A composite transposon 3'' to the cow fetal globin gene binds a sequence specific factor.

Two unusual sequence organizations were found within the beta-globin locus of the cow. Each was a composite, consisting of closely linked Alu-type repeats with a short stretch of genomic non-repetitive sequence, called a lagan, sandwiched between. One lagan was found 3' to the fetal globin gene, while the second lay between the adult globin gene and a globin pseudogene. Southern blot analysis indicated that both lagans appeared twice within the cow haploid genome, with the second copies lying outside the cow beta-globin locus. One of these non-globin locus homologues was cloned and subjected to sequence analysis. Comparison of the DNA sequence data showed that the lagan-Alu composite was transposed as a unit. The lagan 3' to the cow fetal globin gene contains the recognition site for a sequence specific DNA binding factor. This factor was present in extracts from fetal, but not from adult cow tissues.     

Effects of serial anesthesia using ketamine or ketamine/medetomidine on hematology and serum biochemistry values in rhesus macaques (Macaca Mulatta)

Background  This study aimed at determining the cumulative effect of daily anesthesia, using two drug regimens, over hematological and biochemical parameters.
Methods  Blood samples were obtained from rhesus monkeys 20 minutes after intramuscular administration of ketamine or ketamine/medetomidine combination for three consecutive days and results were evaluated to determine their effect on hematological and serum biochemistry values. Statistical significance of drug, day, and interaction of these two variables were evaluated.
Results  Drug effect resulted in a dramatic increase of aspartate aminotransferase and creatine kinase values. Day effect resulted in decreases of RBC, HCT, Hgb, and alkaline phosphatase but an increase of other biochemical parameters evaluated. The drug/day interaction effect was found to be –significant for RBC, platelets, aspartate aminotransferase, alanine aminotransferase, and creatine kinase values.
Conclusion  The results of our study suggest a cumulative effect of serial anesthesia and should be an important consideration when interpreting hematology and serum biochemistry in rhesus macaques.     

Mechanisms of depletion of substance P by capsaicin

Capsaicin is a neurotoxin that can deplete sensory nerves of their content of substance P and interfere with certain sensory functions, such as responses of animals to noxious heat stimuli. In adult guinea pigs, a species that is susceptible to the effects of capsaicin on both substance P content and sensory function, capsaicin induces selective depletion of substance P from dorsal root ganglia and the dorsal spinal cord, sites of the cell bodies and central terminals of primary afferent neurons, respectively. As the onset of thermal analgesia in guinea pigs precedes depletion of substance P, direct neural actions of capsaicin probably account for its effects on sensory function. Capsaicin interferes with the retrograde transport of nerve growth factor (NGF) to the cell bodies of sensory nerves. Decreased availability of NGF at the site of neural protein synthesis leads to decreased synthesis of substance P. After failure of synthesis of substance P, the content of the peptide in sensory nerves gradually decreases until depletion occurs.     

Enrichment and visualization of small replication units from cultured mammalian cells

DNA from cultured Chinese hamster cells has been fractionated to yield a population of DNA enriched for replicating molecules. Molecules containing replication structures were analyzed by electron microscopy, and replicon size was estimated. The enrichment procedure takes advantage of single-stranded regions characteristic of replicating molecules, and the greater affinity of mercuric ion for single-stranded rather than native DNA. After interaction with low concentrations of HgCl2, DNA with bound mercury is separated from the bulk of the DNA by virtue of its increased buoyant density in an isopycnic Cs2SO4 gradient. When DNA from cells labeled with [3H]thymidine for 45 s is interacted with HgCl2 and banded in Cs2SO4, the DNA with the highest specific activity is found in a dense region of the gradient. The high specific activity DNA behaves kinetically like nascent DNA since the radioactivity can be chased into main band if the cells are incubated for a further 2 h in excess unlabeled thymidine. Electron microscope analysis of the DNA in the enriched fraction confirmed that it contains a substantial fraction of molecules with replication structures. The level of enrichment is about 25-fold compared to unfractionated DNA or DNA taken from the main band of the Hg++/Cs2SO4 gradient. Of the replicating molecules visualized, 85% possessed a single replication structure. All molecules with multiple replication forms contained replicon sizes less than 5 micron, ranging from 0.2 to 4.5 micron. Replicon size was determined by measuring the distance from the center of one replication structure to the center of the adjacent replication structure on the same molecule. The replicons observed in this study are far smaller than can be detected by DNA fiber autoradiography and are in the same size range as the very small replication units reported in embryonic systems.     

Motility effects of opioid peptides in dog intestine

Six opioid peptides, like morphine, were found to produce dose-dependent contractions of dog isolated intestine when administered as intraarterial boluses. The increases in incidence and amplitude of intestinal contractions were antagonized by naloxone. The rank order of potency of the opioid agonists tested was D-Ala2-met-enkephalinamide greater than D-Ala2-leu-enkephalinamide greater than met-enkephalin greater than beta-endorphin 1-31 greater than morphine greater than morphiceptin greater than dynorphin 1-13. The contractions induced by two opioid agonists displayed differential sensitivity to blockade by tetrodotoxin (TTX). Met-enkephalin was barely affected by concentrations of TTX that markedly reduced responses to morphiceptin. Some portion of the motility effect of metenkephalin may be exerted directly on intestinal smooth muscle.     

Essential Regulation of Lung Surfactant Homeostasis by the Orphan G Protein-Coupled Receptor GPR116

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Engineered recombinant peanut protein and heat-killed Listeria monocytogenes coadministration protects against peanut-induced anaphylaxis in a murine model

Peanut allergy (PNA) is the major cause of fatal and near-fatal anaphylactic reactions to foods. Traditional immunotherapy using peanut (PN) protein is not an option for PNA therapy because of the high incidence of adverse reactions. We investigated the effects of s.c. injections of engineered (modified) recombinant PN proteins and heat-killed Listeria monocytogenes (HKLM) as an adjuvant on anaphylactic reactions in a mouse model of PN allergy. PN-allergic C3H/HeJ mice were treated s.c. with a mixture of the three major PN allergens and HKLM (modified (m)Ara h 1-3 plus HKLM). The effects on anaphylactic reactions following PN challenge and the association with Ab levels and cytokine profiles were determined. Although all mice in the sham-treated groups exhibited anaphylactic symptoms with a median symptom score of 3, only 31% of mice in the mAra h 1-3 plus HKLM group developed mild anaphylaxis, with a low median symptom score of 0.5. Alterations in core body temperature, bronchial constriction, plasma histamine, and PN-specific IgE levels were all significantly reduced. This protective effect was markedly more potent than in the mAra h 1-3 protein alone-treated group. HKLM alone did not have any protective effect. Reduced IL-5 and IL-13, and increased IFN-gamma levels were observed only in splenocytes cultures from mAra h 1-3 plus HKLM-treated mice. These results show that immunotherapy with modified PN proteins and HKLM is effective for treating PN allergy in this model, and may be a potential approach for treating PNA.     

Structure of the major peanut allergen Ara h 1 may protect IgE-binding epitopes from degradation

In the past decade, there has been an increase in allergic reactions to peanut proteins, sometimes resulting in fatal anaphylaxis. The development of improved methods for diagnosis and treatment of peanut allergies requires a better understanding of the structure of the allergens. Ara h 1, a major peanut allergen belonging to the vicilin family of seed storage proteins, is recognized by serum IgE from >90% of peanut-allergic patients. In this communication, Ara h 1 was shown to form a highly stable homotrimer. Hydrophobic interactions were determined to be the main molecular force holding monomers together. A molecular model of the Ara h 1 trimer was constructed to view the stabilizing hydrophobic residues in the three dimensional structure. Hydrophobic amino acids that contribute to trimer formation are at the distal ends of the three dimensional structure where monomer-monomer contacts occur. Coincidentally, the majority of the IgE-binding epitopes are also located in this region, suggesting that they may be protected from digestion by the monomer-monomer contacts. On incubation of Ara h 1 with digestive enzymes, various protease-resistant fragments containing IgE-binding sites were identified. The highly stable nature of the Ara h 1 trimer, the presence of digestion resistant fragments, and the strategic location of the IgE-binding epitopes indicate that the quaternary structure of a protein may play a significant role in overall allergenicity.