Template-Assisted Enzymatic Synthesis of Oligopeptides from a Polylactide Chain.

Peptides are often attached to polymer materials, as bioactive components, for the control of interactions between the material and its surrounding proteins and cells. However, synthesizing peptides and attaching them to polymers can be challenging and laborious. Herein, we describe the grafting of oligopeptides to an aliphatic polyester, using a one-step chemo-enzymatic synthesis with papain as the biocatalyst.

Short one-pot chemo-enzymatic synthesis of L-lysine and L-alanine diblock co-oligopeptides.

Amphiphilic diblock co-oligopeptides are interesting and functional macromolecular materials for biomedical applications because of their self-assembling properties. Here, we developed a synthesis method for diblock co-oligopeptides by using chemo-enzymatic polymerization, which was a relatively short (30 min) and efficient reaction (over 40% yield). Block and random oligo(L-lysine-co-L-alanine) [oligo(Lys-co-Ala)] were synthesized using activated papain as enzymatic catalyst.

Ultrastructural analysis in preclinical safety evaluation.

The first electron microscopic images of biological specimens were made in the 1940s, and the next 30 years comprised an era of descriptive ultrastructure during which transmission electron microscopy (TEM) was integral to an explosion in cellular and molecular biology. However, when questions could no longer be answered by ultrastructural information alone, the use of TEM in biological research declined. Innovative molecular techniques and newer imaging technologies such as confocal fluorescence microscopy filled the gap, providing faster answers with less rigorous training as a prerequisite to data collection.

The role of the toxicologic pathologist in the biopharmaceutical industry.

Toxicologic pathologists contribute significantly to the development of new biopharmaceuticals, yet there is often a lack of awareness of this specialized role. As the members of multidisciplinary teams, toxicologic pathologists participate in all aspects of the drug development process. This review is part of an initiative by the Society of Toxicologic Pathology to educate scientists about toxicologic pathology and to attract junior scientists, veterinary students, and veterinarians into the field.

Asymptomatic macrothrombocytopenia in a young pure-bred beagle dog: a case report.

During baseline evaluation prior to a preclinical safety study, a 10-month-old male pure-bred Beagle dog was found to have marked thrombocytopenia (6 × 10(3) platelets [PLT]/µL) associated with a mean platelet volume (MPV) of 17.9 fL. Tests for Rickettsia rickettsii, Ehrlichia canis, and Borrelia burgdorferi were negative.

A quantitation method for mass spectrometry imaging.

A new quantitation method for mass spectrometry imaging (MSI) with matrix-assisted laser desorption/ionization (MALDI) has been developed. In this method, drug concentrations were determined by tissue homogenization of five 10 µm tissue sections adjacent to those analyzed by MSI. Drug levels in tissue extracts were measured by liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS).

A rationale for determining, testing, and controlling specific impurities in pharmaceuticals that possess potential for genotoxicity.

The synthesis of pharmaceutical products frequently involves the use of reactive reagents and the formation of intermediates and by-products. Low levels of some of these may be present in the final drug substance and drug product as impurities. Such chemically reactive impurities may have at the same time the potential for unwanted toxicities including genotoxicity and carcinogenicity and hence can have an impact on product risk assessment.

Isolated human hepatocytes in culture display markedly different gene expression patterns depending on attachment status.

In vitro human hepatocyte cultures are a key tool in the investigation of xenobiotic toxicity and metabolism. In most in vitro hepatocyte studies, the cells are allowed to adhere to an extracellular matrix, such as collagen. Unfortunately, the ability of freshly isolated hepatocytes to adhere to collagen varies from donor to donor.

Analysis of two matrix metalloproteinase inhibitors and their metabolites for induction of phospholipidosis in rat and human hepatocytes(1).

ABT-770 [(S)-N-[1-[[4'-trifluoromethoxy-[1,1'-biphenyl]-4-yl]oxy]methyl-2-(4,4-dimethyl-2,5-dioxo-1-imidazolidinyl)ethyl]-N-hydroxyformamide], a matrix metalloproteinase inhibitor (MMPI), produced generalized phospholipidosis in rats. Phospholipid accumulation was accompanied by retention of drug-related material and was associated with increased mortality. Generation of a successful drug candidate depended upon understanding the cause of the phospholipidosis and redesigning the chemical structure accordingly.

Potent inhibition of NFAT activation and T cell cytokine production by novel low molecular weight pyrazole compounds.

NFAT (nuclear factor of activated T cell) proteins are expressed in most immune system cells and regulate the transcription of cytokine genes critical for the immune response. The activity of NFAT proteins is tightly regulated by the Ca(2+)/calmodulin-dependent protein phosphatase 2B/calcineurin (CaN). Dephosphorylation of NFAT by CaN is required for NFAT nuclear localization.

Tenascin synthesis, deposition, and isoforms in monocrotaline-induced pulmonary hypertensive rat lungs.

Monocrotaline (MCT)-induced pulmonary hypertension is characterized by alterations in vascular extracellular matrix and neomuscularization of small blood vessels. Tenascin (TN) is a matrix glycoprotein which modulates cellular attachment, proliferation, and migration. The present study used immunohistochemistry and Northern analyses to examine the hypothesis that treatment of rats with the potent pneumotoxin MCT induces temporal alterations in TN synthesis/deposition in the affected lungs.

Basic fibroblast growth factor alterations during development of monocrotaline-induced pulmonary hypertension in rats.

The chemical signaling pathways which orchestrate lung cell responses in hypertensive pulmonary vascular disease are poorly understood. The present study examined temporal alterations in lung basic Fibroblast Growth Factor (bFGF) in a well characterized rat model of monocrotaline (MCT)-induced pulmonary hypertension. By immunohistochemical analysis, there were progressive increases in bFGF in airway, vascular and gas exchange regions of MCT-treated rat lungs.

Monocrotaline alters type II pneumocyte morphology and polyamine regulation.

Lungs from monocrotaline (MCT)-treated rats exhibit altered polyamine metabolism and content. One of the prominent morphological abnormalities in MCT-treated lungs is a decrease in population density of type II pneumocytes. Against this background, the present study tested the hypothesis that failure to maintain normal population density of type II pneumocytes is associated with MCT-induced derangements in polyamine biosynthesis and/or transmembrane polyamine transport.

Uptake of ferritin and Bordetella avium in bronchus-associated lymphoid tissue of turkeys.

The uptake of macromolecular and particulate materials in bronchus-associated lymphoid tissue (BALT) in turkeys was examined using transmission electron microscopy. Tracer materials used were live and ultraviolet-killed (UV-killed) Bordetella avium and ferritin. Suspensions of bacteria and ferritin were instilled via intratracheal catheterization and allowed to remain in contact with the respiratory surfaces for 0, 10, 30, 60, 90, and 120 min.

Morphogenesis of enteric lesions induced by group D rotavirus in ringneck pheasant chicks (Phasianus colchicus).

Eight day-old male and female ringneck pheasants (Phasianus colchicus) were inoculated with group D rotavirus and necropsied at 4, 7, and 11 days post-inoculation. The intestinal tracts were examined by light and electron microscopic and immunohistochemical methods. By 4 days post-inoculation, 2/3 (66%) inoculated birds were stunted and had diarrhea and dilated intestines.

Structure and development of bronchus-associated lymphoid tissue in conventionally reared broiler chickens.

The development of bronchus-associated lymphoid tissue (BALT) in conventionally reared broiler chickens of 1 day and 1, 2, 3, 4, 6, and 8 weeks of age was studied using light and electron microscopy (scanning and transmission). BALT in these chickens resembled other mucosa-associated lymphoid tissues (MALT) in that it was composed of an altered epithelium overlying a population of lymphocytes and contained potential antigen-presenting cells, such as macrophages and dendritic cells; high endothelial venules were also present. In contrast to other MALT, epithelial cells in chicken BALT were not of the M-cell type; i.

Distribution and quantitation of plasma cells, T lymphocyte subsets, and B lymphocytes in bronchus-associated lymphoid tissue of chickens: age-related differences.

Development of the lymphoid cell compartment in bronchus-associated lymphoid tissue (BALT) of specific pathogen free chickens was examined. Specifically, B lymphocytes, T cell subsets (CD4 and CD8), and IgA-, IgG-, and IgM-producing plasma cells were labeled using immunocytochemical methods. Immunoglobulin-producing cells (IgPC) were quantitated, and comparisons of IgPC numbers were made among chickens of different ages, among immunoglobulin isotypes, and between lymphoid (BALT) and nonlymphoid (non-BALT) areas in the primary bronchus.

Long-segmented filamentous organisms observed in poults experimentally infected with stunting syndrome agent.

One-day-old turkeys were inoculated per os with material shown previously to induce stunting syndrome (SS). Weight gain and feed efficiency of inoculated poults from 1 to 13 days of age were impaired (P less than 0.01) compared with uninoculated poults.

A morphologic study of bronchus-associated lymphoid tissue in turkeys.

Bronchus-associated lymphoid tissue (BALT) in normal turkeys of ages 1 day and 1, 2, 3, 4, 8, and 18 weeks was examined by light microscopy and by scanning and transmission electron microscopy. Turkey BALT resembled other mucosa-associated lymphoid tissues; it was made up of a population of lymphocytes covered by a specialized epithelium different from typical pseudostratified ciliated columnar bronchial epithelium. There were distinct age-related differences in BALT structure.

Cultivation and partial characterization of bovine astrovirus.

Bovine astrovirus serotype 2 (US2) was adapted to primary neonatal kidney cell (NBK) cultures by the addition of 50 micrograms ml-1 of trypsin in the medium. Infectious virus was released from the cells within 7 days post-infection in early passages and within 3 days in later passages. In the absence of trypsin, neither passage of infected cells nor release of infectious virus occurred.

A comparison of outer membrane proteins and surface characteristics of adhesive and non-adhesive phenotypes of Bordetella avium.

The outer membrane protein profiles of four adherent and one reduced-adherence mutant phenotype of Bordetella avium were compared; a non-adherent B. avium-like organism isolated from turkeys was also examined. The organisms were grown on brain-heart infusion agar at 35 C for 36 hours.

Ultrastructural pathology of Bordetella avium infection in turkeys.

One-day-old turkeys were infected intranasally with Bordetella avium, and tracheas were examined by scanning and transmission electron microscopy at 1 to 5 weeks post-inoculation (PI). The predominant ultrastructural lesions were progressive loss of ciliated epithelium with replacement by nonciliated cells, bacterial colonization of ciliated cells, membrane-bound crystalline inclusions in cytoplasma of epithelial cells, depletion of mucous granules, and distortion of tracheal rings and the mucosal surface. Tracheal surface exudates consisted of mucus, necrotic cells, heterophils, and fibrin.

Ultrastructure and stereology of leukocytes and platelets of normal foxes and a fox with a Chediak-Higashi-like syndrome.

Peripheral blood leukocytes and platelets from five normal foxes (Vulpes vulpes) and a fox with phenotypical characteristics of Chediak-Higashi syndrome (CHS) were examined by electron microscopy. Lymphocytes, monocytes, neutrophils, eosinophils, and platelets from the affected fox contained giant membrane-bound granules that resembled lysosomes. In eosinophils and neutrophils from the affected fox and a normal fox, relative cell volume occupied by granules and number of granules per unit area were calculated.

A morphological study of the replication of Breda virus (proposed family Toroviridae) in bovine intestinal cells.

The morphological aspects of Breda virus serotype 2 replication in intestinal cells of gnotobiotic calves were investigated by electron microscopy. Ultrastructural findings suggest a morphogenetic pathway involving cytoplasmic vesicles, the Golgi apparatus and the cell nucleus. Virus uptake probably occurs via a receptor-mediated endocytosis-like mechanism.

Astrovirus and Breda virus infections of dome cell epithelium of bovine ileum.

A bovine enteric virus antigenically related to the United Kingdom isolate of bovine astrovirus was isolated from diarrheic feces, also containing rotavirus, of a calf in Florida. The astrovirus infected cell cultures and the epithelial cells of domes in the ileum, and there was cross-immunofluorescence with antiserum to the United Kingdom astrovirus. Calves infected with astrovirus alone did not develop clinical disease, but when astrovirus was mixed with rotavirus or Breda virus 2, the calves developed severe diarrhea and more extensive astrovirus infection of the dome epithelium.