Use of an Enrichment Broth Cultivation-PCR Combination Assay for Rapid Diagnosis of Swine Erysipelas

We have previously described the creation by Tn916 mutagenesis of avirulent transposition mutants from a highly virulent strain of Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas. In this study, we cloned a 2.2-kb DNA fragment which flanked the Tn916 insertion in an avirulent mutant (strain 33H6) and evaluated the possibility that this region could be used for the specific detection of E. rhusiopathiae. According to the sequences of this region, oligonucleotide primers were designed to amplify a 937-bp fragment of the E. rhusiopathiae chromosome by PCR. The specificity of the PCR was investigated by analyzing 64 strains of Erysipelothrix species and 27 strains of other genera different from Erysipelothrix. A 937-bp DNA fragment could be amplified from all E. rhusiopathiae strains tested, and no amplification was observed by using DNAs from the other species tested. To make a rapid and definite diagnosis of swine erysipelas in slaughterhouses, we developed an enrichment broth cultivation-PCR combination assay, which used a commercially available DNA extraction kit, to identify E. rhusiopathiae in the specimens from swine with arthritis. After samples were enriched in selective broth culture, detection of E. rhusiopathiae was tested by either conventional methods or the PCR. Of 102 samples tested, 15 samples were positive by conventional methods and 12 of the 15 samples were positive by the PCR. The detection limit of the PCR was 10³ CFU per reaction mixture for the PCR-positive samples. These results indicate that this PCR technique could be used as a first-line screening technique for the specific detection of E. rhusiopathiae in specimens.     

Effects of intra- and extracellular H+ and Na+ concentrations on Na+-H+ antiport activity in the lacrimal gland acinar cells

Kinetic properties of the Na⁺-H⁺ antiport in the acinar cells of the isolated, superfused mouse lacrimal gland were studied by measuring intracellular pH (pH_i) and Na⁺ activity (aNa_i) with the aid of double-barreled H⁺- and Na⁺-selective microelectrodes, respectively. Bicarbonate-free solutions were used throughout. Under untreated control conditions, pH_i was 7.12±0.01 and aNa_i was 6.7±0.6 mmol/l. The cells were acid-loaded by exposure to an NH ₄ ⁺ solution followed by an Na⁺-free N-methyl-d-glucamine (NMDG⁺) solution. Intracellular Na⁺ and H⁺ concentrations were manipulated by changing the duration of exposure to the above solutions. Subsequent addition of the standard Na⁺ solution rapidly increased pH_i. This Na⁺-induced increase in pH_i was almost completely inhibited by 0.5 mmol/l amiloride and was associated with a rapid, amiloride-sensitive increase in aNa_i. The rate of pH_i recovery induced by the standard Na⁺ solution increased in a saturable manner as pH_i decreased, and was negligible at pH_i 7.2–7.3, indicating an inactivation of the Na⁺-H⁺ antiport. The apparent K _m for intracellular H⁺ concentration was 105 nmol/l (pH 6.98). The rate of acid extrusion from the acid-loaded cells increased proportionally to the increase in extracellular pH. Depletion of aNa_i to less than 1 mmol/l by prolonged exposure to NMDG⁺ solution significantly increased the rate of Na⁺-dependent acid extrusion. The rate of acid extrusion increased as the extracellular Na⁺ concentration increased following Michaelis-Menten kinetics (V _max was 0.55 pH/min and the apparent K _m was 75 mmol/l at pH_i 6.88). The results clearly showed that the Na⁺-H⁺ antiport activity is dependent on the chemical potential gradient of both Na⁺ and H⁺ ions across the basolateral membrane, and that the antiporter is asymmetric with respect to the substrate affinity of the transport site. The data agree with the current model of activation and inactivation of the antiporter by an intracellular site through changes in the intracellular Na⁺ and H⁺ concentrations.     

Brief Communication: Role of Nuclear Background and in vivo Environment in Variable Segregation Behavior of the Aging-Dependent T414G Mutation at Critical Control Site for Human Fibroblast mtDNA Replication

Previous work had shown a large accumulation (up to 50% of mtDNA) of a noninherited T414G transversion at a critical control site for mtDNA replication in skin fibroblasts from the majority of human subjects above 65 years old, and its absence in younger individuals. In the present studies, long-term in vitro culture of several fibroblasts populations carrying the heteroplasmic T414G mutation revealed an outgrowth of the mutant cells by wild-type cells. This observation supported the previous conclusion that the mutation accumulation is an in vivo phenomenon, while, at the same time, indicating intrinsic physiological differences between mutant and wild-type cells. Furthermore, subcloning experiments revealed a striking mosaic distribution of the mutation in the original fibroblasts populations, as shown by its presence, in heteroplasmic or homoplasmic form, in a fraction (18–32%) of the fibroblasts, and its absence in the others. In other investigations, transfer of mitochondria from mutation-carrying fibroblasts into mtDNA-less 143B.TK^–0 206 cells revealed the persistence of the mosaic distribution of the mutation, however, with a near-complete shift to homoplasmy. The generality of the latter phenomenon would exclude a founder effect by one or few mitochondria in the transformation experiments, and would rather point to the important role of the nuclear background in the in vitro behavior of the T414G mutation. The stability of the homoplasmic mutation in ⁰ cell transformants provides a powerful tool for analyzing its biochemical effects.     

Mechanisms of blood coagulation induced by latex particles and the roles of blood cells

Latex particles with highly negative or positive charges shortened the clotting time of whole blood and platelet-rich plasma and activated platelet factor 3. Platelet-poor plasma was clotted by the particles with a highly negative charge, but not by those with a positive charge, except hydrophobic particles. Blood coagulation by positively-charged particles was attributed to platelet activation. An enhancement of blood coagulation was also observed in the presence of erythrocytes, leucocytes, their cell membranes or negatively charged phospholipids, and phosphatidylserine instead of platelets. Hydrophilic and low-charged particles suppressed blood coagulation.     

Ultrastructural, cytochemical, and biophysical aspects of mechanisms of bone matrix calcification

Primary calcification in embryonic ossification occurs as follows: crystallization within matrix vesicles, formation of calcified nodules, and finally the establishment of expansive calcified matrix. However, the participation of the matrix vesicles in other types of bone calcification, such as bone formation during bone remodeling in adults has not been examined sufficiently. We introduce our recent observations on the presence of matrix vesicles in aged bones. In addition, although it is well known that the extracellular fluid supersaturates the calcification crystal, hydroxyapatite, the specific mechanisms by which bone matrix calcify remain unclear. In order to further approach the mechanisms of bone matrix calcification, we also review ultrastructural and localizational alterations of the matrix organics according to the progression of calcification, and an evaluation of mineral micro-environment in the calcifying sites by energy-filter transmission electron microscopy.     

α2-Adrenergic receptor subtype alterations in the brainstem in the sudden infant death syndrome

Background: The sudden infant death syndrome (SIDS) is still the main cause of postneonatal infant death. However, the causes and mechanisms of SIDS have never been completely elucidated. Catecholamines, via α2-adrenergic receptor (α2-AR) interactions, are known to influence brainstem autonomic and respiratory activity. Aims: To examine the catecholaminergic system abnormalities in SIDS victims, we investigated the alterations of α2-AR subtypes. Subjects and methods: We examined the developmental changes of α2-AR subtypes in the brainstem, especially in cardiorespiratory nuclei, in 21 SIDS victims and 17 age-matched controls by means of immunohistochemical methods. For statistical analysis, the χ²-test or Fisher’s exact probability test was performed. Results: There was a significant decrease in α2A-AR immunoreactivity in the solitary nucleus and ventrolateral medulla (VLM) in the medulla oblongata in SIDS victims compared with in control cases, but there were no significant differences of the α2B and α2C-AR immunoreactivity in the brainstem between SIDS victims and controls. Conclusion: α2A-AR immunoreactivity was selectively decreased in the solitary nucleus and VLM in the medulla oblongata in SIDS victims, so there was no possibility that it was secondary to chronic hypoxia or repeated ischemia. It may be related to some impairment of the cardiorespiratory neuronal system. Therefore, SIDS victims may be vulnerable to asphyxia, hypoxia, and/or hypercapnia, and fail to exhibit brainstem responses.     

Flow-cytometric analysis of immune cell populations in human decidua from various types of first-trimester pregnancy.

We undertook an investigation in which flow cytometry was used to characterize immune cell populations in the decidua of first-trimester normal pregnancies, spontaneous abortions, and ectopic pregnancies in comparison to the nonpregnant endometrium to demonstrate how the proportions of immunocompetent cell populations at the fetomaternal interface are influenced by the presence and state of a fetoplacental allograft. No significant differences were found in the decidua of the different types of first-trimester pregnancy in the proportions of the CD45+, CD14+, CD3+, CD4+, CD8+, CD19+, CD3-/CD16+ and/or CD56+, CD3+/CD16+ and/or CD56+, CD4+/Leu-8+, CD4+/Leu-8-, CD8+/CD11b+, CD8+/CD11b-, and CD3+/HLA-DR- decidual leukocyte subsets. However, the percentage of decidual CD3+/HLA-DR+ cells, which are characteristic of activated T cells, was significantly higher in spontaneous abortions than in normal pregnancies (p less than 0.05). This suggests that the accumulation of decidual leukocytes may be regulated mainly by hormones and/or cytokines rather than by the presence and state of an intrauterine conceptus and that on/off-switching of activation of decidual T cells may be associated with successful maintenance of the implanted embryo.     

Molecular cloning and expression analysis of a macrophage-colony stimulating factor receptor-like gene from rainbow trout, Oncorhynchus mykiss

The M-CSF and its receptor (M-CSFR, CSF-1R or c-fms proto-oncogene) system were initially implicated as essential in mammals for normal monocyte development as well as for pregnancy. To allow a comparison with the M-CSF and M-CSFR system of an oviparous animal, we cloned a M-CSFR-like gene from rainbow trout (Oncorhynchus mykiss). The gene was cloned from a cDNA library of head kidney. It contained an open reading frame encoding 967 amino acids with a predicted size of 109 kDa. The putative amino acid sequence of rainbow trout M-CSFR showed 54% amino acid identity to fugu (Takifugu rubripes) M-CSFR, 52% to zebrafish (Danio rerio) M-CSFR and 40% to mouse (Mus musculus) and human (Homo sapiens) M-CSFR. The M-CSFR-like gene was constitutively expressed in head kidney, kidney, intestine, spleen and blood. The gene was detected especially in the ovary of immature female rainbow trout. These results suggest that a M-CSFR-like receptor may be involved in female reproductive tracts even in an oviparous animal like fish.