Pentafluorophenyl ester-functionalized phosphorylcholine polymers: preparation of linear, two-arm, and grafted polymer-protein conjugates

Novel pentafluorophenyl (PFP)-ester-functionalized phosphorylcholine (PC) polymers of different architectures were prepared and conjugated to lysozyme as a model protein. Linear and two-arm poly(2-methacryloyloxyethyl phosphorylcholine) (polyMPC) structures containing PFP functionality at the chain-end were prepared by atom transfer radical polymerization (ATRP) from novel initiators. Additional conjugates were prepared from phosphorylcholine-substituted cyclooctene (PC-COE) polymers containing PFP-ester bearing comonomers. The polymer-protein conjugates were characterized by HPLC, FPLC, and DLS and were seen to retain most (~80% or greater) of their native enzymatic activity. Pharmacokinetic profiles of the polymer-protein conjugates were studied in mice and found to increase the circulation half-life compared with lysozyme alone.     

Long-term effects of diagnostic ultrasound during fetal period on postnatal development and adult behavior of mouse

Pregnant Swiss albino mice were exposed to diagnostic levels of ultrasound (3.5 MHz, intensity 65 mW, I(SPTP) = 1 W/cm(2), I(SATA) = 240 W/cm(2)) for 10, 20 and 30 minutes on day 14 of gestation. Sham exposed controls were maintained for comparison. Fifteen pregnant mice were exposed for each group. Exposed as well as control animals were left to complete gestation and parturition. Ultrasound induced changes in maternal vaginal temperature was recorded. The changes in the physiological reflexes and postnatal mortality up to 6 weeks of age were recorded. The litters were subjected to behavioral tests for locomotor activity, learning and memory at 4 month and 1 year of age. Neither the physiological reflexes nor the postnatal mortality was affected by ultrasound exposure. However, there was a noticeable impairment in both locomotor and learning behavior even after a 10 min exposure, which further increased with increases in exposure time. Thus the present study demonstrates the neurotoxicity of diagnostic ultrasound and the high susceptibility of early fetal brain to induction of lasting detrimental changes by ultrasound exposure.     

The fetal cleft palate: III. Ultrastructural and functional analysis of palatal development following in utero repair of the congenital model

The role of fetal surgery in the management of congenital anomalies and intrauterine abnormalities is appropriately restricted on the basis of feasibility and risk-to-benefit analyses of intrauterine intervention. Recently, the authors demonstrated that in utero cleft palate repair of the congenital caprine model is technically feasible and results in scarless healing of the mucoperiosteum and velum, with subsequent development of a potentially functional bilaminar palate with distinct oral and nasal mucosal layers, following single-layer repair of the fetal mucoperiosteal flaps. A slight indentation at the site of repair was the only remaining evidence of a cleft. At 6 months of age, normal palatal architecture, including that of mucosal, muscular, and glandular elements, was seen grossly and histologically. The present work investigated the ultrastructural and functional aspects of the palate following in utero cleft repair to determine what benefits might be derived from fetal intervention. Six goats pregnant with twins were gavaged twice daily for 10 days (gestational days 32 to 41; term, 145 days) with dry, ground Nicotiana glauca plant delivering between 2.4 and 14 mg/kg per day of anabasine, doses that were adjusted in response to mater-nal toxicity. At 85 days' gestation, six fetuses underwent in utero palatoplasty using a modified von Langenbeck technique with elevation of bilateral mucoperiosteal flaps and lateral relaxing incisions. A single-layer repair of the mucoperiosteal flaps was performed using interrupted 6-0 Vicryl sutures. Six fetuses remained as unrepaired clefted controls. Six months after in utero palatoplasty, each group of goats underwent nasoendoscopy to evaluate palatal function; two unclefted 6-month-old goats served as controls. Subsequently, soft palate muscle was harvested from each of the goats and was evaluated by light and electron microscopy. Velar muscle was also harvested from the unclefted control goats and was similarly studied. Nasoendoscopy demonstrated functional palates capable of dynamic velopharyngeal closure following in utero cleft repair; this motion was similar to that observed in unclefted animals. Unrepaired clefted goats did not demonstrate any evidence of velar motion or velopharyngeal closure. Soft palate muscle from this group demonstrated evidence of myofibril degeneration, atrophy, and loss compared with unclefted control velar muscle. Ultrastructural changes included sarcomere "scalloping, " partial Z-line degeneration and loss, and progressive I-band degeneration and loss. Repaired clefted soft palate muscle was remarkably similar to unclefted control muscle. Significantly less myofibril, Z-line, and I-band degeneration and loss were observed with minimal evidence of sarcomere scalloping. In utero cleft palate repair results in a functional soft palate with restoration of ultrastructural architecture of the velum. These findings were attributed to reconstitution of the velar muscular sling, which is disrupted during the clefting process and remains abnormally inserted into the posterior edge of the palatal bone and along the bony cleft. Although repaired velar muscle does demonstrate some evidence of ultrastructural change compared with control muscle, these findings are significantly less pronounced than those observed in the unrepaired clefted muscle.     

Human complement proteins D, C2, and B. Active site mapping with peptide thioester substrates

The specificity and reactivity of complement serine proteases D, B, Bb, C2, and C2a were determined using a series of peptide thioester substrates. The rates of thioester hydrolysis were measured using assay mixtures containing the thiol reagent 4,4'-dithiodipyridine at pH 7.5. Each substrate contained a P1 arginine residue, and the effect of various groups and amino acids in the P2, P3, P4, and P5 positions was determined using kcat/Km values to compare reactivities. Among peptide thioesters corresponding to the activation site sequence in B, dipeptide thioesters containing a P2 lysine residue were the best substrates for D. Extending the chain to include a P3 or P4 amino acid resulted in loss of activity, and neither the tripeptide nor the tetrapeptide containing the cleavage sequence of B was hydrolyzed. Overall, D cleaved fewer substrates and was 2-3 orders of magnitude less reactive than C1s against some thioester substrates. C2 and fragment C2a had comparable reactivities and hydrolyzed peptides containing Leu-Ala-Arg and Leu-Gly-Arg, which have the same sequence as the cleavage sites of C3 and C5, respectively. The best substrates for C2 and C2a were Z-Gly-Leu-Ala-Arg-SBzl and Z-Leu-Gly-Leu-Ala-Arg-SBzl, respectively, where Bzl is benzyl. B was the least reactive among these complement enzymes. The best substrate for B was Z-Lys-Arg-SBzl with a kcat/Km value of 1370 M-1 s-1. The catalytic fragment of B, Bb, had higher activity toward these peptide thioester substrates. The best substrate for Bb was Z-Gly-Leu-Ala-Arg-SBzl with a kcat/Km similar to C2a and 10 times higher than the value for B. Both C2a and Bb were considerably more reactive against C3-like than C5-like substrates. Bovine trypsin hydrolyzed thioester substrates with kcat/Km approximately 10(3) higher than the complement enzymes. These thioester substrates for D, B, and C2 should be quite useful in kinetic and active site studies of the purified enzymes.     

Cutting edge: impairment of dendritic cells and adaptive immunity by Ebola and Lassa viruses

Acute infection of humans with Ebola and Lassa viruses, two principal etiologic agents of hemorrhagic fevers, often results in a paradoxical pattern of immune responses: early infection, characterized by an outpouring of inflammatory mediators such as TNF-alpha, IL-1 beta, and IL-6, vs late stage infections, which are associated with poor immune responses. The mechanisms underlying these diverse outcomes are poorly understood. In particular, the role played by cells of the innate immune system, such as dendritic cells (DC), is not known. In this study, we show that Ebola and Lassa viruses infect human monocyte-derived DC and impair their function. Monocyte-derived DC exposed to either virus fail to secrete proinflammatory cytokines, do not up-regulate costimulatory molecules, and are poor stimulators of T cells. These data represent the first evidence for a mechanism by which Ebola and Lassa viruses target DC to impair adaptive immunity.     

A review of the Pterogeniidae (Goleoptera)

The Pterogeniidae, a family of beetles from Indoaustralia, are revised. They comprise five genera and 24 species. Three genera and 17 species are described as new and one species is synonymized. It is shown that the male and particularly the female genitalia provide useful means for species definition. The phylogenetic relationships are discussed based on a cladistic analysis of 23 morphological characters using PAUP. The analysis resulted in a single cladogram with following grouping: ( Kryptogenius + ( Tychogenius + ( Katagenius + ( Plerogenins + Histanocerus )))). For rooting the cladogram and polarizing the characters, Sivacrypticus indicus (Archeocrypticidae) was used as an outgroup. The majority of the species is restricted to insular tropical Asia and Oceania but four of them extend their range onto the Malayan Peninsula. Another four species are known only from continental Asia, i.e. two species from South India and one each from Malayan Peninsula and Vietnam respectively. Species of Kryptogenius, Pterogenius, Katagenius and Tychogenius are highly endemic and could therefore potentially be useful for analysing areas of endemism. For this, however, the cladistic relationships should be resolved at species level. Species of Histanocerus are more widely distributed but none is found on both sides of Wallace's line.     

Habitat barriers limit gene flow and illuminate historical events in a wide-ranging carnivore, the American puma

We examined the effects of habitat discontinuities on gene flow among puma (Puma concolor) populations across the southwestern USA. Using 16 microsatellite loci, we genotyped 540 pumas sampled throughout the states of Utah, Colorado, Arizona, and New Mexico, where a high degree of habitat heterogeneity provides for a wide range of connective habitat configurations between subpopulations. We investigated genetic structuring using complementary individual- and population-based analyses, the latter employing a novel technique to geographically cluster individuals without introducing investigator bias. The analyses revealed genetic structuring at two distinct scales. First, strikingly strong differentiation between northern and southern regions within the study area suggests little migration between them. Second, within each region, gene flow appears to be strongly limited by distance, particularly in the presence of habitat barriers such as open desert and grasslands. Northern pumas showed both reduced genetic diversity and greater divergence from a hypothetical ancestral population based on Bayesian clustering analyses, possibly reflecting a post-Pleistocene range expansion. Bayesian clustering results were sensitive to sampling density, which may complicate inference of numbers of populations when using this method. The results presented here build on those of previous studies, and begin to complete a picture of how different habitat types facilitate or impede gene flow among puma populations.     

A genetic technique to identify the diet of cownose rays,Rhinoptera bonasus: analysis of shellfish prey items from North Carolina and Virginia

Cownose rays are implicated in the consumption of commercially important shellfish on the U.S. East Coast. We tested this assumption by developing a molecular technique for species identification from cownose ray gut contents. Digestive tracts sampled from 33 rays in Pamlico Sound, NC and Chesapeake Bay, VA contained pieces of partially-digested tissue, well-digested tissue, fluid, and minute shell fragments which made visual identification to the species level nearly impossible. We sequenced the cytochrome oxidase subunit I (COI) for seven locally acquired bivalve species, chosen for their commercial and ecological importance in NC and VA. Sequences were used to design species-specific primers for each bivalve species to amplify polymerase chain reaction (PCR) products. We designed primers such that PCR products were sufficiently different in size to be distinguishable from one another when resolved on an agarose gel, and multiplexing of several species in one reaction was possible. Digestive tract sample testing revealed that cownose rays in Chesapeake Bay ate stout tagelus and soft shell clams. There was no evidence of the rays in the study consuming commercially important oysters, hard clams, and bay scallops. Further sampling over an extended period of time and additional locations is required to confirm these results. Our diagnostic tests could easily be expanded to elucidate the impact of cownose ray predation on prey populations.     

Osmolyte controlled fibrillation kinetics of insulin: New insight into fibrillation using the preferential exclusion principle

Amyloid proteins are converted from their native‐fold to long β‐sheet‐rich fibrils in a typical sigmoidal time‐dependent protein aggregation curve. This reaction process from monomer or dimer to oligomer to nuclei and then to fibrils is the subject of intense study. The main results of this work are based on the use of a well‐studied model amyloid protein, insulin, which has been used in vitro by others. Nine osmolyte molecules, added during the protein aggregation process for the production of amyloid fibrils, slow‐down or speed up the process depending on the molecular structure of each osmolyte. Of these, all stabilizing osmolytes (sugars) slow down the aggregation process in the following order: tri > di > monosaccharides, whereas destabilizing osmolytes (urea, guanidium hydrochloride) speed up the aggregation process in a predictable way that fits the trend of all osmolytes. With respect to kinetics, we illustrate, by adapting our earlier reaction model to the insulin system, that the intermediates (trimers, tetramers, pentamers, etc.) are at very low concentrations and that nucleation is orders of magnitude slower than fibril growth. The results are then collated into a cogent explanation using the preferential exclusion and accumulation of osmolytes away from and at the protein surface during nucleation, respectively. Both the heat of solution and the neutral molecular surface area of the osmolytes correlate linearly with two fitting parameters of the kinetic rate model, that is, the lag time and the nucleation rate prior to fibril formation. These kinetic and thermodynamic results support the preferential exclusion model and the existence of oligomers including nuclei and larger structures that could induce toxicity. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009