Roles of male reproductive tract extracellular vesicles in reproduction.

Extracellular vesicles (EVs) are secreted cell-derived membrane structures present in all organisms across animal, bacterial, and plant phyla. These vesicles play important roles in cell-cell communication in many processes integral to health and disease. Recent studies demonstrate that EVs and their cargo have influential and conserved roles in male reproduction. While EVs have been isolated from virtually all specialized tissues comprising the male reproductive tract, they are best characterized in the epididymis (epididymosomes) and seminal fluid (seminal fluid extracellular vesicles or prostasomes). Broadly speaking, EVs promote reproductive success through supporting sperm development and function, as well as influencing the physiology of female reproductive tract cells after mating. In this review, we present current knowledge on the composition and function of male reproductive tract EV populations in both normal physiology and pathology, and argue that their functions identify them as critical regulators of fertility and fecundity.

Deconstructing compassionate conservation.

Compassionate conservation focuses on 4 tenets: first, do no harm; individuals matter; inclusivity of individual animals; and peaceful coexistence between humans and animals. Recently, compassionate conservation has been promoted as an alternative to conventional conservation philosophy. We believe examples presented by compassionate conservationists are deliberately or arbitrarily chosen to focus on mammals; inherently not compassionate; and offer ineffective conservation solutions. Compassionate conservation arbitrarily focuses on charismatic species, notably large predators and megaherbivores. The philosophy is not compassionate when it leaves invasive predators in the environment to cause harm to vastly more individuals of native species or uses the fear of harm by apex predators to terrorize mesopredators. Hindering the control of exotic species (megafauna, predators) in situ will not improve the conservation condition of the majority of biodiversity. The positions taken by so-called compassionate conservationists on particular species and on conservation actions could be extended to hinder other forms of conservation, including translocations, conservation fencing, and fertility control. Animal welfare is incredibly important to conservation, but ironically compassionate conservation does not offer the best welfare outcomes to animals and is often ineffective in achieving conservation goals. Consequently, compassionate conservation may threaten public and governmental support for conservation because of the limited understanding of conservation problems by the general public.

Deconstrucción de la Conservación Compasiva Resumen La conservación compasiva se enfoca en cuatro principios: no causar daño; los individuos importan; la integración de los animales individualmente; y la coexistencia pacífica entre los humanos u los animales. Recientemente, la conservación compasiva ha sido promovida como una alternativa a la filosofía convencional de la conservación. Creemos que los ejemplos presentados por los conservacionistas compasivos han sido elegidos arbitraria o deliberadamente por estar enfocados en los mamíferos; por ser inherentes y no compasivos; y por ofrecer soluciones de conservación poco efectivas. La conservación compasiva se enfoca arbitrariamente en las especies carismáticas, principalmente los grandes depredadores y los megaherbívoros. La filosofía no es compasiva cuando deja que los depredadores invasores dentro del ambiente causen daño a un vasto número de individuos nativos o usa el miedo al daño por superdepredadores para aterrorizar a los mesodepredadores. El entorpecimiento del control de especies exóticas (megafauna, depredadores) in situ no mejorará las condiciones de conservación de la mayoría de la biodiversidad, incluso si los conservacionistas compasivos no dañan a los individuos exóticos. Las posiciones que toman los llamados conservacionistas compasivos sobre especies particulares y sobre las acciones de conservación podrían extenderse para entorpecer otros tipos de conservación, incluyendo las reubicaciones, el encercado para la conservación y el control de la fertilidad. El bienestar animal es increíblemente importante para la conservación e irónicamente, la conservación compasiva no ofrece los mejores resultados de bienestar para los animales y comúnmente es poco efectiva en el logro de los objetivos de conservación. Como consecuencia, la conservación compasiva puede poner en peligro el apoyo público y del gobierno que tiene la conservación debido al entendimiento poco limitado que tiene el público general sobre los problemas de conservación.

Major pathologic response on biopsy (MPRbx) in patients with advanced melanoma treated with anti-PD-1: evidence for an early, on-therapy biomarker of response.

BACKGROUND: With increasing anti-PD-1 therapy use in patients with melanoma and other tumor types, there is interest in developing early on-treatment biomarkers that correlate with long-term patient outcome. An understanding of the pathologic features of immune-mediated tumor regression is key in this endeavor. MATERIALS AND METHODS: Histologic features of immune-related pathologic response (irPR) following anti-PD-1 therapy were identified on hematoxylin and eosin (H&E)-stained slides in a discovery cohort of pre- and on-treatment specimens from n = 16 patients with advanced melanoma. These features were used to generate an irPR score [from 0 = no irPR features to 3 = major pathologic response on biopsy (MPRbx, ≤10% residual viable tumor)]. This scoring system was then tested for an association with objective response by RECIST1.1 and overall survival in a prospectively collected validation cohort of pre- and on-treatment biopsies (n = 51 on-treatment at 4-week timepoint) from melanoma patients enrolled on the nivolumab monotherapy arm of CA209-038 (NCT01621490). RESULTS: Specimens from responders in the discovery cohort had features of immune-activation (moderate-high TIL densities, plasma cells) and wound-healing/tissue repair (neovascularization, proliferative fibrosis) compared to nonresponders, (P ≤ 0.021, for each feature). In the validation cohort, increasing irPR score associated with objective response (P = 0.009) and MPRbx associated with increased overall survival (n = 51; HR 0.13; 95%CI, 0.054-0.31, P = 0.015). Neither tumoral necrosis nor pretreatment histologic features were associated with response. Eight of 16 (50%) of patients with stable disease showed irPR features, two of which were MPRbx, indicating a disconnect between pathologic and radiographic features at the 4-week on-therapy timepoint for some patients. CONCLUSIONS: Features of immune-mediated tumor regression on routine H&E-stained biopsy slides from patients with advanced melanoma correlate with objective response to anti-PD-1 and overall survival. An on-therapy biopsy may be particularly clinically useful for informing treatment decisions in patients with radiographic stable disease. This approach is inexpensive, straightforward, and widely available.

Pathologic features of response to neoadjuvant anti-PD-1 in resected non-small-cell lung carcinoma: a proposal for quantitative immune-related pathologic response criteria (irPRC).

Background: Neoadjuvant anti-PD-1 may improve outcomes for patients with resectable NSCLC and provides a critical window for examining pathologic features associated with response. Resections showing major pathologic response to neoadjuvant therapy, defined as ≤10% residual viable tumor (RVT), may predict improved long-term patient outcome. However, %RVT calculations were developed in the context of chemotherapy (%cRVT). An immune-related %RVT (%irRVT) has yet to be developed. Patients and methods: The first trial of neoadjuvant anti-PD-1 (nivolumab, NCT02259621) was just reported. We analyzed hematoxylin and eosin-stained slides from the post-treatment resection specimens of the 20 patients with non-small-cell lung carcinoma who underwent definitive surgery. Pretreatment tumor biopsies and preresection radiographic 'tumor' measurements were also assessed. Results: We found that the regression bed (the area of immune-mediated tumor clearance) accounts for the previously noted discrepancy between CT imaging and pathologic assessment of residual tumor. The regression bed is characterized by (i) immune activation-dense tumor infiltrating lymphocytes with macrophages and tertiary lymphoid structures; (ii) massive tumor cell death-cholesterol clefts; and (iii) tissue repair-neovascularization and proliferative fibrosis (each feature enriched in major pathologic responders versus nonresponders, P < 0.05). This distinct constellation of histologic findings was not identified in any pretreatment specimens. Histopathologic features of the regression bed were used to develop 'Immune-Related Pathologic Response Criteria' (irPRC), and these criteria were shown to be reproducible amongst pathologists. Specifically, %irRVT had improved interobserver consistency compared with %cRVT [median per-case %RVT variability 5% (0%-29%) versus 10% (0%-58%), P = 0.007] and a twofold decrease in median standard deviation across pathologists within a sample (4.6 versus 2.2, P = 0.002). Conclusions: irPRC may be used to standardize pathologic assessment of immunotherapeutic efficacy. Long-term follow-up is needed to determine irPRC reliability as a surrogate for recurrence-free and overall survival.

Trap Height Affects Capture of Lady Beetles (Coleoptera: Coccinellidae) in Pecan Orchards.

There is scarce information regarding the vertical stratification of predaceous Coccinellidae in tall trees. Although numerous studies have been done in orchards and forests, very few studies have assessed the occurrence of predaceous Coccinellidae high in tree canopies. The objective of this study was to examine the abundance of Coccinellidae at different heights in mature pecan, Carya illinoinensis (Wangenh.) K. Koch, orchards with tall trees. From spring through late fall during 2013 and 2014, yellow pyramidal Tedders traps were suspended in the pecan canopy at 6.1 and 12.2 m, in addition to being placed on the ground (0 m). The exotic species Harmonia axyridis and Coccinella septempunctata accounted for a high percentage of trap capture during this study. Except for Olla v-nigrum, low numbers of native species (Hippodamia convergens, Coleomegilla maculata, Cycloneda munda, Scymnus spp., and Hyperaspis spp.) were captured. However, significantly more were captured in ground traps rather than in canopy traps with the exception of O. v-nigrum. Similar to most native species, significantly more C. septempunctata were captured in ground traps than canopy traps. This contrasts sharply with H. axyridis captured similarly at all trap heights. The ability to exploit resources across vertical strata, unlike many intraguild predators, may be an underestimated factor helping to explain the invasiveness of H. axyridis.

Trap Cropping Systems and a Physical Barrier for Suppression of Stink Bugs (Hemiptera: Pentatomidae) in Cotton.

Euschistus servus (Say), Nezara viridula (L.), and Chinavia hilaris (Say) (Hemiptera: Pentatomidae) are economic pests of cotton in the coastal plain of the southeastern United States. The objective of this 2-yr study was to determine the ability of trap cropping systems, pheromone-baited stink bug traps, and a synthetic physical barrier at the peanut-to-cotton interface to manage stink bugs in cotton. The physical barrier was the most effective management tactic. Stink bug density in cotton was lowest for this treatment. In 2010, boll injury was lower for the physical barrier compared to the other treatments except for soybean with stink bug traps. In 2011, boll injury was lower for this treatment compared to the control. Soybean was an effective trap crop, reducing both stink bug density in cotton and boll injury regardless if used alone or in combination with either stink bug traps or buckwheat. Incorporation of buckwheat in soybean enhanced parasitism of E. servus egg masses by Telenomus podisi Ashmead in cotton. The insertion of eyelets in the lid of the insect-collecting device of a stink bug trap allowed adult stink bug parasitoids, but not E. servus, to escape. Stand-alone stink bug traps were not very effective in deterring colonization of cotton by stink bugs or reducing boll injury. The paucity of effective alternative control measures available for stink bug management justifies further full-scale evaluations into these management tactics for control of these pests in crops.

Effect of Lures and Colors on Capture of Lady Beetles (Coleoptera: Coccinellidae) in Tedders Pyramidal Traps.

Purposeful attraction and aggregation of adult Coccinellidae at target sites would be useful for sampling purposes and pest suppression. We field-tested 1) lures in yellow and black pyramidal traps and 2) pyramidal traps that had been painted one or two colors (without lures) to determine if lures or trap color affected capture of adult Coccinellidae. In only one experiment with lures did a single rate of limonene increase trap capture, whereas no other lure ever did. Yellow traps, regardless of using a lure, always captured significantly more lady beetles than black traps. When single-color red, orange, yellow, green, blue, purple, black, and white traps (without lures) were tested, yellow traps captured significantly more lady beetles. Of all species of Coccinellidae captured in these single-color traps, 95% were the exotic species Harmonia axyridis (Pallas) and Coccinella septempunctata L. H. axyridis alone dominated trap capture comprising 74.1% of all lady beetles. Two-color traps (yellow-green, yellow-orange, yellow-white, and yellow-black) never captured more than single-color yellow traps. These results demonstrate that yellow pyramidal traps can be used to purposefully attract, and when used without a collection device, possibly aggregate adult Coccinellidae at targeted field sites.

Effect of field edges on dispersal and distribution of colonizing stink bugs across farmscapes of the southeast USA.

Stink bugs (Heteroptera: Pentatomidae), including Nezara viridula (L.), Euschistus servus (Say), and Chinavia hilaris (Say), are economic pests in farmscapes where they move within and between closely associated crop and non-crop habitats. Thus, field edges in these farmscapes include not only crop-to-crop interfaces but also those edges adjoining non-crop habitats. We examined the influence of field edges on colonization of stink bugs in southeastern USA farmscapes composed of typical combinations of corn, peanut, and cotton. For E. servus and N. viridula, egg-to-adult development and presence of both sexes on all crops indicated that the crops served as reproductive plants. Adult C. hilaris were rarely found on corn and on crops associated with it, and they were present mainly in cotton in peanut-cotton farmscapes. Mature crop height was significantly higher for corn than for cotton and significantly higher for cotton over peanut, and an edge effect in dispersal of stink bugs into a crop was detected up to 4.6, 8.2, and 14.6 m from the crop-to-crop interface in corn, cotton, and peanut, respectively. These results suggest that stink bug dispersal into a crop decreases as crop height increases. The first stink bug-infested crop at the crop-to-crop interface was the most significant contributor of colonizing stink bugs to an adjacent crop. An edge effect in dispersal of stink bug adults was detected in corn next to non-woodlands and woodlands and in cotton adjacent to woodlands. Edge effects were never detected in side edges of peanut. Overall, our results indicate that both plant height and host plant suitability can influence edge-mediated dispersal of stink bugs at field edges.

Reliability and validity of the velotron racermate cycle ergometer to measure anaerobic power.

This study assessed the reliability and validity of the Velotron Racermate™ cycle ergometer to assess anaerobic power. Men (9 cyclists and 13 recreationally-active) and women (17 recreationally-active and 1 cyclist) (age=24.7±4.2 yr) performed 2 Wingate tests on the Velotron or 3 Wingate tests (2 on the Velotron and 1 on the Monark Peak Bike) over a 7-14 day period. Peak power, mean power, minimum power, fatigue index, heart rate, and peak and minimum cadence were assessed. Results revealed significant test-retest reliability for mean power (r=0.90, p<0.01), minimum power (r=0.79, p<0.05) and peak power (r=0.70, p<0.05) with repeated bouts on the Velotron. Peak power was significantly higher (p<0.05) on the Velotron (9.95±1.39 W/kg) vs. the Monark (9.13±1.26 W/kg); however, mean power was higher (p<0.05) on the Monark (6.95±0.89 W/kg) vs. the Velotron (6.11±0.52 W/kg and 6.25±0.59 W/kg). Data reveal significant reliability for mean and peak power from the Velotron Racermate, yet multiple variables differ between the Velotron and the Monark mechanically-braked cycle ergometer.

Laboratory virulence and orchard efficacy of entomopathogenic nematodes against the lesser peachtree borer (Lepidoptera: Sesiidae).

The lesser peachtree borer, Synanthedon pictipes (Grote & Robinson) (Lepidoptera: Sesiidae), is indigenous to eastern North America. It is a pest of commercially grown Prunus spp., especially to southeastern peach orchards where earlier regulatory changes affected pesticide use on peach leading to increased S. pictipes damage. Pest management practices are now having a positive effect toward control of this pest, but cost-competitive biological control solutions that promote environmental stewardship are needed. Here, we tested four Steinernema species and five Heterorhabditis species of entomopathogenic nematodes against larval S. pictipes. Included were four strains of S. carpocapsae (All, DD136, Sal, and Hybrid2) and three strains of S. riobrave (3-8b, 7-12, and 355). Larvae treated with any strain of S. carpocapsae always resulted in <20% survival, whereas larval survival was always >50% when treated with any other Steinernema or Heterorhabditis spp. These differences were always significant for the Hybrid2 strain of S. carpocapsae and similarly for other tested S. carpocapsae strains except for when larvae were treated with the 3-8b strain of S. riobrave. In addition, we determined the susceptibility of different size S. pictipes larvae, because they occur simultaneously in orchards, and we found that larvae rated as "medium" and "large" were significantly more susceptible than "small" larvae. Last, we demonstrated that moisture-retaining covers (placed over S. pictipes-infested wounds on peach limbs) increased efficacy of nematode treatments against larval S. pictipes. Even when using highly virulent nematodes against S. pictipes, it is likely that an aboveground application will require an environmental modification to remain efficacious.

Lesser peachtree borer (Lepidoptera: Sesiidae) oviposition on Prunus germplasm.

The lesser peachtree borer, Synanthedon pictipes (Grote and Robinson) (Lepidoptera: Sesiidae), is a serious pest of peach, Prunus persica (L.) Batsch, across the southeastern United States. We examined oviposition by S. pictipes on field-grown Prunus scion and rootstock cultivars and two endemic Prunus spp. when sawn limbs, not roots, were assayed in the laboratory. A choice test compared oviposition on the peach scion 'Harvester', peach rootstock 'Guardian', plum×peach hybrid rootstock 'MP-29', and the plum hybrid rootstock 'Sharpe'. A significantly lower percentage of eggs occurred on limbs of Sharpe rootstock than other choices. A choice test using two endemic hosts, black cherry (P. serotina Ehrh.) and Chickasaw plum (P. angustifolia Marsh.), along with Sharpe rootstock, found a lower percentage of eggs on limbs of Sharpe than either endemic host. However, when only limbs of Sharpe and a decoy were used, almost all eggs were laid on Sharpe. Interestingly, when Harvester and Sharpe limbs were paired side by side, a higher percentage of eggs were recovered from the Harvester limb than from the Sharpe limb. An analysis of volatiles from Sharpe may identify why fewer eggs were laid on it. Because S. pictipes attacks host trees above ground and Sharpe rootstock on grafted trees grows below ground, this rootstock might be a management option against the congeneric, root-attacking peachtree borer, S. exitiosa (Say). Our results suggest that high budding a peach scion onto Sharpe rootstock, thus allowing the rootstock to serve as the trunk, warrants further investigation against S. exitiosa under orchard conditions.

Pheromone attraction and cross-attraction of Nezara, Acrosternum, and Euschistus spp. stink bugs (Heteroptera: Pentatomidae) in the field.

Detecting infestations of stink bugs (Heteroptera: Pentatomidae) using pheromones remains problematic, particularly so in the United States for the exotic stink bug, Nezara viridula L., and our native stink bug, Acrosternum hilare (Say). Therefore, we conducted a 2-yr on-farm study to examine the attractiveness and possible cross-attraction of the reported pheromones for N. viridula and A. hilare and those previously discovered for Euschistus servus (Say) and Plautia stali Scott to N. viridula, A. hilare, and E. servus. The attractiveness of selected pentatomid pheromones to tachinid parasitoids of stink bugs was also examined. We showed for the first time under field conditions that N. viridula can be trapped with its reported pheromone, a 3:1 trans- to cis-(Z)-alpha-bisabolene epoxide blend. In fact, attraction of N. viridula increased with higher pheromone doses. Traps baited with a 5:95 trans- to cis-(Z)-alpha-bisabolene epoxide blend, the reported male-produced A. hilare attractant pheromone, failed to attract significantly more A. hilare than did unbaited control traps. Instead A. hilare was significantly cross-attracted to the P. stali pheromone [methyl (E,E,Z)-2,4,6-decatrienoate]. The E. servus pheromone [methyl (E,Z)-2,4-decadienoate], either alone or in combination with P. stali pheromone, was more attractive to E. servus than to N. viridula, P. stali, or A. hilare pheromones. In general, tachinid parasitoids were found responsive to the male-specific volatiles of their known hosts, including the attractiveness of Trichopoda pennipes (F.) to sesquiterpenoid blends characteristic of A. hilare and N. viridula. A tachinid parasitoid of E. servus, Cylindromyia sp., seemed to be attracted to E. servus pheromone. In conclusion, our results indicate that stink bug traps baited with lures containing N. viridula pheromone blend, P. stali pheromone, and E. servus pheromone have the greatest potential for detecting populations of N. viridula, A. hilare, and E. servus, respectively, in diversified agricultural landscapes.

Influence of Prunus spp., peach cultivars, and bark damage on oviposition choices by the lesser peachtree borer (Lepidoptera: Sesiidae).

An examination of oviposition choices by the lesser peachtree borer, Synanthedon pictipes (Grote and Robinson) (Lepidoptera: Sesiidae), showed that wounded peach, Prunus persica (L.) Batsch, bark was attractive to females for oviposition. Females responded to bark that was injured mechanically (e.g., hammer blows, knife cuts, pruning wounds), infested by lesser peachtree borer larvae or injured by disease. In fact, there was no difference in female oviposition response to knife cut wounds and knife cut wounds infested with lesser peachtree borer larvae. Oviposition on wounded bark from three different high chill peach cultivars was similar and strongly suggests that the narrow genetic base of high chill peach cultivars grown in the southeastern United States has little inherent resistance to the lesser peachtree borer. In stark contrast, when provided different Prunus spp., i.e., exotic peach and the native species P. angustifolia and P. serotina, the exotic peach was highly preferred for oviposition by the native lesser peachtree borer.

Development and application of a competitive enzyme-linked immunosorbent assay for the detection of serum antibodies to porcine circovirus type 2.

We report the development of a competitive enzyme-linked immunosorbent assay (c-ELISA) for the detection of antibodies to porcine circovirus type 2 (PCV2), the agent associated with the recently described postweaning multisystemic wasting syndrome in pigs. At present, no method has been published describing a c-ELISA for the detection of antibodies to PCV2, and currently employed tests are impractical for use in some laboratories. The assay described here uses a cell culture isolate of porcine circovirus type 2 as antigen and a PCV2-specific monoclonal antibody as the competing reagent. Evaluation of the ELISA was performed by comparison with results obtained using an indirect immunofluorescent test on 484 sera from pig herds in the United Kingdom, Canada, France, and the USA and serial bleeds from pigs experimentally infected with porcine circoviruses. The sensitivity and specificity of the ELISA were determined as 99.58% and 97.14%, respectively, at 2 standard deviations (SD) from the mean or 95.81% and 100% at 3 SD from the mean. Using this ELISA, a serologic survey of 461 sera collected from commercial pig herds in Northern Ireland between 1973 and 1999 was undertaken. Analysis of the results of this survey demonstrated that the number of ELISA-positive sera detected in an individual year during this period ranged from 55% to 100%. This c-ELISA has applications for large-scale rapid diagnosis of PCV2 infection in pig populations worldwide and for immunoscreening of sera from other species for antibodies to PCV2.

Volatile components of roots, stems, leaves, and flowers of Echinacea species.

The headspace volatile components of roots, stems, leaves, and flowers of Echinacea angustifolia,E. pallida, and E. purpurea were analyzed by capillary gas chromatography/mass spectrometry (GC/MS). Over 70 compounds were identified in the samples. All plant tissues, irrespective of the species, contain acetaldehyde, dimethyl sulfide, camphene, hexanal, beta-pinene, and limonene. The main headspace constituents of the aerial parts of the plant are beta-myrcene, alpha-pinene, limonene, camphene, beta-pinene, trans-ocimene, 3-hexen-1-ol, and 2-methyl-4-pentenal. The major headspace components of root tissue are alpha-phellandrene (present only in the roots of E. purpurea and E.angustifolia), dimethyl sulfide, 2-methylbutanal, 3-methylbutanal, 2-methylpropanal, acetaldehyde, camphene, 2-propanal, and limonene. Aldehydes, particularly butanals and propanals, make up 41-57% of the headspace of root tissue, 19-29% of the headspace of the leaf tissue, and only 6-14% of the headspace of flower and stem tissues. Terpenoids including alpha- and beta-pinene, beta-myrcene, ocimene, limonene, camphene, and terpinene make up 81-91% of the headspace of flowers and stems, 46-58% of the headspace of the leaf tissue, and only 6-21% of the roots. Of the 70 compounds identified, >50 are reported in Echinacea for the first time.

Solving the complementarity dilemma: evolving strategies for simultaneous hermaphroditism.

We introduce a complementarity dilemma, a two-player, binary response game in which the playoffs are highest when the two players respond differently. Using the classifier EvA, we determine the evolutionary dynamics and structure of strategies that evolve to play an iterated version of this game, and we relate the results to the evolution of major types of sexual reproduction, particularly simultaneous hermaphroditism. We find that complementarity strategies consistently evolve under a broad range of conditions, but that those most consistent with the simultaneous hermaphroditism can predominate only when a substantial cost of repeatedly adopting the female role is imposed. The cost is analogous to the fecundity reduction to be expected when a single partner must repeatedly produce the eggs in sexual reproduction.

The sole lysine residue in porcine pepsin works as a key residue for catalysis and conformational flexibility.

Pepsin contains a single lysine residue which protrudes from the enzyme's surface, behind the active site cleft, on the C-terminal domain. Mutations of pepsin by site-directed mutagenesis of the Lys-319 residue were generated to study the structure-function relationships. Kinetic parameters, pH activity profiles, along with conformational analysis using circular dichroism (CD), and molecular modelling were examined for the wild-type (non-mutant) and mutant enzymes. The pepsin mutations, Lys-319-->Met and Lys-319-->Glu, resulted in a progressive increase in the Km and similar decrease in kcat, respectively, as well as being denatured at a lower pH than the wild-type pepsin. CD analysis indicated that mutations at Lys-319 resulted in changes in secondary structure fractions which were reflected in changes in enzymatic activity as compared to the wild-type pepsin, i.e. kinetic data and pH denaturation study. Molecular modelling of mutant enzymes indicated differences in flexibility in the flap loop region of the active site, the region around the entrance of the active site cleft, sub-site regions for peptide binding, and in the subdomains of the C-terminal domain when compared to the wild-type enzyme. The results suggest that Lys-319, which is distal to the active site, is important to the flexibility/stability of the enzyme, as well as to its catalytic machinery.

Palmitate incorporation in the lungs of dogs with granulomatous disease.

Moderate hypoxia did not influence the pulmonary incorporation of an intravenous dose of [1-14C]palmitate either in dogs with experimentally produced granulomatous disease or in normal controls. The lung weight in the diseased animals, was, on the average, double that of the controls. There was a proportionate increase in uptake of the radioactive label at 1 hr after infusion in the diseased lungs, hence the specific activity of labeled palmitate (counts per minute per gram of phospholipid) was no different in the two groups. Moreover, half the radioactivity of the phospholipids was recovered in palmitate separated from the phosphatidyl choline fraction in both diseased and normal lungs. Anatomic studies demonstrated increased numbers of Type II pneumocytes lining all alveolar air spaces in the diseased lung. Autoradiographic studies indicated the presence of labeled palmitate in the Type II cells, but not in the inflammatory cells of the granulomata. We conclude that the increased palmitate uptake in this disease is accounted for by the metabolic activity of the Type II pneumocytes.

The relationship between right duct lymph flow and extravascular lung water in dogs given alpha-naphthylthiourea.

The relationship between right duct lymph flow and extravascular lung water was studied in 3 normal dogs and 15 dogs with pulmonary edema induced by alpha-naphthylthiourea (ANTU). Right duct lymph was collected in a pouch created by ligating jugular, subclavian, and brachiocephalic veins. Extravascular lung water was measured in vivo by double indicator dilution and post-mortem by weighting lungs before and after drying. Cardiac output, pulmonary artery and pulmonary artery wedge pressures, and the concentration of protein and electrolytes in plasma and right duct lymph were determined. Eight lungs were examined by light and electron microscopy. There was a direct relationship between right duct lymph flow (RDLF in milliters per hour per gram dry lung) and extravascular lung water (Qwl in milliliters per gram dry lung) which was best described by the equation RDLF=0.75-0.26 Qwl+0.03 (Qwl).2 Dogs with severe ANTU-induced edema had extensive lung capillary endothelial destruction but only mild interstitial swelling and no visible damage to type I alveolar epithelial cells. Cardiac output, pulmonary artery and wedge pressures, and protein and electrolyte concentrations did not correlate with either extravascular water or right duct flow. Thus, in ANTU-induced pulmonary edema right duct lymph flow was directly related to extravascular lung water with the highest flows occurring with severe edema. The absence of a rapid increase in lymph flow with small increases in extravascular water may be due to early sequestration of fluid in the alveolar space. Hemodynamic changes did not account for changes in lung water or lymph flow. The pulmonary interstitial factors relating increased extravascular water to lymph drainage remain to be determined.