Localization of alkaline phosphatase and osteopontin during matrix mineralization in the developing cartilage of coccygeal vertebrae

We observed the manner in which alkaline phosphatase (ALPase) and osteopontin were localized in the cartilage and intramembranous bone of coccygeal vertebrae during matrix mineralization, shedding considerable light on the manner in which they develop. In the cartilage matrix of coccygeal vertebrae, we observed the localization of ALPase activity in the boundary of the proliferative and the hypertrophic zones. Granular nodules of mineralization were consistently found in the boundary of both zones, and increased in size when close to the hypertrophic zone. While osteopontin was rarely present in the early stages of mineralization, its localization along the margins of mineralized matrices in the hypertrophic zone was prominent. In contrast to cartilage, mineralized nodules in the intramembranous bone in the mid-portion of the vertebra displayed osteopontin-immunoreactivity, indicating its early synthesis and subsequent accumulation to early-stage mineralized nodules. When blood vessels, accompanied by osteoblastic and osteoclastic cell populations, invaded the cartilage, osteopontin was localized in the lower region of the hypertrophic zone, despite its maintaining the localization of ALPase and early-stage mineralization. Thus, our investigation demonstrated ALPase activity consistent with early-stage mineralization in the cartilage matrix. However, the fact that osteopontin-localization could not be pinpointed might account for its multifunctionality as concerns both the regulation of mineralization and the attachment of migrating osteogenic and osteoclastic cells to the mineralized matrix.     

Spatiotemporal changes of coxsackievirus and adenovirus receptor in rat hearts during postnatal development and in cultured cardiomyocytes of neonatal rat

Coxsackievirus B is the most common cause of viral myocarditis and is particularly virulent in neonates and children. Adenovirus is also a leading cause of the disease. The determinant of tropism for both viruses is considered to be the expression of coxsackievirus and adenovirus receptor (CAR) in target organs. However, developmental change and physiological localization of CAR in the heart are unknown. We examined expression levels of CAR in rat hearts by quantitative real-time polymerase chain reaction and Western blot analysis and found that CAR decreased gradually during postnatal development, although CAR was detectable, even in adults. Immunohistochemistry revealed CAR on the whole surface of cardiomyocytes in immature rat hearts. In contrast, CAR was detected predominantly on intercalated disks in the adult heart and was accumulated especially at the contact point between the cultured cardiomyocytes, even though they were prepared from the neonatal rat heart. In conclusion, CAR was expressed abundantly on the whole surface of cardiomyocytes in immature rat hearts. Both the expression level and the localization of CAR are possible determinants of the susceptibility to viral myocarditis of neonates and children.     

Immunohistochemical detection of hepatocellular carcinoma in the setting of ongoing necrosis after radiofrequency ablation.

After radiofrequency ablation (RFA), hepatocellular carcinoma undergoes complete necrosis and an ongoing necrosis that is irreversible and characterized histologically by disrupted cell outlines, homogenous cytoplasmic eosinophilia, and preserved nuclear staining, with the cells appearing quite distinct from viable cancer cells. Antibody to detect single-stranded DNA (ssDNA) specifically labeled nuclei in the setting of ongoing necrosis, but not viable tumor cells, whereas human mitochondrial antibody labeled the cytoplasm of viable cells but not cells of ongoing necrosis. The results demonstrate that RFA causes denaturation of both DNA and proteins and that the immunohistochemistry of ssDNA and mitochondrial protein is useful in detection of ongoing necrosis after RFA and provides pathological information on the validity of this procedure.     

The effect of acetylcholine on chloride transport across the mouse lacrimal gland acinar cell membranes

The mechanisms of Cl^– transport and the effects of acetylcholine (ACh) and electrochemical Cl^– potential changes across the basolateral plasma membrane on intracellular Cl^– activity in the acinar cells of isolated mouse lacrimal glands were studied using double-barreled Cl^–-selective microelectrodes. In the resting state, the basolateral membrane potential (V _m) was about –40 mV and intracellular Cl^– activity was about 35 mmol/l. Addition of ACh (10^–910^–6 mol/l) hyperpolarizedV _m and decreased the Cl^– activity in a dose-dependent manner. ACh (10^–6 mol/l) hyperpolarizedV _m by 20 mV and decreased the cytosolic Cl^– activity with an initial rate of 16.0 mmol/l · min. Reduction of the perfusate Cl^– concentration to 1/9 control depolarizedV _m and decreased cytosolic Cl^– activity at a rate of 1.9 mmol/l · min. AV _m hyperpolarization of 20 mV produced by DC injection to the adjacent cell decreased Cl^– activity at a rate of 4.6 mmol/l · min. DIDS (1 mmol/l) hyperpolarizedV _m by 8 mV with little change in Cl^– activity and increased the input resistance of the cells by 25%. DIDS decreased the rate of change in Cl^– activity induced by low-Cl^– Ringer to 35% of control, but had no effect on the ACh-evoked decrease in the Cl^– activity. Furosemide (1 mmol/l) slightly hyperpolarizedV _m and decreased Cl^– activity at a slow rate but affected Cl^– movements induced by ACh or low-Cl^– Ringer only slightly. Cl^– uptake into the cells was inhibited partially by furosemide. The present results showed that ACh induces an increase in the Cl^– permeability across the luminal plasma membrane and that the basolateral membrane possesses a DIDS-sensitive Cl^– conductance pathway and a furosemide-sensitive Cl^– uptake mechanism.     

Lethal anemia caused by interferon-beta produced in mouse embryos carrying undigested DNA

The livers of DNase II-deficient mouse embryos contain many macrophages carrying undigested DNA, and the embryos die in utero. Here we report that erythroid precursor cells underwent apoptosis in the livers of DNase II-deficient embryos and that in the liver, interferon-beta mRNA was expressed by the resident macrophages. When the DNase II-deficient mice were crossed with mice deficient in type I interferon receptor, the resultant 'double-mutant' mice were born healthy. The double-mutant embryos expressed interferon-beta mRNA, but the expression of a subset of the interferon-responsive genes dysregulated in DNase II-deficient embryos was restored to normal. These results indicate that the inability to degrade DNA derived from erythroid precursors results in interferon-beta production that induces expression of a specific set of interferon-responsive genes associated with embryonic lethality in DNase II-deficient mice.     

Quantitative studies on the neutralization reaction between African horse-sickness virus and antiserum

Summary Kinetic neutralization curves formed with African horse-sickness virus were studied by the plaque reduction technique. There was no recovery phase in surviving virus titer during a 60-minute incubation period at 37°C when antiserum was diluted 1100 or more.Factors affecting the accuracy of the serum neutralization test in tube cultures of monkey kidney stable (MS) cells were studied to ascertain optimal conditions. Dose-response patterns, variations within and between tests, and the relationship between the amount of virus and serum titers were examined to develop a test procedure showing accuracy of less than a 3-fold dilution deviation under ordinary circumstances.With antisera against 8 different types of AHS virus, it was demonstrated that there is linear relationship between the amount of serum antibody and amount of virus neutralized. When tested in MS cell tube cultures, neutralization slopes of all AHS virus types ranged between 1.33 and 1.59. A decrease in the slope was noticed when tested in mice.This work was undertaken at the Near East Animal Health Institute, a project established by the United Nations Development Program Special Fund through the Food and Agriculture Organization in cooperation with the Ministry of Agriculture of Iran.     

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.     

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.