Effects of continuous and pulsatile PTH treatments on rat bone marrow stromal cells

Bone marrow stromal cells (MSCs) differentiation and proliferation are controlled by numerous growth factors and hormones. Continuous parathyroid hormone (PTH) treatment has been shown to decrease osteoblast differentiation, whereas pulsatile PTH increases osteoblast differentiation. However, the effects of PTH treatments on MSCs have not been investigated. This study showed continuous PTH treatment in the presence of dexamethasone (DEX) promoted osteogenic differentiation of rat MSCs in vitro, as demonstrated by increased alkaline phosphatase (ALP) activity, number of ALP expressing cells, and up-regulation of PTH receptor-1, ALP, and osteocalcin mRNA expressions. In contrast, pulsatile PTH treatment was found to suppress osteogenesis of rat MSCs, possibly by promoting the maintenance of undifferentiated cells. Additionally, the observed effects of PTH were strongly dependent on the presence of DEX. MSC proliferation however was not influenced by PTH independent of treatment regimen and presence or absence of DEX. Furthermore, our work raised the possibility that PTH treatment may modulate stem/progenitor cell activity within MSC cultures.     

Photoacoustically‐guided photothermal killing of mosquitoes targeted by nanoparticles

In biomedical applications, nanoparticles have demonstrated the potential to eradicate abnormal cells in small localized pathological zones associated with cancer or infections. Here, we introduce a method for nanotechnology‐based photothermal (PT) killing of whole organisms considered harmful to humans or the environment. We demonstrate that laser‐induced thermal, and accompanying nano‐ and microbubble phenomena, can injure or kill C. elegans and mosquitoes fed carbon nanotubes, gold nanospheres, gold nanoshells, or magnetic nanoparticles at laser energies that are safe for humans. In addition, a photoacoustic (PA) effect was used to control nanoparticle delivery. Through the integration of this technique with molecular targeting, nanoparticle clustering, magnetic capturing and spectral sharpening of PA and PT plasmonic resonances, our laser‐based PA‐PT nano‐theranostic platform can be applied to detection and the physical destruction of small organisms and carriers of pathogens, such as malaria vectors, spiders, bed bugs, fleas, ants, locusts, grasshoppers, phytophagous mites, or other arthropod pests, irrespective of their resistance to conventional treatments. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

De Novo Loss-of-Function Mutations in SETD5, Encoding a Methyltransferase in a 3p25 Microdeletion Syndrome Critical Region,Cause Intellectual Disability

To identify further Mendelian causes of intellectual disability (ID), we screened a cohort of 996 individuals with ID for variants in 565 known or candidate genes by using a targeted next-generation sequencing approach. Seven loss-of-function (LoF) mutations—four nonsense (c.1195A>T [p.Lys399^∗], c.1333C>T [p.Arg445^∗], c.1866C>G [p.Tyr622^∗], and c.3001C>T [p.Arg1001^∗]) and three frameshift (c.2177_2178del [p.Thr726Asnfs^∗39], c.3771dup [p.Ser1258Glufs^∗65], and c.3856del [p.Ser1286Leufs^∗84])—were identified in SETD5, a gene predicted to encode a methyltransferase. All mutations were compatible with de novo dominant inheritance. The affected individuals had moderate to severe ID with additional variable features of brachycephaly; a prominent high forehead with synophrys or striking full and broad eyebrows; a long, thin, and tubular nose; long, narrow upslanting palpebral fissures; and large, fleshy low-set ears. Skeletal anomalies, including significant leg-length discrepancy, were a frequent finding in two individuals. Congenital heart defects, inguinal hernia, or hypospadias were also reported. Behavioral problems, including obsessive-compulsive disorder, hand flapping with ritualized behavior, and autism, were prominent features. SETD5 lies within the critical interval for 3p25 microdeletion syndrome. The individuals with SETD5 mutations showed phenotypic similarity to those previously reported with a deletion in 3p25, and thus loss of SETD5 might be sufficient to account for many of the clinical features observed in this condition. Our findings add to the growing evidence that mutations in genes encoding methyltransferases regulating histone modification are important causes of ID. This analysis provides sufficient evidence that rare de novo LoF mutations in SETD5 are a relatively frequent (0.7%) cause of ID.     

Structure–function relationships of the peptide Paulistine: A novel toxin from the venom of the social wasp Polybia paulista

Background

The peptide Paulistine was isolated from the venom of wasp Polybia paulista. This peptide exists under a natural equilibrium between the forms: oxidised — with an intra-molecular disulphide bridge; and reduced — in which the thiol groups of the cysteine residues do not form the disulphide bridge. The biological activities of both forms of the peptide are unknown up to now.

Methods

Both forms of Paulistine were synthesised and the thiol groups of the reduced form were protected with the acetamidemethyl group [Acm-Paulistine] to prevent re-oxidation. The structure/activity relationships of the two forms were investigated, taking into account the importance of the disulphide bridge.

Results

Paulistine has a more compact structure, while Acm-Paulistine has a more expanded conformation. Bioassays reported that Paulistine caused hyperalgesia by interacting with the receptors of lipid mediators involved in the cyclooxygenase type II pathway, while Acm-Paullistine also caused hyperalgesia, but mediated by receptors involved in the participation of prostanoids in the cyclooxygenase type II pathway.

Conclusion

The acetamidemethylation of the thiol groups of cysteine residues caused small structural changes, which in turn may have affected some physicochemical properties of the Paulistine. Thus, the dissociation of the hyperalgesy from the edematogenic effect when the actions of Paulistine and Acm-Paulistine are compared to each other may be resulting from the influence of the introduction of Acm-group in the structure of Paulistine.

General significance

The peptides Paulistine and Acm-Paulistine may be used as interesting tools to investigate the mechanisms of pain and inflammation in future studies.     

The development of gluconeogenesis in rat liver. Experiments in vivo

1. The injection of substrate amounts of lactate into newborn rats produced an increase in the concentration of phosphoenolpyruvate in liver. Similar experiments with foetal rats showed no increase in phosphoenolpyruvate concentration although pyruvate formation was observed. 2. The administration of pyruvate to foetal rats was also without effect on the hepatic phosphoenolpyruvate concentration, although a 20-fold increase in this was observed when pyruvate was injected into newborn animals. 3. Analogous experiments with aspartate produced qualitatively similar differences between foetal and newborn rats. 4. When [(14)C]-lactate, -pyruvate or -aspartate was injected into foetal or newborn rats incorporation of radioactivity into liver glucose was observed only in the newborn animals. 5. Lactate/pyruvate ratios of 213 in foetal liver and 13.5 in the livers of newborn rats indicated a relatively reduced environment in the cytosol of foetal liver. This difference in redox state was illustrated experimentally by a greater conversion of pyruvate into lactate and an increased formation of malate in foetal liver. 6. Although both the substrate-loading and tracer experiments indicated a block in gluconeogenesis in foetal liver at the stage of conversion of oxaloacetate into phosphoenolpyruvate, gluconeogenesis was also hindered by a highly reduced environment.     

Short-term effects of urea amended with the urease inhibitor N-(n-butyl) thiophosphoric triamide on perennial ryegrass

The current study investigated the short-term physiological implications of plant nitrogen uptake of urea amended with the urease inhibitor N-(n-butyl) thiophosphoric triamide (nBTPT) under both greenhouse and field conditions. ¹⁵N labelled urea amended with 0.0, 0.01, 0.1 and 0.5% nBTPT (w/w) was surface applied at a rate equivalent to 100 kg N ha^–1 to perennial ryegrass in a greenhouse pot experiment. Root, shoot and soil fractions were destructively harvested 0.75, 1.75, 4, 7 and 10 days after fertilizer application. Urease activity was determined in each fraction together with ¹⁵N recovery and a range of chemical analyses. The effect of nBTPT amended urea on leaf tip scorch was evaluated together with the effect of the inhibitor applied on its own on plant urease activity.nBTPT-amended urea dramatically reduced shoot urease activity for the first few days after application compared to unamended urea. The higher the nBTPT concentration the longer the time required for shoot activity to return to that in the unamended treatment. At the highest inhibitor concentration of 0.5% shoot urease activity had returned to that of unamended urea by 10 days. Root urease activity was unaffected by nBTPT in the presence of urea but was affected by nBTPT in the absence of urea.Transient leaf tip scorch was observed approximately 7–15 days after nBTPT + urea application and was greatest with high concentrations of nBTPT and high urea-N application rates. New developing leaves showed no visual sign of tip necrosis.Urea hydrolysis of unamended urea was rapid with only 1.3% urea-N remaining in the soil after 1.75 days. N uptake and metabolism by ryegrass was rapid with ¹⁵N recovery from unamended urea, in the plant (shoot + root) being 33% after 1.75 days. Most of the ¹⁵N in the soil following the urea+0.5% nBTPT application was still as urea after 1.75 days, yet ¹⁵N plant recovery at this time was 25% (root+shoot). This together with other evidence, suggests that if urea hydrolysis in soil is delayed by nBTPT then urea can be taken up by ryegrass as the intact molecule, albeit at a significantly slower initial rate of uptake than NH₄ ⁺-N. Protein and water soluble carbohydrate content of the plant were not significantly affected by amending urea with nBTPT however, there was a significant effect on the composition of amino acids in the roots and shoots, suggesting a difference in metabolism.Although nBTPT-amended urea affected plant urease activity and caused some leaf-tip scorch the effects were transient and short-lived. The previously reported benefit of nBTPT in reducing NH₃ volatilization of urea would appear to far outweigh any of the observed short-term effects, as dry-matter production of ryegrass is increased.     

External surface area determination of lipid vesicles using trinitrobenzene sulfonate and ultraviolet/visible spectrophotometry

The lamellarity of liposomes is an important parameter to be controlled in liposomal delivery–release applications. A practical estimate of the degree of liposome lamellarity can be obtained by measuring the relative external surface area of the liposomes using a chemical assay. All such assays are based on a signal change caused by exposed marker lipids on reaction with a specific externally added reagent. However, a quantitative determination is often distorted by background reactions and contributions of internal lipid labeling. In the so-called TNBS assay, the marker lipid is phosphatidylethanolamine (PE) and the externally added reagent is TNBS (2,4,6-trinotrobenzene sulfonate). Mechanistic aspects of the TNBS assay were considered for improving the assay. Internal lipid labeling via PE flip-flop and/or TNBS permeation was minimal not only in cholesterol-containing liposomes but also in cholesterol-free liposomes if in the latter case membrane fluidity was decreased by slightly increasing the PE content. Compared with earlier versions of the TNBS assay, the amount of marker lipid and the time for analysis could be reduced considerably. The elaborated protocol was also applied to liposomes prepared from lipidic egg yolk isolates, offering a simple and inexpensive method for the development and in-process control of new liposome formation technologies.     

The conjugative plasmid SLP2 of Streptomyces lividans is a 50 kb linear molecule

The SLP2 plasmid had previously been demonstrated genetically to exist In Streptomyces lividans by its ability to promote conjugation and to elicit‘pocks’on recipient (SLP2^?) cultures, but it had not been physically detected. Using pulsed-field gel electrophoresis, a 50kb linear DNA was isolated from SLP2⁺ but not SLP2^? strains of S. lividans, and from Streptomyces coelicolor and Streptomyces parvulus strains to which SLP2 had been transferred by conjugation or transformation. We conclude that this linear DNA is SLP2. The terminal fragments of SLP2 were cloned. The determined sequences revealed a 44 bp imperfect terminal inverted repeat. The terminal 12 bp sequence of SLP2 was identical to those of two other Streptomyces linear plasmids, pSLA2 and pSCL, and similar to the terminal sequences of another Streptomyces linear plasmid, SCP1. The termini of SLP2 DNA were resistant to digestion by λ exonuclease and ExoIII. A truncated (probably crippled) copy of Tn4811 is present on the plasmid. While the SLP2 plasmid exists as a tree form in the host, a 15.7 kb sequence corresponding to the segment of SLP2 from Tn4811 to the right terminus is also present (at a copy number similar to the free form) elsewhere in the genome of S. lividans. Furthermore, SLP2 is partially homologous to a newly discovered 650 kb linear plasmid in S. parvulus.