A highly sensitive sandwich-type enzyme immunoassay system for the assay of rat creatine kinase (CK) MM, MB and BB isozymes was developed by use of β-D-galactosidase from Escherichia coli as label and the purified antibodies raised in rabbits by injecting CK-MM or CK-BB. The antibodies specific to each subunit were purified from the immunoglobulin G (IgG) fractions of antisera by immunoaffinity chromatography with a column ofrespective antigen (CK-MM or CK-BB)-coupled Sepharose. The CK-MM and CK-BB assays were performed with the respective antibody F(ab’)2 fragments-immobilized polystyrene balls and the same antibody Fab’ fragments labeled with β-D-galactosidase from Escherichia coli. The CK-MB was assayed with the polystyrene balls with either antibody (anti-CK-M or anti-CKB) and another antibody (anti-CK-B and anti-CK-M, respectively) labeled with galactosidase. The minimum detection limit of each assay was 1 to 10 pg or (10 to 100 amol) per tube. The assays for CK-MM and CK-BB were cross-reacted about 12% with CK-MB, but the assay for CK-MB was specific showing no cross-reactivity with and no interference by CK-MM and CK-BB under the limited conditions. Therefore, the concentrations of CK-MM and CK-BB were able to estimate by subtracting the cross-reacting CK-MB in the samples. The concentrations of three forms of CK in various rat tissues were determined by the present assay system. High concentrations of CK-MM were observed in the heart and tissues composed of striated muscle or chondrocytes. CK-MB was also present at relatively high levels in the above-mentioned tissues, while CK-BB was mostly located in the central nervous system and tissues composed of smooth muscle. These results are in line with the previous reports on the distribution of CK isozymes in human tissues.
The present study was undertaken to investigate the involvement of actin-linked regulatory proteins (caldesmon and tropomyosin) in concanavalin A (Con A) receptor capping of mouse splenic T-lymphocytes. Caldesmons with a high molecular weight (130-150 kDa) as well as those with a relatively low molecular weight (70-77 kDa) are shown to be present in a wide range of tissues and cells. By a technique of immunoblotting, we demonstrated that T-lymphocytes contain exclusively the low molecular weight form of caldesmon in addition to tropomyosin with a nonmuscle type. Using immunofluorescence microscopy, caldesmon and tropomyosin are revealed to redistribute into subcaps during Con A-receptor capping of T-lymphocytes. Such redistributions of caldesmon and tropomyosin and the capping of Con A-receptor are inhibited by calmodulin antagonists. These observations suggest that the calmodulin-caldesmon-tropomyosin system is involved in the capping processes of Con A receptors mediated through the control of the actinmyosin interaction.
The time course of production of catecholamine neurons in the mouse brain stem was examined by [3H]thymidine autoradiography and peroxidase-antiperoxidase (PAP)-immunocytochemistry applied simultaneously to the same tissue section. To identify catecholamine neurons, an antiserum for tyrosine hydroxylase (TH) was used. The period of neurogenesis of TH-immunoreactive neurons was embryonic day 9-11 (E9-E11) in the lateroventral reticular formation of the medulla oblongata (A1), E9-E12 in the nucleus tractus solitarii complex (A2), E9-E11 in the locus coeruleus (A6), E10-E13 in the substantia nigra (A9) and the ventral tegmental area (A10), and E11-E15 in the hypothalamic arcuate nucleus (A12). The peak of neurogenesis was seen in E10 for Al and A6, in E11 for A2, A9 and A10, and in E13-E14 for A12. The results indicate that noradrenergic neurons (A1, A2, A6) begin to be produced earlier than dopaminergic neurons (A9, A10, A12). Among the noradrenergic neurons, A2 cells differentiated later and took a longer period than A1 or A6 cells. Most of the hypothalamic A12 cells were produced after the midbrain dopaminergic cells (A9, A10) had differentiated. The data indicate that the neurogenesis of catecholamine neurons occurs in varying time sequence according to their location in brain and begins several days earlier than the phenotypic expression of catecholamines.
The effects of the venom of the male Sydney funnel-Web spider (Atrax robustus) on pancreatic exocrine secretion were studied in an isolated rat pancreas preparation perfused with modified Krebs-Henseleit solution. The venom increased pancreatic juice flow and protein concentration. The protein output depended on the presence of extracellular Ca2+ ion and was dose-dependent over a wide venom concentration (6 ng/ml-10 μg/ml). No ultrastructural changes were seen in the venom-treated glands. The mechanism of the stimulatory effect of the venom remains to be elucidated.
Based on the predicted amino acid sequence of human renal preprorenin (HRPR), two peptides corresponding to the prorenin-related (21-64) and renin/proreninrelated (337-360) sequences were synthesized by a solid-phase technique. The synthetic peptides were used as immunogens, which elicited antibodies in rabbits. Both anti-HRPR (21-64) serum R0066 and anti-HRPR (337-360) serum R0417 regularly immunostained cells in the juxtaglomerular apparatus in human adult and fetal renal tissues. The immunoreactive cells revealed by antiserum R0066 were identical with those detected by antiserum R0417, but the stainings with antiserum R0066 were less frequent and significantly weaker than those with antiserum R0417 and more strictly confined in the narrow perinuclear area of the cytoplasm. The present observations support the presence of prorenin or/and its amino-terminal fragment(s) in the Golgi apparatus in cells and suggest enzymatic degradation of the (21-64) portion in a rather early stage of prorenin processing. The results also validate the use of these antisera in further detailed studies on the prorenin-renin system.
Effect of β-adrenergic blockade on hypoxia-induced stimulation of anaerobic metabolism in rats was investigated. 1) Administration of the β-adrenergic blocker propranolol inhibited the formation of lactate and pyruvate in rats exposed to 8,000 m-simulated altitude, but showed no effect on the increase in uric acid level. 2) Blood glucose increased after the rats were returned to sea-level, and propranolol inhibited the increase completely. 3) β-Blockade did not affect the increase in lactate and pyruvate in rats to which glucose had been administered before exposed to hypoxia. β-Adrenergic effect is responsible for supply of the glycolytic substrate through the degradation of glycogen in muscle under highly hypoxic conditions such as an exposure to 8,000 m altitude, but does not participate in the stimulation of glycolysis when blood glucose can be readily utilized as a substrate.
Long-term intraperitoneal administration of chloroquine to rats induced autolysosomes in liver. Autolysosomes were isolated by Percoll density gradient equilibrium fractionation of a mitochondrial-lysosomal (ML) fraction. Electron microscopy revealed that this fraction was rich in small vesicles containing membranous dense materials. Various lysosomal enzymes including acid proteases such as cathepsin B, H, and L were rich in the ML fraction from chloroquine-treated rat liver. Autolysosomes produced by a chronic chloroquine treatment have lower density in Percoll gradient and higher acid protease activities than those in the control. In addition, several lysosomal enzyme activities were rich in postmicrosomal supernatant, suggesting fragility of lysosomal membranes in chloroquine-treated rat liver. Intralysosomal pH was elevated by a single chloroquine administration, but not by chronic administration. Therefore, proteolysis in autolysosomes may not be inhibited by chronic chloroquine treatment. The production of autolysosomes in the liver of rats treated with chloroquine may provide an experimental model for studies on the mechanism of intralysosomal protein degradation.
A sandwich-type immunoassay method for measurement of the α subunit of S100 protein in human biological fluids was developed by use of affinity-purified antibodies raised in rabbits with human S100a0 (αα form ofS100 protein) as immunogen. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab’ fragments labeled with β-D-galactosidase from Escherichia coli. The minimum detection limit of the assay was 3 pg S100a0 per assay tube. The assay cross-reacted about 20% with S100a (αβ form of S100 protein), which contain an α subunit in the molecule, but showed no cross-reactivity with S100b (ββ form of S100 protein), indicating that the assay was specific to the α subunit of S100 protein (S100-α). Coefficients of variation in within-run and between-run precision studies for serum and urine S100-α were <15%. Concentrations of serum S100-α in healthy adults ranged from <0.060—0.464 ng/ml, and were significantly higher in male than in female. Concentrations in urine S100-α in normal adults ranged from 0.033 to 0.623 ng/ml showing no significant difference between the both sexes. Parotid saliva also contained a relatively high level (average value of 0.411 ng/ml) of S100-α, which was probably derived from serous acini of parotid gland. A preliminary determination of S100-α in urine samples from patients with kidney diseases showed that the S100-α concentrations were enhanced in some patients with renal cell carcinoma, nephritis and nephrosis.
The distribution of substance P (SP)-immunoreactive fibers and terminals in the rat spinal cord during postnatal development was investigated by peroxidase-antipe roxidase (PAP) immunocytochemistry, and the effect of neonatal androgen exposure on their distribution was studied. In the male rats, a dense network of SP-immunoreactive fibers and terminals, and terminals was detected in the ventral column of the rostral lumber segments with a density and extent different from the other segmental levels during the postnatal period between neonates and adults. These fibers and terminals were accumulated within and around the nucleus centromedialis lumbaris (CM) (15) of the L1 and L2 segments (L1-2). However, in the female rats, SP-immunoreactive fibers and terminals in their densities during the postnatal period. Neonatal androgen exposure resulted in an increase in the SP-immunoreactivity in the ventral column of the rostral lumber segments of the female rats. These results indicate that the ventral horn of the L1-2 Shows sexual dimorphism in the distributional pattern of SP-immunoreactive fibers and terminals, and suggest that postnatal development of the densely distributed SP-immunoreactivity in these areas is partly due to androgenic rugulation to the central nervous system.
The myogenic growth-stimulating activity in chicken breast muscle during regeneration following cold injury has been studied. Muscle extracts prepared at various stages of regeneration were added to the culture medium and [3H]thymidine incorporation in intact chicken breast muscle cells in culture was measured. The extract of muscles on days 2-6 of regeneration contained a growth-stimulating factor identified as transferrin (Tf). The occurrence of an additional growth-stimulating factor (or factors) which required the presence of Tf was suggested in extracts of muscles on days 2-4 of regeneration. Tf and a Tf-dependent growth factor (or factors) may play important roles for the initiation of muscle regeneration in situ.