Immobilization of Escherichia coli cells by use of the antimicrobial peptide cecropin P1

An immobilization scheme for bacterial cells is described, in which the antimicrobial peptide cecropin P1 was used to trap Escherichia coli K-12 and O157:H7 cells on microtiter plate well surfaces. Cecropin P1 was covalently attached to the well surfaces, and E. coli cells were allowed to bind to the peptide-coated surface. The immobilized cells were detected colorimetrically with an anti-E. coli antibody-horseradish peroxidase conjugate. Binding curves were obtained in which the signal intensities were dependent upon the cell concentration and upon the amount of peptide attached to the well surface. After normalization for the amount of peptide coupled to the surface and the relative binding affinity of the antibody for each strain, the binding data were compared, which indicated that there was a strong preference for E. coli O157:H7 over E. coli K-12. The cells could be immobilized reproducibly at pH values ranging from 5 to 10 and at ionic strengths up to 0.50 M.     

Tracking extranigral degeneration in animal models of Parkinson's disease: quest for effective therapeutic strategies

Sporadic Parkinson's disease (PD) is now interpreted as a complex nervous system disorder in which the projection neurons are predominantly damaged. Such an interpretation is based on mapping of Lewy body and Lewy neurite pathology. Symptoms of the human disease are much widespread, which span from pre-clinical non-motor symptoms and clinical motor symptoms to cognitive discrepancies often seen in advanced stages. Existing symptomatic treatments further complicate with overt drug-irresponsive symptoms. PD is better understood by assimilation of extranigral degenerative pathways with nigrostriatal degenerative mechanisms. The term 'extranigral' appeared first in the 1990s to more rigorously define the nigral pathology by process of elimination. However, as clinicians progressively identified PD symptoms unresponsive to the gold standard drug l-DOPA, definitions of PD symptoms were redefined. Non-motor symptoms prodromal to motor symptoms just as pre-clinical to clinical, and conjointly emerged the concept of nigral versus extranigral degeneration in PD. While nigrostriatal degeneration is responsible for the neurobiological substrates of extrapyramydal motor features, extranigral degeneration corroborates a vast majority of other changes in discrete central, peripheral, and enteric nervous system nuclei, which together account for global symptoms of the human disease. As an extranigral site, spinal cord degeneration has also been implicated in PD progression. Interconnected to the upper CNS structures with descending and ascending pathways, spinal neurons participate in movement and sensory circuits, controlling movement and reflexes. Several clinical and in vivo studies have demonstrated signs of parkinsonism-related degenerative processes in spinal cord, which led to recent consideration of spinal cord as an area of potential therapeutic target. In a nutshell, this review explores how the existing animal models can actually reflect the human disease in order to facilitate PD research. Evolution of extranigral degeneration studies has been succinctly revisited, followed by a survey on animal models in light of recent findings in clinical PD. Together, it may help to develop effective therapeutic strategies for PD.     

Development of HIV-1 Rectal-Specific Microbicides and Colonic Tissue Evaluation

The gastrointestinal tract is structurally and functionally different from the vagina. Thus, the paradigm of topical microbicide development and evaluation has evolved to include rectal microbicides (RMs). Our interest was to create unique RM formulations to safely and effectively deliver antiretroviral drugs to mucosal tissue. RMs were designed to include those that spread and coat all surfaces of the rectum and distal colon rapidly (liquid) and those that create a deformable, erodible barrier and remain localized at the administration site (gel). Tenofovir (TFV) (1%) was formulated as an aqueous thermoreversible fluid and a carbopol-based aqueous hydrogel. Lipid-based liquid and gel formulations were prepared for UC781 (0.1%) using isopropyl myristate and GTCC (Caprylic/Capric Triglycerides), respectively. Formulations were characterized for pH, viscosity, osmolality, and drug content. Pre-clinical testing incorporated ex vivo colonic tissue obtained through surgical resections and flexible sigmoidoscopy (flex sig). As this was the first time using tissue from both sources side-by-side, the ability to replicate HIV-1 was compared. Efficacy of the RM formulations was tested by applying the products with HIV-1 directly to polarized colonic tissue and following viral replication. Safety of the formulations was determined by MTT assay and histology. All products had a neutral pH and were isoosmolar. While HIV-1_BaL and HIV-1_JR-CSF alone and in the presence of semen had similar replication trends between surgically resected and flex sig tissues, the magnitude of viral replication was significantly better in flex sig tissues. Both TFV and UC781 formulations protected the colonic tissue, regardless of tissue source, from HIV-1 and retained tissue viability and architecture. Our in vitro and ex vivo results show successful formulation of unique RMs. Moreover, the results of flex sig and surgically resected tissues were comparable suggesting the incorporation of both in pre-clinical testing algorithms.     

The relationship of diet to growth and ammonium excretion in salt marsh fish

Synopsis Detritus is an abundant but poor quality food source for consumers in salt marsh ecosystems. Here we present results of feeding experiments to determine the ability of Fundulus heteroclitus, Cyprinodon variegatus, and Mugil cephalus, three major detritivores in Great Sippewissett Marsh, Massachussetts, to assimilate detritus and use it for growth. C. variegatus, the sheepshead minnow, gained weight on a detrital diet, but F. heteroclitus, the marsh killifish, and juvenile M. cephalus, the striped mullet, lost weight and suffered high mortality on detrital diets. C. variegatus is a herbivore with morphological adaptations for ingesting plant material. F. heteroclitus is a carnivore poorly suited to effectively assimilate detritus from the diet. Although adult M. cephalus are adapted for ingesting and assimilating detrital material, the young may lack these adaptations and thus do not assimilate detritus. C. variegatus excretes ammonium at a lower rate than F. heteroclitus when fed animal food, or when starved for short periods of time. This protein sparing effect could be crucial for survival when detritus is the only food available at certain times of the year. While both C. variegatus and M. cephalus are visitors in the marsh and may leave to exploit food sources elsewhere, F. heteroclitus is a year-round resident and is the most abundant species of fish in the marsh. Yet when high quality food sources become scarce in late summer and fall, detritus, although plentiful, is apparently not a suitable alternative.To whom all correspondences should be addressed.     

Growth of Enveloped RNA Viruses in a Line of Chinese Hamster Ovary Cells with Deficient N-Acetyl-glucosaminyltransferase Activity

Sindbis and vesicular stomatitis viruses were grown in a line (termed 15B) of Chinese hamster ovary (CHO) cells that is deficient in a specific UDP-N-acetylglucosamine:glycoprotein N-acetylglucosaminyltransferase. Both viruses replicated normally in the cell line, but the glycoproteins of the released virus migrated faster on sodium dodecyl sulfate-polyacrylamide gels than did glycoproteins of virus grown in parent CHO cells. Digestion of the viral glycoproteins with Pronase followed by gel filtration demonstrated that the glycopeptides of Sindbis-15B virus were much smaller than the glycopeptides of Sindbis-CHO virus. In addition, Sindbis-15B viral glycopeptides but not Sindbis-CHO viral glycopeptides contained terminal α-mannose residues as shown by their susceptibility to α-mannosidase digestion. These findings demonstrate that the oligosaccharide units of the glycoproteins of vesicular stomatitis and Sindbis viruses are altered when the viruses are grown in 15B cells. We conclude that the N-acetylglucosaminyltransferase that is missing in 15B cells normally participates in the biosynthesis of the oligosaccharide units of the viral glycoproteins, and in the absence of this enzyme incomplete oligosaccharide chains are produced. Viruses released from 15B cells appear to retain full infectivity; Sindbis-15B virus, however, showed a significant decrease in hemagglutination titer compared with that of Sindbis-CHO virus.     

A genome-wide association study of osteochondritis dissecans in the Thoroughbred

Osteochondrosis is a developmental orthopaedic disease that occurs in horses, other livestock species, companion animal species, and humans. The principal aim of this study was to identify quantitative trait loci (QTL) associated with osteochondritis dissecans (OCD) in the Thoroughbred using a genome-wide association study. A secondary objective was to test the effect of previously identified QTL in the current population. Over 300 horses, classified as cases or controls according to clinical findings, were genotyped for the Illumina Equine SNP50 BeadChip. An animal model was first implemented in order to adjust each horse's phenotypic status for average relatedness among horses and other potentially confounding factors which were present in the data. The genome-wide association test was then conducted on the residuals from the animal model. A single SNP on chromosome 3 was found to be associated with OCD at a genome-wide level of significance, as determined by permutation. According to the current sequence annotation, the SNP is located in an intergenic region of the genome. The effects of 24 SNPs, representing QTL previously identified in a sample of Hanoverian Warmblood horses, were tested directly in the animal model. When fitted alongside the significant SNP on ECA3, two of these SNPs were found to be associated with OCD. Confirmation of the putative QTL identified on ECA3 requires validation in an independent sample. The results of this study suggest that a significant challenge faced by equine researchers is the generation of sufficiently large data sets to effectively study complex diseases such as osteochondrosis.     

Detecting low level sequence variantsin recombinant monoclonal antibodies

A systematic analytical approach combining tryptic and chymotryptic peptide mapping with a Mascot Error Tolerant Search (ETS) has been developed to detect and identify low level protein sequence variants, i.e., amino acid substitutions, in recombinant monoclonal antibodies. The reversed-phase HPLC separation with ultraviolet (UV) detection and mass spectral acquisition parameters of the peptide mapping methods were optimized by using a series of model samples that contained low levels (0.5–5.0%) of recombinant humanized anti-HER2 antibody (rhumAb HER2) along with another unrelated recombinant humanized monoclonal antibody (rhumAb A). This systematic approach’s application in protein sequence variant analysis depends upon time and sensitivity constraints. An example of using this approach as a rapid screening assay is described in the first case study. For stable CHO clone selection for an early stage antibody project, comparison of peptide map UV profiles from the top four clone-derived rhumAb B samples quickly detected two sequence variants (M83R at 5% and P274Tat 42% protein levels) from two clones among the four. The second case study described in this work demonstrates how this approach can be applied to late stage antibody projects. A sequence variant, L413Q, present at 0.3% relative to the expected sequence of rhumAb C was identified by a Mascot-ETS for one out of four top producers. The incorporation of this systematic sequence variant analysis into clone selection and the peptide mapping procedure described herein have practical applications for the biotechnology industry, including possible detection of polymorphisms in endogenous proteins.Key words: recombinant monoclonal antibody, cell line development, sequence variants, HPLC-UV/MS/MS, tryptic peptide mapping, Mascot error tolerant search     

Indirect effects of habitat disturbance on invasion: nutritious litter from a grazing resistant plant favors alien over native Collembola

Biological invasions are major threats to biodiversity, with impacts that may be compounded by other forms of environmental change. Observations of high density of the invasive springtail (Collembola), Hypogastrura manubrialis in heavily grazed renosterveld vegetation in the Western Cape, South Africa, raised the question of whether the invasion was favored by changes in plant litter quality associated with habitat disturbance in this vegetation type. To examine the likely mechanisms underlying the high abundance of H. manubrialis, cages with three types of naturally occurring litter with different nutrient content were placed out in the area and collected after different periods of time. Hypogastrura manubrialis was mainly found in the nutrient‐rich litter of the yellowbush (Galenia africana), which responds positively to disturbance in the form of overgrazing. This suggests that invasion may have been facilitated by a positive interaction with this grazing resistant plant. By contrast, indigenous Collembola were least abundant in yellowbush litter. Negative correlations between high abundance of H. manubrialis and the abundance and diversity of other species suggest that competitive interactions might underlie low abundance of these other species at the patch level. Group behavior enables H. manubrialis to utilize efficiently this ephemeral, high quality resource, and might improve its competitive ability. The results suggest that interactions among environmental change drivers may lead to unforeseen invasion effects. H. manubrialis is not likely to be very successful in un‐grazed renosterveld, but in combination with grazing, favoring the nutrient‐rich yellowbush, it may become highly invasive. Field manipulations are required to fully verify these conclusions.