Pancreatic trypsin activates human promatrix metalloproteinase-2

In contrast to the prevalent view in the literature hitherto, the present study shows that pancreatic trypsin can activate human promatrix metalloproteinase-2 (proMMP-2). It is shown that trypsin's ability to activate proMMP-2 is dependent on various environmental factors such as the level of exogenously added Ca(2+) and Brij-35, temperature, as well as trypsin concentration. The activation occurred as a sequential processing of the proenzyme, initially generating an active 62kDa species. This was followed by successive truncation of the C-terminal domain, giving rise to active species of 56kDa, 52kDa and 50kDa. Tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) prevented the trypsin-mediated C-terminal truncation, without affecting the generation of the 62kDa species, while the presence of EDTA increased the rate of the trypsin-mediated activation of proMMP-2. MALDI-TOF MS analysis of the 50kDa form indicated that trypsin generated active forms with either Lys87 or Trp90 as the N-terminal residue and Arg538 as a C-terminal residue. The trypsin-activated MMP-2 was active in solution against both synthetic and physiologic substrates, and the steady-state kinetic coefficients k(cat), K(m) and k(cat)/K(m) were determined for the enzyme activated either by APMA, membrane-type 1 matrix metalloproteinase (MT1-MMP) or trypsin. The trypsin-activated MMP-2 exhibited slightly lower k(cat) and k(cat)/K(m) values as well as a slightly higher K(i) value against TIMP-1 compared to the enzyme activated by APMA or MT1-MMP. Docking studies of TIMP-1 revealed that the slightly weaker binding of the inhibitor to the trypsin-activated MMP-2 could be attributed to its shorter N terminus (Lys87/Trp90 versus Tyr81), as Phe83 and Arg86 interacted directly with the inhibitor. Our results suggest that the trypsin-activated MMP-2 possesses the catalytic and regulatory potential to be of significance in vivo.     

The K15 protein of Kaposi's sarcoma-associated herpesvirus recruits the endocytic regulator intersectin 2 through a selective SH3 domain interaction

Kaposi's sarcoma-associated herpesvirus, also known as human herpesvirus 8, is closely associated with several cancers including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. The rightmost end of the KSHV genome encodes a protein, K15, with multiple membrane-spanning segments and an intracellular carboxy-terminal tail that contains several conserved motifs with the potential to recruit interaction domains (i.e., SH2, SH3, TRAF) of host cell proteins. K15 has been implicated in downregulating B cell receptor (BCR) signaling through its conserved motifs and may thereby play a role in maintaining viral latency and/or preventing apoptosis of the infected B cells. However, K15's mode of action is largely unknown. We have used mass spectrometry, domain and peptide arrays, and surface plasmon resonance to identify binding partners for a conserved proline-rich sequence (PPLP) in the K15 cytoplasmic tail. We show that the PPLP motif selectively binds the SH3-C domain of an endocytic adaptor protein, Intersectin 2 (ITSN2). This interaction can be observed both in vitro and in cells, where K15 and ITSN2 colocalize to discrete compartments within the B cell. The ability of K15 to associate with ITSN2 suggests a new role for the K15 viral protein in intracellular protein trafficking.     

Behavioural responses in a net restraint test predict interrenal reactivity in Arctic charr Salvelinus alpinus

In this study, a 1 min net restraint test was evaluated as a method to predict stress‐coping style in Arctic charr Salvelinus alpinus, by investigating the relationship between behaviour during the test and levels of plasma cortisol sampled after 30 min confinement. In two separate groups of S. alpinus, general linearized model revealed significant correlations between cortisol levels and principal component scores extracted from principal component analysis, combining measures of activity in the tests. With the use of glmulti, the model selection ruled out any effects of size, sex and order of capture on interrenal reactivity. In general, S. alpinus that were more active in the net restraint test also had low levels of circulating cortisol, suggesting a proactive coping style. The results from two repeated runs were not correlated, but both runs, performed eight days apart, show a negative correlation between post‐stress cortisol level and activity in the net. The lack of consistency could be explained by different treatments before each run and individual differences in behavioural plasticity. The net restraint test is thus predictive of stress‐coping style in S. alpinus, and has the benefit of being less time‐consuming than the commonly used confinement stress test.     

Male Courtship Pheromones Affect Female Behaviour in the Swordtail Characin (Corynopoma riisei)

Pheromones constitute an important cue used by both males and females during courtship. Here, we investigate the effect of male pheromones on female behaviour in the swordtail characin (Corynopoma riisei), a species of fish where males have a caudal pheromone gland which has been suggested to affect female behaviour during courtship. We subjected female C. riisei to male courtship pheromones and investigated the effect on both female behaviour and brain serotonergic activity levels compared to a control group. While no difference in serotonergic activity was found, the pheromone‐treated females showed lower stress levels compared to the control group. Furthermore, pheromone‐treated females increased locomotor activity over time, while a decrease in locomotor activity was observed in the control group. These results suggest that the male courtship pheromones may serve to reduce female stress and increase female activity, possibly to aid males in gaining access to females and facilitating sperm transfer.     

Temporal patterns of nucleotide misincorporations and DNA fragmentation in ancient DNA

DNA that survives in museum specimens, bones and other tissues recovered by archaeologists is invariably fragmented and chemically modified. The extent to which such modifications accumulate over time is largely unknown but could potentially be used to differentiate between endogenous old DNA and present-day DNA contaminating specimens and experiments. Here we examine mitochondrial DNA sequences from tissue remains that vary in age between 18 and 60,000 years with respect to three molecular features: fragment length, base composition at strand breaks, and apparent C to T substitutions. We find that fragment length does not decrease consistently over time and that strand breaks occur preferentially before purine residues by what may be at least two different molecular mechanisms that are not yet understood. In contrast, the frequency of apparent C to T substitutions towards the 5'-ends of molecules tends to increase over time. These nucleotide misincorporations are thus a useful tool to distinguish recent from ancient DNA sources in specimens that have not been subjected to unusual or harsh treatments.     

Increased heat resistance in mycelia from wood fungi prevalent in forests characterized by fire: a possible adaptation to forest fire

Forest fires have been the major stand-replacing/modifying disturbance in boreal forests. To adapt to fire disturbance, different strategies have evolved. This study focuses on wood fungi, and a specific adaptation to forest fire: increased heat resistance in their mycelia. Fifteen species of wood fungi were selected and a priori sorted in two groups according to their prevalence in fire-affected environments. The fungi were cultivated on fresh wood and exposed to 100, 140, 180, 220 °C for 5, 10, 15, 20 and 25 min. under laboratory conditions. A clear difference was found among the two groups. Species prevalent in fire-affected habitats had a much higher survival rate over all combinations of time and temperature compared to species associated with other environments. Thus, the results indicate that fire adaptation in terms of increased heat resistance in mycelia occurs in some species of wood fungi. Such adaptation will influence the ecology and population dynamics of wood fungi, as well as having implications for best practices during restoration fires.     

Nuclear gene sequences from a late pleistocene sloth coprolite

The determination of nuclear DNA sequences from ancient remains would open many novel opportunities such as the resolution of phylogenies, the sexing of hominid and animal remains, and the characterization of genes involved in phenotypic traits. However, to date, single-copy nuclear DNA sequences from fossils have been determined only from bones and teeth of woolly mammoths preserved in the permafrost. Since the best preserved ancient nucleic acids tend to stem from cold environments, this has led to the assumption that nuclear DNA would be retrievable only from frozen remains. We have previously shown that Pleistocene coprolites stemming from the extinct Shasta sloth (Nothrotheriops shastensis, Megatheriidae) contain mitochondrial (mt) DNA from the animal that produced them as well as chloroplast (cp) DNA from the ingested plants. Recent attempts to resolve the phylogeny of two families of extinct sloths by using strictly mitochondrial DNA has been inconclusive. We have prepared DNA extracts from a ground sloth coprolite from Gypsum Cave, Nevada, and quantitated the number of mtDNA copies for three different fragment lengths by using real-time PCR. We amplified one multicopy and three single-copy nuclear gene fragments and used the concatenated sequence to resolve the phylogeny. These results show that ancient single-copy nuclear DNA can be recovered from warm, arid climates. Thus, nuclear DNA preservation is not restricted to cold climates.     

Chlorosis caused by two recessively interacting genes reveals a role of RNA helicase in hybrid breakdown in Arabidopsis thaliana

Hybrids often differ in fitness from their parents. They may be superior, translating into hybrid vigour or heterosis, but they may also be markedly inferior, because of hybrid weakness or incompatibility. The underlying genetic causes for the latter can often be traced back to genes that evolve rapidly because of sexual or host–pathogen conflicts. Hybrid weakness may manifest itself only in later generations, in a phenomenon called hybrid breakdown. We have characterized a case of hybrid breakdown among two Arabidopsis thaliana accessions, Shahdara (Sha, Tajikistan) and Lövvik‐5 (Lov‐5, Northern Sweden). In addition to chlorosis, a fraction of the F₂ plants have defects in leaf and embryo development, and reduced photosynthetic efficiency. Hybrid chlorosis is due to two major‐effect loci, of which one, originating from Lov‐5, appears to encode an RNA helicase (AtRH18). To examine the role of the chlorosis allele in the Lövvik area, in addition to eight accessions collected in 2009, we collected another 240 accessions from 15 collections sites, including Lövvik, from Northern Sweden in 2015. Genotyping revealed that Lövvik collection site is separated from the rest. Crosses between 109 accessions from this area and Sha revealed 85 cases of hybrid chlorosis, indicating that the chlorosis‐causing allele is common in this area. These results suggest that hybrid breakdown alleles not only occur at rapidly evolving loci, but also at genes that code for conserved processes.     

A new approach to genome mapping and sequencing: slalom libraries

We describe here an efficient strategy for simultaneous genome mapping and sequencing. The approach is based on physically oriented, overlapping restriction fragment libraries called slalom libraries. Slalom libraries combine features of general genomic, jumping and linking libraries. Slalom libraries can be adapted to different applications and two main types of slalom libraries are described in detail. This approach was used to map and sequence (with ~46% coverage) two human P1-derived artificial chromosome (PAC) clones, each of ~100 kb. This model experiment demonstrates the feasibility of the approach and shows that the efficiency (cost-effectiveness and speed) of existing mapping/sequencing methods could be improved at least 5–10-fold. Furthermore, since the efficiency of contig assembly in the slalom approach is virtually independent of length of sequence reads, even short sequences produced by rapid, high throughput sequencing techniques would suffice to complete a physical map and a sequence scan of a small genome.