Increase in transglutaminase and its extracellular products in response to an inflammatory stimulus by lipopolysaccharide

Transglutaminase (TGase) activities were measured in rat tissues 1-7 days after intraperitoneal injection of saline or lipopolysaccharide (LPS) and in the cells and media from pre-confluent human fibroblasts cultured for two days in the presence or absence of LPS. (-glutamyl)lysine and [3H]putrescine-labelled -glutamyl derivatives in extracellular and cellular fibroblast proteins were also measured. Three effects of LPS were observed. Firstly, total TGase activity is greater in the tissues from the LPS-injected animals, with the maximum increase occurring at 1 day in dermis, epidermis and liver, at 5 days in the aorta and, after a decrease at 2-5 days, at 7 days in the panniculus muscle. Secondly, the fraction of the total activity which is buffer-extractable is greater on days 1 and/or 2 in all the tissues from the LPS-injected rats. Thirdly, in cultures of human fibroblasts, LPS increases that fraction of bound [3H]putrescine and of TGase and its -glutamylamine products which occurs in the extracellular medium. In addition, a higher concentration of TGase-derived crosslinks was found in extracellular as opposed to intracellular proteins. In conjunction with previous findings in skin wound healing and in atherosclerosis these results support the concept of an extracellular function for tissue TGase and indicate that there is a widespread association of increases in TGase and its extracellular products with inflammation and the healing or fibrotic processes which follow it.     

Cleavable ester-linked magnetic nanoparticles for labeling of solvent-exposed primary amine groups of peptides/proteins

To study the solvent-exposed lysine residues of peptides/proteins, we previously reported disulfide-linked N-hydroxysuccinimide ester-modified silica-coated iron oxide magnetic nanoparticles (NHS–SS–SiO₂@Fe₃O₄ MNPs). The presence of a disulfide bond in the linker limits the use of disulfide reducing agent during protein digestion and allows unwanted disulfide formation between the MNPs and protein. In the current work, the disulfide bond was replaced with a cleavable ester group to synthesize NHS ester-modified SiO₂@Fe₃O₄ MNPs. Use of the cleavable ester group provides an improved method for protein labeling and allows the use of disulfide reducing agents during protein digestion.     

Plant progesterone 5beta-reductase is not homologous to the animal enzyme. Molecular evolutionary characterization of P5betaR from Digitalis purpurea

Plants of the genus Digitalis produce cardiac glycosides, i.e. digoxin, which are widely used for congestive heart failure. Progesterone 5beta-reductase (P5betaR) is a key enzyme in the biosynthesis of these natural products. Here, we have carried out the purification and partial amino acid sequencing of the native P5betaR from foxglove (Digitalis purpurea), and isolated a cDNA encoding this enzyme. Similarly to other steroid 5beta-reductases, the recombinant P5betaR catalyzes the stereospecific reduction of the Delta(4)-double bond of several steroids with a 3-oxo,Delta(4,5) structure. The gene encoding P5betaR is expressed in all plant organs, and maximally transcribed in leaves and mature flowers. P5betaR belongs to the short-chain dehydrogenase/reductase (SDR) superfamily, bearing no structural homology to its mammalian counterpart, which is a member of the aldo-keto reductase (AKR) superfamily. A similar situation occurs with 3beta-hydroxy-Delta(5)-steroid dehydrogenase (3betaHSD), the gene immediately preceding P5betaR in the cardenolide pathway, which suggests that the entire route has evolved independently in animals and plants. P5betaR is retained only in plants, where it is ubiquitous, and a few distantly related bacterial lineages after its diversification from the last universal common ancestor. Evolutionary conserved changes in its putative active site suggest that plant P5betaR is a member of a novel subfamily of extended SDRs, or a new SDR family.     

Fragment-based screening of programmed death ligand 1 (PD-L1)

The PD-1 immune checkpoint pathway is a highly validated target for cancer immunotherapy. Despite the potential advantages of small molecule inhibitors over antibodies, the discovery of small molecule checkpoint inhibitors has lagged behind. To discover small molecule inhibitors of the PD-1 pathway, we have utilized a fragment-based approach. Small molecules were identified that bind to PD-L1 and crystal structures of these compounds bound to PD-L1 were obtained.     

Experimental Determination of the Membrane Topology of the Plasmodium Protease Plasmepsin V

The malaria parasite exports hundreds of proteins into its host cell. The majority of exported proteins contain a Host-Targeting motif (also known as a Plasmodium export element) that directs them for export. Prior to export, the Host-Targeting motif is cleaved by the endoplasmic reticulum-resident protease Plasmepsin V and the newly generated N-terminus is N-α-acetylated by an unidentified enzyme. The cleaved, N-α-acetylated protein is trafficked to the parasitophorous vacuole, where it is translocated across the vacuole membrane. It is clear that cleavage and N-α-acetylation of the Host-Targeting motif occur at the endoplasmic reticulum, and it has been proposed that Host-Targeting motif cleavage and N-α-acetylation occur either on the luminal or cytosolic side of the endoplasmic reticulum membrane. Here, we use self-associating ‘split’ fragments of GFP to determine the topology of Plasmepsin V in the endoplasmic reticulum membrane; we show that the catalytic protease domain of Plasmepsin V faces the endoplasmic reticulum lumen. These data support a model in which the Host-Targeting motif is cleaved and N-α-acetylated in the endoplasmic reticulum lumen. Furthermore, these findings suggest that cytosolic N-α-acetyltransferases are unlikely to be candidates for the N-α-acetyltransferase of Host-Targeting motif-containing exported proteins.     

Biocontrol of Escherichia coli O157 with O157-Specific Bacteriophages

Escherichia coli O157 antigen-specific bacteriophages were isolated and tested to determine their ability to lyse laboratory cultures of Escherichia coli O157:H7. A total of 53 bovine or ovine fecal samples were enriched for phage, and 5 of these samples were found to contain lytic phages that grow on E. coli O157:H7. Three bacteriophages, designated KH1, KH4, and KH5, were evaluated. At 37 or 4°C, a mixture of these three O157-specific phages lysed all of the E. coli O157 cultures tested and none of the non-O157 E. coli or non-E. coli cultures tested. These results required culture aeration and a high multiplicity of infection. Without aeration, complete lysis of the bacterial cells occurred only after 5 days of incubation and only at 4°C. Phage infection and plaque formation were influenced by the nature of the host cell O157 lipopolysaccharide (LPS). Strains that did not express the O157 antigen or expressed a truncated LPS were not susceptible to plaque formation or lysis by phage. In addition, strains that expressed abundant mid-range-molecular-weight LPS did not support plaque formation but were lysed in liquid culture. Virulent O157 antigen-specific phages could play a role in biocontrol of E. coli O157:H7 in animals and fresh foods without compromising the viability of other normal flora or food quality.     

Microsatellite variation in simulated and natural founder populations of the Laysan finch (Telespiza cantans)

Historical and demographic data were used in a computer model tosimulate neutral genetic change in populations of the Laysanfinch (Telespiza cantans), an insular passerine bird that hasundergone documented founder events at Pearl and Hermes reef(PHR). Measures of genetic variation in the natural PHRpopulations generally matched those in the simulated populations,except that heterozygosity on Southeast Island was lower than themodel predicted, and the heterozygote excess in the naturalpopulations had a low probability of occurrence in the simulatedpopulations. The estimate of effective population size (N _e) fromthe stochastic demographic model matched the estimate fromgenetic data for two populations, but the demographic estimatewas higher than the genetic estimate for Southeast Island. Smallfounder number was rejected as a possible explanation for thereduced genetic variation on Southeast. We suggest that N _e wasoverestimated in part because we assumed seasonal variance inreproductive success. Additional variance components need to bemeasured in the field and incorporated into the model. Accounting for the heterozygote excess also requires furthertheoretical and field investigations. Possible explanations forthe excess include inbreeding depression, incest avoidance, andthe effect of polygyny on heterozygote excess in smallpopulations. We concluded that the Pearl and Hermes reefpopulation will continue to lose genetic variation at a highrate, and translocations from the native population on Laysan maybe required to maintain a viable population on the reef.     

A new and permanent staining method for starch granules using fluorescence microscopy

A fluorescence technique has been developed for observing starch granules in plant tissues. Sections are stained with a mixture of dyes which we have named F.A.S.G.A. from the initials of the Spanish names of its components (fucsina, alcian blue, safranina, glicerina, agua), and viewed by epifluorescence microscopy. The starch granules fluoresce greenish yellow, allowing the degradative state to be observed. Cell structures which do not fluoresce are also differentiated. The stain permits identification of other structures when examined by visible light microscopy and is relatively resistant to fading over time.     

Temperature-dependence of cardiac output and regional blood flow in rainbow trout, Salmo gairdneri Richardson

Cardiac output, blood flow distribution and regional perfusion were determined in free-swimming rainbow trout acclimated to 6, 12 and 18°C, using the indicator dilution and microsphere methods. Cardiac output (ml min⁻¹ kg⁻¹) increased linearly with increasing temperature, while circulation time decreased. Blood flow distribution (% of cardiac output) to the spleen, liver, kidney, gall bladder and gastro-intestinal tract was significantly reduced at 18°C relative to 6°C-acclimated fish. White muscle received the largest fraction of cardiac output, and blood flow distribution to white muscle increased significantly with increasing acclimation temperature. Blood perfusion (ml h⁻¹ g⁻¹) of various organs and red muscle was not influenced by acclimation temperature, while white muscle perfusion increased with increasing temperature. These results demonstrate physiological adaptation of the cardiovascular system of rainbow trout to changes in acclimation temperature.