Automated high-performance liquid chromatographic method for determination of furosemide in dog plasma

An automated high-performance liquid chromatographic method for the determination of the diuretic drug furosemide has been established. Dog plasma was injected directly into a two-column system with a BSA—ODS (ODS column coated with bovine serum albumin) precolumn and a C₁₈ analytical column for the separation of furosemide. The two columns were automatically switched. Furosemide remained trapped on the precolumn while proteins were eluted to waste. After column switching, furosemide was washed onto the analytical column and analysed without interference. The greatest advantage of the method is its easy performance without manual sample preparation; it requires no extraction or deproteinization. The method allows determination of 0.1–10 μg/ml of furosemide with accuracy and precision comparable with previously reported values. The coefficients of variation obtained from replicate measurements of 1 μg/ml and 5 μg/ml samples were 1.65% and 2.40%, respectively. This method was used to measure the plasma levels of furosemide in beagle dogs to whom the drugs was administered, as a reference, in a toxicological study.     

Profilin 1 mutants form aggregates that induce accumulation of prion-like TDP-43

Mutations in the profilin 1 (PFN1) gene have been identified as a cause of familial amyotrophic lateral sclerosis (ALS), and neuropathological studies indicate that TDP-43 is accumulated in brains of patients with PFN1 mutation. Here, we investigated the role of PFN1 mutations in the formation of prion-like abnormal TDP-43. Expression of PFN1 with pathogenic mutations resulted in the formation of cytoplasmic aggregates positive for p62 and ubiquitin, and these aggregates sequestered endogenous TDP-43. TDP-43 accumulation was facilitated in the presence of proteasome or lysosome inhibitor. Co-expression of mutant PFN1 and TDP-43 increased the levels of detergent-insoluble and phosphorylated TDP-43, and this increase required the C-terminal region of TDP-43. Moreover, detergent-insoluble fractions prepared from cells expressing ALS-linked mutant PFN1 induced seed-dependent accumulation of TDP-43. These findings indicate that expression of PFN1 mutants induces accumulation of TDP-43, and promotes conversion of normal TDP-43 into an abnormal form. These results provide new insight into the mechanisms of TDP-43 proteinopathies and other diseases associated with amyloid-like protein deposition.     

The role of hapten-reactive T lymphocytes in the induction of autoimmunity in mice. I. Establishment of the experimental conditions for the termination of immunological tolerance to human gamma globulin.

In order to study the role of hapten-reactive helper T cells in the induction of autoimmunity in mice, an attempt was made to establish an experimental model for the development of hapten-reactive helper T cells and the termination of immunological tolerance against heterologous proteins. Spleen cells taken from mice which were immunized with hapten-isologous protein conjugates (PAB-MGG) demonstrated helper activity for the anti-DNP antibody response of DNP-primed B cells responding to DNP and PAB-conjugated protein, but spleen cells from hapten-heterologous protein conjugate (PAB-HGG)-primed mice could not respond to PAB-determinant. Thus, hapten-reactive helper T cells can develop in mice by the immunization with hapten-isologous protein conjugate, but not with hapten-heterologous protein conjugate. However, spleen cells from mice which had been rendered tolerant by treatment with 2.5 or 0.2 mg of DHGG and then immunized with PAB-HGG could demonstrate helper activity responding to PAB-determinant. This helper activity was PAB-specific, because these spleen cells did not demonstrate helper activity if PAB-determinant was omitted in the primary and the secondary antigen. This helper activity was abrogated by the treatment of spleen cells with anti-θ serum and complement. Thus, hapten-reactive helper T cells were successfully induced by the challenge with hapten-heterologous protein conjugate in carrier-protein tolerant mice. When mice were treated with 2.5 or 0.2 mg of DHGG, no anti-HGG antibody response was induced by the challenge with HGG or PAB-HGG. However, the termination of HGG-tolerance was demonstrated only when the mice were preimmunized with PAB-MGG to raise PAB-rcactive helper T cells, treated with 0.2 mg of DHGG, and then challenged with PAB-HGG. This termination of immunological tolerance was not observed when the mice were preimmunized with PAB-BαA to raise PAB-specific B cells and anti-PAB antibody, or when the mice were treated with 2.5 mg of DHGG. Thus, if HGG-specific B cells remain intact in mice such as treated with low dose of DHGG, these B cells can be activated by some bypass mechanisms in the presence of PAB-reactive helper T cells through the PAB-determinant even in the absence of HGG-reactive helper T cells. These data clearly showed the role of hapten-reactive helper T cells in the termination of immunological tolerance and provide experimental supports to the hypothesis on the termination mechanism proposed by Weigle. The cellular mechanism for the development of hapten-reactive helper T cells in tolerant animals and the cellular mechanism of autoantibody production were discussed on the basis of T-B cell collaboration.     

Identity of mitochondrial and cytosolic glycerate kinases in rat liver and regulation of their intracellular localization by dietary protein

Glycerate kinase (ATP : D-glycerate 2-phosphotransferase EC 2.7.1.31) is a key enzyme of gluconeogenesis from serine via hydroxypyruvate. A differential centrifugation of rat liver homogenate and an analysis of the particle fraction by sucrose density gradient centrifugation indicated that 72% and 26% of glycerate kinase are present in mitochondria and cytosol, respectively. A study on the intramitochondrial localization of the enzyme suggested that the mitochondrial glycerate kinase was present in inner membrane and/or matrix. It was found that dietary protein selectively induced mitochondrial glycerate kinase. This result suggested that mitochondrial glycerate kinase had a physiological function for gluconeogenesis from serine. However, the metabolic significance of the cytoplasmic enzyme was still unclear. The properties of solubilized-mitochondrial and cytosolic glycerate kinases were compared. However, no difference between the two enzymes could be found in the kinetic properties, thermal stability, molecular size or electrochemical properties. These results suggested that both enzymes originate from common genetic information. In order to elucidate the regulatory mechanism of the intracellular distribution of glycerate kinase in rat liver, the responses of mitochondrial and cytosolic glycerate kinases to an alteration of dietary protein were studied. The result suggested that an alteration of dietary protein content may regulate the distribution and the translocation of glycerate kinase to mitochondria and cytosol as well as the total amount of glycerate kinase.     

A new species of Trichogramma (Hymenoptera: Trichogrammatidae) parasitic on eggs of the alderfly Sialis melania (Neuroptera: Sialidae) from Japan,with comments on its phylogeny and male wing polymorphism

A new species of Trichogramma that parasitizes Sialis melania eggs is described as Trichogramma tajimaense Yashiro, Hirose and Honda, sp. nov. from Japan. Its phylogenetic position is based on a DNA‐based analysis, and data regarding its male wing polymorphism are also presented. The view that T. tajimaense is closely related to T. semblidis, another parasitoid of Sialis eggs, is supported by the results of a phylogenetic analysis, as well as by the biological and morphological similarities between both species. Trichogramma tajimaense is also similar in male wing polymorphism to T. kurosuae, a gregarious egg parasitoid of the lepidopteran Ivela auripes, as both Trichogramma species exhibit male wing trimorphism (fully alate, brachypterous and apterous forms) in contrast to the male wing dimorphism (fully alate and apterous forms) of T. semblidis. However, no phylogenetic analysis reveals a close relationship between T. tajimaense and T. kurosuae, and a difference exists between these two species in the mean percentage of flightless (brachypterous and apterous) males that emerge from a host egg mass; 96% of T. tajimaense males are incapable of flight, whereas about 50% of T. kurosuae males are flightless. Because all or almost all males of T. semblidis parasitizing Sialis eggs are apterous, T. tajimaense is more similar to T. semblidis than to T. kurosuae in the proportion of flightless males. In addition, male wing polymorphisms of Trichogramma in relation to mating systems could also show a similarity between T. tajimaense and T. semblidis when considering both species as quasi‐gregarious parasitoids of Sialis eggs.     

Histochemical study of jaw muscle fibers in the American alligator (Alligator mississippiensis)

The ontogenetic development of caudal vertebrae and associated skeletal elements of salmonids provides information about sequence of ossification and origin of bones that can be considered as a model for other teleosts. The ossification of elements forming the caudal skeleton follows the same sequence, independent of size and age at first appearance. Dermal bones like principal caudal rays ossify earlier than chondral bones; among dermal bones, the middle principal caudal rays ossify before the ventral and dorsal ones. Among chondral bones, the ventral hypural 1 and parhypural ossify first, followed by hypural 2 and by the ventral spine of preural centrum 2. The ossification of the dorsal chondral elements starts later than that of ventral ones. Three elements participate in the formation of a caudal vertebra: paired basidorsal and basiventral arcocentra, chordacentrum, and autocentrum; appearance of cartilaginous arcocentra precedes that of the mineralized basiventral chordacentrum, and that of the perichordal ossification of the autocentrum. Each ural centrum is mainly formed by arcocentral and chordacentrum. The autocentrum is irregularly present or absent. Some ural centra are formed only by a chordacentrum. This pattern of vertebral formation characterizes basal teleosts and primitive extant teleosts such as elopomorphs, osteoglossomorphs, and salmonids. The diural caudal skeleton is redefined as having two independent ural chordacentra plus their arcocentra, or two ural chordacentra plus their autocentra and arococentra, or only two ural chordacentra. A polyural caudal skeleton is identified by more than two ural centra, variably formed as given for the diural condition. The two ural centra of primitive teleosts may result from early fusion of ural centra 1 and 2 and of ural centra 3 and 4, or 3, 4, and 5 (e.g., elopomorphs), respectively. The two centra may corespond to ural centrum 2 and 4 only (e.g., salmonids). Additionally, ural centra 1 and 3 may be lost during the evolution of teleosts. Additional ural centra form late in ontogeny in advanced salmonids, resulting in a secondary polyural caudal skeleton. The hypural, which is a haemal spine of a ural centrum, results by growth and ossification of a single basiventral ural arococentrum and its haemal spine. The proximal part of the hypural always includes part of the ventral ural arcocentrum. The uroneural is a modification of a ural neural arch, which is demonstrated by a cartilaginous precursor. The stegural of salmonids and esocids originates from only one paired cartilaginous dorsal arcocentrum that grows anteriorly by a perichondral basal ossification and an anterodorsal membranous ossification. The true epurals of teleosts are detached neural spines of preural and ural neural arches as shown by developmental series; they are homologous to the neural spines of anterior vertebrae. Free epurals without any indication of connection with the dorsal arococentra are considered herein as an advanced state of the epural. Caudal distal radials originate from the cartilaginous distal portion of neural and haemal spines of preural and ural (epurals and hypurals) vertebrae. Therefore, they result from distal growth of the cartilaginous spines and hypurals. Cartilaginous plates that support rays are the result of modifications of the plates of connective tissue at the posterior end of hypurals (e.g., between hypurals 2 and 3 in salmonids) and first preural haemal spines, or from the distal growth of cartilaginous spines (e.g., epural plates in Thymallus). Among salmonids, conditions of the caudal skeleton such as the progressive loss of cartilaginous portions of the arcocentra, the progressive fusion between the perichondral ossification of arcocentra and autocentra, the broadening of the neural spines, the enlargement and interdigitation of the stegural, and other features provide evidence that Prosopium and Thymallus are the most primitive, and that Oncorhynchus and Salmo are the most advanced salmonids respectively. This interpretation supports the current hypothesis of phylogenetic relationships of salmonids. © 1992 Wiley-Liss, Inc.