Fluorescence resonance energy transfer between the nucleotide binding site and Cys-10 in G-actin and F-actin

Intramonomer fluorescence resonance energy transfer between the donor epsilon-ATP bound to the nucleotide site and the acceptor N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) or 4-dimethylaminophenyl-azophenyl-4'-maleimide bound to Cys-10 in G-actin was measured. The donor-acceptor distance was calculated to be about 40 A. The intermonomer energy transfer in F-actin occurring between epsilon-ADP and DABMI was also measured. The radial coordinate of Cys-10 was calculated to be 25 A based on the helical symmetry of F-actin and the recently calculated radial coordinate of the nucleotide binding site in F-actin i.e. 25 A (Miki, M., Hambly, B. and dos Remedios, C.G. (1986) Biochim. Biophys. Acta 871, 137-141). (The assumption has been made in calculating these distances that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime.) Corresponding distances separating the donor nucleotide in one monomer from acceptors on Cys-10 in the first and second nearest neighbours in F-actin are 39-40 A and 41-43 A.     

Embryonic development of adriatic sturgeon,Acipenser naccarii (Bonaparte 1836), in farming conditions: a guide

The sequence and timing of the embryonic development of Adriatic sturgeon (Acipenser naccarii) are described at a constant temperature (17 ± 1 °C), from fertilisation to hatch in a hatchery. Fertilised eggs, obtained from one female and two males, were held in a flow‐through system. On the first day, embryos were sampled every 20 min during the first 12 h, and every 40 min in the following 12 h. Embryos were sampled hourly on the second day. On the third and fourth days, embryos were sampled every 2 h, and every 3 h on the fifth and sixth days. The first cleavage furrow appeared at 1 h 40 min postfertilisation. Blastulation concluded at 12 HPF (hours postfertilisation), when the dorsal blastopore lip formed. The completion of gastrulation was at 18 HPF, closing of the neural tube at 23 HPF and formation of the s‐shape heart at 31 HPF. The mass hatch occurred at 120 HPF. A. naccarii is a relatively common sturgeon species in aquaculture and possesses similar developmental patterns to those of other sturgeons; hence, we can use this species as a model for further studies about sturgeon species which are more difficult to breed artificially. Information about the embryonic development of the Adriatic sturgeon could assist restocking programmes.     

Peripartum cardiomyopathy: a global effort to find the cause and cure for the rare and little understood disease

In this review, we present our current understanding of peripartum cardiomyopathy (PPCM) based on reports of the incidence, diagnosis and current treatment options. We summarise opinions on whether PPCM is triggered by vascular and/or hormonal causes and examine the influence of comorbidities such as preeclampsia. Two articles published in 2021 strongly support the hypothesis that PPCM may be a familial disease. Using large cohorts of PPCM patients, they summarised the available genomic DNA sequence data that are expressed in human cardiomyocytes. While PPCM is considered a disease predominately affecting the left ventricle, there are data to suggest that some cases also involve right ventricular failure. Finally, we conclude that there is sufficient evidence to warrant an RNAseq investigation and that this would be most informative if performed at the cardiomyocytes level rather than analysing genomic DNA from the peripheral circulation. Given the rarity of PPCM, the combined resources of international human heart tissue biobanks have assembled 30 ventricular tissue samples from PPCM patients, and we are actively seeking to enlarge this patient base by collaborating with human heart tissue banks and research laboratories who would like to join this endeavour.     

Two opposite effects of cofilin on the thermal unfolding of F-actin: a differential scanning calorimetric study

Differential scanning calorimetry was used to examine the effects of cofilin on the thermal unfolding of actin. Stoichiometric binding increases the thermal stability of both G- and F-actin but at sub-saturating concentrations cofilin destabilizes F-actin. At actin:cofilin molar ratios of 1.5-6 the peaks corresponding to stabilized (66-67 degrees C) and destabilized (56-57 degrees C) F-actin are observed simultaneously in the same thermogram. Destabilizing effects of sub-saturating cofilin are highly cooperative and are observed at actin:cofilin molar ratios as low as 100:1. These effects are abolished by the addition of phalloidin or aluminum fluoride. Conversely, at saturating concentrations, cofilin prevents the stabilizing effects of phalloidin and aluminum fluoride on the F-actin thermal unfolding. These results suggest that cofilin stabilizes those actin subunits to which it directly binds, but destabilizes F-actin with a high cooperativity in neighboring cofilin-free regions.     

Unbalanced collagenases/TIMP-1 expression and epithelial apoptosis in experimental lung fibrosis

In this study, we examined the sequential expression of several matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and growth factors as well as the presence of apoptosis in a model of pulmonary fibrosis induced in rats with paraquat and hyperoxia. Animals showing neither clinical nor morphological changes with this double aggression were classified as "resistant". Rats were killed at 1, 2, 3, and 6 wk, and lungs were used for collagen content, gene expression by real-time PCR, gelatinolytic activity by zymography, apoptosis by in situ DNA fragmentation, and protein localization by immunohistochemistry. Our results showed a significant decrease of collagenases MMP-8 and MMP-13, with an increase of TIMP-1 and transforming growth factor-beta. Immunoreactive TIMP-1 was increased in experimental rats and primarily localized in alveolar macrophages. Expression of gelatinases MMP-2 and MMP-9 mRNAs was not affected, but lung zymography revealed an increase in progelatinase B, progelatinase A, and its active form. Epithelial apoptosis was evident from the first week, whereas at later periods, interstitial cell apoptosis was also noticed. Resistant animals behave as controls. These findings suggest that an imbalance between collagenases and TIMPs, excessive gelatinolytic activity, and epithelial apoptosis participate in the fibrotic response in this experimental model.     

A technique for microsecond heating and cooling of a thin (submicron) biological sample

Temperature excursions of short duration are useful in exploring the effects of stress on biological systems. Stress will affect the conformation of biological molecules such as proteins, which will lead to an effect on their function. The feasibility of generating such temperature excursions is demonstrated by solving the heat diffusion equation for an aqueous layer on a silicon wafer. The silicon is rapidly heated by a laser pulse and also acts as a heat sink to quench the temperature rise. An oxide layer was used to limit the maximum temperature attained by the sample. We show that exposures above a 50 degrees C benchmark can be confined to times less than 5 micros.     

Effects of phosphinotricin treatment on glutamine synthetase isoforms in Scots pine seedlings

The occurrence of GS isoenzymes has been investigated in Scots pine (Pinus sylvestris) seedlings. A transient increase of glutamine synthetase (GS, EC 6.3.1.2) activity was observed in the cotyledon whorl of plants treated with the herbicide phosphinotricin (PPT). The increase in GS activity was accompanied by a parallel accumulation of GS1 protein, which remained at high levels throughout the PPT treatment. Two-dimensional SDS-PAGE western analysis showed that pine extracts contained two GS1 polypeptides which differ in their corresponding isoelectric points. Analysis of crude extracts by ion-exchange chromatography led to the separation of two GS isoforms. The first peak (GS1-a) eluted from the columns at a low ionic strength (0.15-0.18 M KCl), whereas the second one (GS1-b) was detected at 0.5 M KCl. A detailed molecular study of both GS holoenzymes confirmed that their subunits were similar in size (about 41 kDa) but different in charge. All these data clearly demonstrate the presence of two GS1 forms in Scots pine cotyledons. Moreover, a comparison of isolated GS isoproteins with the recombinantly expressed Scots pine cytosolic subunit suggests that GS1-a corresponds to the previously characterized cDNA (pGSP114) whereas GS1-b is a minor GS isoenzyme with increased relative abundance in phosphinotricin treated plants.     

Best practice BioBanking of human heart tissue

This review provides a guide to researchers who wish to establish a biobank. It also gives practical advice to investigators seeking access to samples of healthy or diseased human hearts. We begin with a brief history of the Sydney Heart Bank (SHB) from when it began in 1989, including the pivotal role played by the late Victor Chang. We discuss our standard operating procedures for tissue collection which include cryopreservation and the quality assurance needed to maintain the long-term molecular and cellular integrity of the samples. The SHB now contains about 16,000 heart samples derived from over 450 patients who underwent isotopic heart transplant procedures and from over 100 healthy organ donors. These enable us to provide samples from a wide range of categories of heart failure. So far, we have delivered heart samples to more than 50 laboratories over two decades, and we answer their most frequently asked questions. Other SHB services include the development of tissue microarrays (TMA). These enable end users to perform preliminary examinations of the expression and localisation of target molecules in diseased or aging donor hearts, all in a single section of the TMA. Finally, the processes involved in managing tissue requests from external users and logistics considerations for the shipment of human tissue are discussed in detail.

Electronic supplementary material

The online version of this article (doi:10.1007/s12551-015-0182-6) contains supplementary material, which is available to authorized users.     

Overexpression of MMP9 in macrophages attenuates pulmonary fibrosis induced by bleomycin

Pulmonary fibrosis is a common response to a variety of lung injuries, characterized by fibroblast/myofibroblast expansion and abnormal accumulation of extracellular matrix. An increased expression of matrix metalloprotease 9 (MMP9) in human and experimental lung fibrosis has been documented, but its role in the fibrotic response is unclear. We studied the effect of MMP9 overexpression in bleomycin-driven lung fibrosis using transgenic mice expressing human MMP9 in alveolar macrophages (hMMP9-TG). At 8 weeks post-bleomycin, the extent of fibrotic lesions and OH-proline content were significantly decreased in the TG mice compared to the WT mice. The decreased fibrosis in hMMP9-TG mice was preceded by a significant reduction of neutrophils and lymphocytes in bronchoalveolar lavage (BAL) at 1 and 4 weeks post-bleomycin, respectively, as well as by significantly less TIMP-1 than the WT mice. From a variety of cytokines/chemokines investigated, we found that BAL levels of insulin-like growth factor binding protein-3 (IGFBP3) as well as the immunoreactive protein in the lungs were significantly lower in hMMP9-TG mice compared with WT mice despite similar levels of gene expression. Using IGFBP-3 substrate zymography we found that BAL from TG mice at 1 week after bleomycin cleaved IGFBP-3. Further, we demonstrated that MMP9 degraded IGFBP-3 into lower molecular mass fragments. These findings suggest that increased activity of MMP9 secreted by alveolar macrophages in the lung microenvironment may have an antifibrotic effect and provide a potential mechanism involving IGFBP3 degradation.