The milk protein promoter is a useful tool for developing a rat with tolerance to a human protein

Biopharmaceuticals intended for humans are immunogenic in animals. Antibodies associated with their administration make it difficult to perform repeated-dose pharmacology and toxicology studies in animals. Despite suggestions to solve this problem with transgenic animal technology, an effective strategy has not yet been reported. The objective of the present study was to provide an efficient strategy to develop rats tolerant to biopharmaceuticals such as human gene-based proteins. The present study used transgenic rat lines (lines 311-6, 308-5, and 305-1) carrying a fusion gene designed to express the human growth hormone (hGH) gene under the control of the bovine S1 casein gene promoter. Three lactating females with the transgene, produced approximately 4mg/ml, 300g/ml, and 10ng/ml in their milk. Male 8-week-old rats from these three lines were immunized with hGH three times (week 0, 1, and 3 ) and the production of antibodies against hGH in their sera were examined at week 4. While the hGH serum antibody titers increased over 1000-fold in wild-type control rats, there was no detectable antibody against hGH in the sera of these three transgenic lines. Human growth hormone in their sera was undetectable (lines 308-5 and 305-1) or much lower than the endogenous biologic level of rat growth hormone (line 311-6). Importantly, lines 308-5 and 305-1 developed tolerance to hGH without detectable hGH in their sera and these lines will be very useful for the repeated dose pharmacology and toxicology studies. These results suggest that a milk protein promoter can be a useful tool to develop transgenic rats that are tolerant to biopharmaceuticals intended for humans.     

Synthesis of sulfoquinovosylacylglycerols, inhibitors of eukaryotic DNA polymerase alpha and beta

Sulfoquinovosyldiacylglycerols (SQDGs) and sulfoquinovosylmonoacylglycerols (SQMGs), bearing diverse fatty acids, were synthesized from D-glucose, and were examined for enzymatic inhibitions of DNA polymerase alpha and beta. These results indicated that the carbon numbers of the fatty acids were highly related to the activities, at least in vitro, of eukaryotic DNA polymerase inhibition.     

Specific inhibitor of puromycin-sensitive aminopeptidase with a homophthalimide skeleton: identification of the target molecule and a structure-activity relationship study

2-(2,6-Diethylphenyl)-1,2,3,4-tetrahydroisoquinoline-1,3-dione (2: PIQ-22) was found to be a potent and specific inhibitor of puromycin-sensitive aminopeptidase (PSA). Lineweaver-Burk plot analysis showed that PSA is inhibited by PIQ-22 in a non-competitive manner. Structure -activity relationship studies indicated that tautomerism of the imidobenzoylketone group in the cyclic imide moiety of the PIQ-22 skeleton is important for the inhibitory activity.     

Upregulation of ET-1 and its receptors and remodeling in small pulmonary veins under hypoxic conditions

Pulmonary veins show greater sensitivity to endothelin (ET)-1-induced vasoconstriction than pulmonary arteries, and remodeling was observed in pulmonary veins under hypoxic conditions. We examined, using an immunohistochemical method, the expression of Big ET-1, ET-converting enzyme (ECE), and ET(A) and ET(B) receptors in rat pulmonary veins under normoxic and hypoxic conditions. In control rats, Big ET-1 and ECE were coexpressed in the intima and media of the pulmonary veins, with an even distribution along the axial pathway. ET(A) and ET(B) receptors were expressed in the pulmonary veins, with a predominant distribution in the proximal segments. The expression of Big ET-1 was more abundant in the pulmonary veins than in the pulmonary arteries. After exposure to hypoxia for 7 or 14 days, the expression of Big ET-1, ECE, and ET receptors increased in small pulmonary veins. Increases in the medial thickness, wall thickness, and immunoreactivity for alpha-smooth muscle actin were also observed in the small pulmonary veins under hypoxic conditions. The upregulation of ET-1 and ET receptors in the small pulmonary veins is associated with vascular remodeling, which may lead to the development of hypoxic pulmonary hypertension.     

In vitro abzyme evolution to optimize antibody recognition for catalysis

Enzymes have evolved their ability to use binding energies for catalysis by increasing the affinity for the transition state of a reaction and decreasing the affinity for the ground state. To evolve abzymes toward higher catalytic activity, we have reconstructed an enzyme-evolutionary process in vitro. Thus, a phage-displayed combinatorial library from a hydrolytic abzyme, 6D9, generated by the conventional in vivo method with immunization of the transition-state analog (TSA), was screened against a newly devised TSA to optimize the differential affinity for the transition state relative to the ground state. The library format successfully afforded evolved variants with 6- to 20-fold increases in activity (kcat) as compared with 6D9. Structural analysis revealed an advantage of the in vitro evolution over the in vivo evolution: an induced catalytic residue in the evolved abzyme arises from double mutations in one codon, which rarely occur in somatic hypermutation in the immune response.     

Hormone-specific combinations of isoforms of adenylyl cyclase and phosphodiesterase in the rat liver

Since many isoforms of adenylyl cyclase and adenosine 3', 5'-monophosphate (cAMP) phosphodiesterase have been cloned, it is likely that receptors of each hormone have a specific combination of these isoforms. Types I, III and VIII adenylyl cyclases are reported to be stimulated by Ca(2+)-calmodulin, type I phosphodiesterase by Ca(2+)-calmodulin, but types IV and VII (cAMP-specific) phosphodiesterases by Co2+. In the present study, we examined different effects of Ca2+ and Co2+ on hormone-induced cAMP response in the isolated perfused rat liver.The removal of Ca2+ from the perfusion medium (0 mM CaCl(2 ) + 0.5 mM EGTA) did not affect glucagon (0.1 nM)-responsive cAMP but reduced secretin (1 nM)-, vasoactive intestinal polypeptide (VIP, 1-10 nM)- and forskolin (1 microM)-responsive cAMP considerably. The addition of 1 mM CoCl2 reduced glucagon- and secretin-responsive cAMP considerably, forskolin-responsive cAMP partly, did not affect 1 nM VIP-responsive cAMP, but enhanced 10 nM VIP-responsive cAMP. Forskolin- and VIP-responsive cAMP was greater in the combination (0 mM CaCl(2) + 0.5 mM EGTA + 3 mM CoCl2) than in the Ca(2+)-free perfusion alone.These results suggest that secretin, VIP1 and VIP2 receptors are linked to Ca(2+)-calmodulin-sensitive adenylyl cyclase; glucagon receptor to Ca(2+)-calmodulin-insensitive adenylyl cyclase; VIP1 receptor to Ca(2+)-calmodulin-dependent phosphodiesterase; glucagon, secretin and VIP2 receptors to cAMP-specific phosphodiesterase, respectively, in the rat liver.     

Epidemiological survey of Orientia tsutsugamushi distribution in field rodents in Saitama Prefecture, Japan, and discovery of a new type

There are various antigenic variants of Orientia tsutsugamushi which are distinguished by immunological and molecular genetic methods targeted at the antigenic diversity of 56-kDa type-specific antigen proteins. The present study was performed to analyze 15 strains successfully isolated from rodents in Saitama Prefecture, Japan, by 56-kDa gene sequence homologies, reactivities with type-specific monoclonal antibodies and polymerase chain reaction (PCR) using type-specific primer-pairs. We demonstrated the presence of a new type of O. tsutsugamushi among the isolates. This new type, designated as the Saitama type, was located in the branch of Karp type in the phylogenetic tree based on 56-kDa gene sequences, but distant from the known Karp types, such as Karp, JP-1 and JP-2, showing less than 90% homology. Strains of this type could not be distinguished by immunological methods from Karp type strains, but a new primer-pair for PCR which specifically amplifies the DNA of this new type strain was designed. This primer-pair may serve to find this strain type in future studies.     

Measurement of mitochondrial pH in situ

In this article, we describe the advantages and disadvantages of procedures for monitoring mitochondrial pH in situ using optical microscopic techniques. The first method employs the combination of the fluorescent pH-sensitive indicator carboxy-SNARF and laser scanning confocal microscopy. Manipulation of the loading and post-loading conditions enables relatively specific accumulation of carboxy-SNARF into mitochondria. With the use of a mitochondrial-specific marker, mitochondrial pH can be accurately monitored. More recently, mitochondrial-targeted, pH-sensitive probes have been used to monitor mitochondrial pH. In particular, mitochondrial targeting of the yellow fluorescent protein (YFP) mutant of green fluorescent protein (GFP) combines the advantages of specific mitochondrial localization, high-fluorophore quantum yield, and extinction coefficient with an appropriate pKa for measuring mitochondrial pH. The use of dual-excitation ratiometry with mitochondrially targeted YFP increases the dynamic range of mitochondrial pH measurements and corrects for differences in the amount of expression of mitochondrially targeted YFP at the level of individual mitochondria.