factors, such as climate variability, vegetation composition, and soil properties may influence this relationship over larger scales.     

Binding hot spots in the TEM1-BLIP interface in light of its modular architecture

Proteins bind one another in aqua's solution to form tight and specific complexes. Previously we have shown that this is achieved through the modular architecture of the interaction network formed by the interface residues, where tight cooperative interactions are found within modules but not between them. Here we extend this study to cover the entire interface of TEM1 beta-lactamase and its protein inhibitor BLIP using an improved method for deriving interaction maps based on REDUCE to add hydrogen atoms and then by evaluating the interactions using modifications of the programs PROBE, NCI and PARE. An extensive mutagenesis study of the interface residues indeed showed that each module is energetically independent on other modules, and that cooperativity is found only within a module. By solving the X-ray structure of two interface mutations affecting two different modules, we demonstrated that protein-protein binding occur via the structural reorganization of the binding modules, either by a "lock and key" or an induced fit mechanism. To explain the cooperativity within a module, we performed multiple-mutant cycle analysis of cluster 2 resulting in a high-resolution energy map of this module. Mutant studies are usually done in reference to alanine, which can be regarded as a deletion of a side-chain. However, from a biological perspective, there is a major interest to understand non-Ala substitutions, as they are most common. Using X-ray crystallography and multiple-mutant cycle analysis we demonstrated the added complexity in understanding non-Ala mutations. Here, a double mutation replacing the wild-type Glu,Tyr to Tyr,Asn on TEM1 (res id 104,105) caused a major backbone structural rearrangement of BLIP, changing the composition of two modules but not of other modules within the interface. This shows the robustness of the modular approach, yet demonstrates the complexity of in silico protein design.     

Processing of artificial peptide-DNA-conjugates by the mitochondrial intermediate peptidase (MIP).

Import of DNA from the cytoplasm into the mitochondrial matrix is an obligatory step for an in organello site-directed mutagenesis or gene therapy approach on mitochondrial DNA diseases. In this context, we have developed an artificial DNA translocation vector that is composed of the mitochondrial signal peptide of the ornithine transcarbamylase (OTC) and a DNA moiety. While this vector is capable of directing attached passenger molecules to the mitochondrial matrix, the recognition of this artificial molecule by the endogenous mitochondrial signal peptide processing machinery as well as the cleavage of the peptide plays a pivotal role in the release of the attached DNA. To study the proteolytic processing of the artificial vector, various signal peptide-DNA-conjugates were treated with purified mitochondrial intermediate peptidase. When the leader peptide is directly linked to the DNA moiety without an intervening spacer, MIP processing is prevented. Cleavage of the peptide can be restored, however, when the first ten amino acid residues of the mature part of OTC are appended at the carboxy-terminal end of the signal peptide. Our results show that artificial peptide-DNA-conjugates are recognized by the mitochondrial proteolytic machinery, and therefore an interference of the peptide with the DNA function can be excluded.     

Characterization of vesicular stomatitis virus mutants by partial proteolysis

Structural proteins of temperature-sensitive (ts) mutants of vesicular stomatitis virus, Indiana serotype, were compared with those of wild-type and revertant virions by electrophoresis on polyacrylamide gels of partial digests with Staphylococcus aureus V8 protease. Mutants of complementation groups III (tsG31 and tsG33), II (tsG22), and IV (tsG41) differed from the wild-type virion in peptide profiles of their M, NS, and N proteins, respectively. The differences were only detectable over a narrow range of enzyme-substrate ratios and were due to peptides transiently generated during incomplete digestion. Proteins of revertants to tsG31, tsG22, and tsG41 exhibited the wild-type virion peptide pattern, indicating that reversion had restored their original conformation. However, in the case of tsG22, the NS peptide profile reverted to the wild-type phenotype only partially, suggesting that a silent mutation might have taken place during either the original chemical mutagenesis or the following repeated laboratory passages. The apparent alteration in protein conformation and its restoration upon reversion of the mutants indicated that the lesions of groups III and IV were located in the M and N proteins, respectively. Moreover, for the first time, the site of mutation of group II could be positively identified as the NS protein cistron.     

Enzyme activity analyses along ragged-red and normal single muscle fibres

Summary Mitochondrial myopathies are morphologically characterized by ragged-red fibres (RRF). Serial cross-section revealed that the ragged-red appearance was only focal. This is in agreement with a partial cytochromec oxidase (COX) deficiency in chronic progressive external ophthalmoplegia (CPEO). Since most of these patients show deletions of the mitochondrial genome single fibre analyses were performed determining COX and succinate dehydrogenase (SDH) in serial muscle sections from two patients with CPEO. High SDH activity was demonstrated in RRF; in contrast COX activity was lower in RRF in a patient, possibly representing a focal assembly of mitochondria with deletions in their genomes. The variation of enzyme activities along the muscle fibre was especially high in RRF. This study presents the first quantitative evidence that enzyme activities vary considerably along fibres in muscle from patients with a mitochondrial myopathy.     

Chromosomal banding patterns in human large bowel adenomas

Summary The human thyroglobulin gene was mapped by in situ hybridization whereby a ³H-labeled recombinant plasmid DNA containing a fragment of 2.3 kilobases of human thyroglobulin gene was hybridized to human chromosome preparations. A high proportion (25%) of hybridized metaphases exhibited silver grains at the distal portion of the long arm of chromosome 8. Analysis of the grain position at this site indicated that the chromosomal localization of the human thyroglobulin gene was 8q242-8q243.     

Haemopoietic chimaerism: a complication in heterozygote detection tests for inherited defects in cattle

Several observations are documented which illustrate that haemopoietic chimaerism is a potential source of error when using assays of cellular components of blood to determine genotype for inherited defects in cattle. Acidic α-glucosidase activity in peripheral mononuclear cells of a twin Brahman bull that had sired calves affected with generalized glycogenosis was similar to that in cells from homozygous normal animals. Activity in fibroblasts from this bull was similar to that in heterozygotes. α-mannosidase activity in fibroblasts of a twin Murray Grey bull with low activity in peripheral granulocytes but high activity in plasma was similar to that in animals homozygous normal for α-mannosidosis. Normal argininosuccinate synthetase nucleotide sequence was detected in leucocytes from two calves affected with citrullinaemia and mutant sequence detected in leucocytes from their homozygous normal co-twins.     

Perfusion of the psoas muscle of the rabbit. Metabolism of a homogeneous muscle composed of "fast glycolytic" fibres

A method was developed for the hemoglobin-free perfusion of the rabbit psoas muscle in situ. This muscle consists almost exclusively of fast-twitch (type IIb) glycolytic fibres and was therefore used as a model of a homogeneous muscle of the glycolytic, metabolic type.     

Proteolytic cleavage inactivates the Staphylococcus aureus lipoteichoic acid synthase

Lipoteichoic acid (LTA) is a crucial cell envelope component in Gram-positive bacteria. In Staphylococcus aureus, the polyglycerolphosphate LTA molecule is synthesized by LtaS, a membrane-embedded enzyme with five N-terminal transmembrane helices (5TM domain) that are connected via a linker region to the C-terminal extracellular enzymatic domain (eLtaS). The LtaS enzyme is processed during bacterial growth, and the eLtaS domain is released from the bacterial membrane. Here we provide experimental evidence that the proteolytic cleavage following residues 215Ala-Leu-Ala217 is performed by the essential S. aureus signal peptidase SpsB, as depletion of spsB results in reduced LtaS processing. In addition, the introduction of a proline residue at the +1 position with respect to the cleavage site, a substitution known to inhibit signal peptidase-dependent cleavage, abolished LtaS processing at this site. It was further shown that the 5TM domain is crucial for enzyme function. The observation that the construction of hybrid proteins between two functional LtaS-type enzymes resulted in the production of proteins unable to synthesize LTA suggests that specific interactions between the 5TM and eLtaS domains are required for function. No enzyme activity was detected upon expression of the 5TM and eLtaS domains as separate fragments, indicating that the two domains cannot assemble postsynthesis to form a functional enzyme. Taken together, our data suggest that only the full-length LtaS enzyme is active in the LTA synthesis pathway and that the proteolytic cleavage step is used as a mechanism to irreversibly inactivate the enzyme.