Comparison of proteoglycans synthesized by porcine normal and polycystic renal tubular epithelial cells in vitro

Newly synthesized porcine tubular epithelial cell proteoglycans were labeled in vitro with Na2[35S]SO4. At the beginning of the labeling period (24 h) [35S] sulfate incorporated into macromolecules was measured following PD-10 chromatography. There was a significant reduction in the amount of 35S-labeled macromolecules isolated from polycystic cells compared to that from normal cells. The distribution of recovered radiolabeled material among the medium, cell surface, and intracellular fractions was similar for both normal and polycystic cells. Analysis of the proteoglycans in polycystic cells demonstrated that 86 and 73% of 35S-labeled macromolecules were of the heparan sulfate type in polycystic and normal cells, respectively. The remainder was chondroitin sulfate. Proteoglycans were characterized using DEAE-Sephacel ion-exchange chromatography, chondroitinase ABC, heparitinase, and nitrous acid digestion followed by Sepharose CL-4B gel permeation chromatography. The majority of radiolabeled material in the medium, cell surface, and intracellular fractions eluted between 0.35 and 0.39 M NaCl. However, a second peak (peak II) that eluted at 0.25 M NaCl was found in the medium from polycystic cells. This peak accounted for 27% of the total macromolecules secreted into the medium. Proteoglycans in the major peak were susceptible to nitrous acid and chondroitinase ABC digestion. A similar proportion of peak II was degraded by chondroitinase ABC. However, the remainder was only slightly susceptible to treatment with nitrous acid or heparitase. In normal cells a small amount of material eluted at a similar low charge; the proteoglycans were the same as those found in the major peak and appeared as a shoulder on this peak.     

Multiple forms of Go alpha mRNA: analysis of the 3'-untranslated regions

Go, a guanine nucleotide binding protein found predominantly in neural tissues, interacts in vitro with rhodopsin, muscarinic, and other receptors and has been implicated in the regulation of ion channels. Despite the virtual identity of reported cDNA sequences for the alpha subunit of Go (Go alpha), multiple molecular weight forms of mRNA have been identified in tissues from all species examined. To investigate the molecular basis for the size heterogeneity of Go alpha mRNAs, four cDNA clones were isolated from the same retinal lambda gt10 cDNA library that was used earlier to isolate lambda GO9, a clone encompassing the complete coding region of Go alpha. These clones were identified as Go alpha clones based on nucleotide sequence identity with lambda GO9 in the coding region; they diverge, however, from lambda GO9 in the 3'-untranslated region 28 nucleotides past the stop codon. An oligonucleotide probe complementary to a portion of the 3'-untranslated region of lambda GO9 that differs from the newly isolated clones hybridized with 3.0- and 4.0-kb mRNAs present in bovine brain and retina whereas a similar probe for the unique region of the new clones hybridized with a 4.0-kb mRNA in both tissues and with a 2.0-kb mRNA found predominantly in retina. A similar hybridization pattern was observed when brain poly(A+) RNA from other species was hybridized with the different 3'-untranslated region probes. It appears that differences in the 3'-untranslated regions could, in part, be the basis for the observed heterogeneity in Go alpha mRNAs.     

Uptake of Exogenous Glutamate and Aspartate by Circumventricular Organs but Not Other Regions of Brain

Abstract: Glutamate (Glu) and aspartate (Asp) concentrations in blood and selected regions of brain were measured at sequential intervals over a 3-h period following subcutaneous administration of Glu, Asp, or Glu plus Asp (2 mg/g body wt) to 4-day-old mouse or rat pups. Marked serum elevations of the administered amino acids (peak values exceeding 200 times control levels) were detected within 1 h. In circumventricular organ (CVO) regions of brain, which are thought to have no blood-brain barriers, a sharp and steady increase in tissue concentrations of the administered amino acids (peak values 4–10 times higher than control levels) occurred during a 15–120 min interval, whereas no appreciable increases were detected in other brain regions. When 2 mg/g Glu plus 2 mg/g Asp were administered, CVO tissue concentrations of each amino acid rose to approximately the same level obtained when the individual amino acids were given. It is concluded that blood-brain barriers preventing net entry of Glu or Asp into brain proper are relatively well established by the 4th postnatal day in rodents, but that CVO brain regions lack such barriers; selective access of blood-borne Glu or Asp to CVO neurons explains why these neurons are selectively destroyed by systemic administration of these neurotoxic amino acids.     

1,25-Dihydroxyvitamin D3 increases serum levels of the vitamin K-dependent bone protein

1,25-dihydroxyvitamin D₃ increases serum levels of bone Gla protein (BGP). The maximal increase occurs 12 h after injection and is given by 350 ng 1,25(OH)₂D₃ per 180 g body weight. In both 2 and 11 month-old male rats, the maximal increase is about 3 times the normal level, while in 2 month old female rats, the maximal increase is 2 times the normal level. These effects of 1,25(OH)₂D₃ in rats parallel the previously described effects of the vitamin on BGP secretion by rat osteosarcoma cells in culture.BGP is the first bone-specific protein whose synthesis in animals is dramatically increased by 1,25(OH)₂D₃. The possible functions of BGP in the biological actions of 1,25(OH)₂D₃ on bone are discussed.     

Genic variation in the deer mouse,Peromyscus maniculatus,in a small geographic area

Genic variation was examined with starch and polyacrylamide gel electrophoresis at 14 loci in 12 populations of Peromyscus maniculatus nubiterrae from a small part of the Appalachians in eastern Tennessee and western North Carolina. Polymorphism was observed at eight loci with no significant correlations between frequency of common genotypes or alleles and altitude. Average individual heterozygosity (\-H) values were low for P. maniculatus whether using only slow evolving loci (mean = 3.0%) or both slow and fast evolving loci (mean = 4.9%). No significant correlation was present between altitude and \-H. Interlocality variation of \-H was as great in this study as previously reported for P. maniculatus over larger geographic areas. Rogers' coefficients of genetic similarity of paired combinations of populations based on slow evolving loci (range of 0.941 to 0.997) or based on slow and fast evolving loci (range of 0.858 to 0.974) showed all populations to be highly similar. Ranges of similarity values observed in the present study were as great as those previously reported for P. maniculatus over a larger geographic area.     

Heat capacity microcalorimetry of the in vitro reconstitution of calf brain microtubules.

The self-assembly of calf brain tubulin, purified by the modified Weisenberg procedure, was examined in an adiabatic differential heat capacity microcalorimeter. Tubulin solutions at concentrations between 6 and 17 mg/mL were heated from 8 to 40 degrees C at heating rates between 0.1 and 1.0 deg/min in a pH 7.0 phosphate buffer containing 1 X 10(-3) M GTP, 1.6 X 10(-2) M MgCl2, and 3.4 M glycerol. The heat capacity change, deltaCp of the microtubule growth reaction was found to be -1600 +/- 500 cal/(deg mol) per 110 000 molecular weight tubulin dimer incorporated into microtubules, in agreement with the reported van't Hoff deltaCp value of -1500 cal/(deg mol) [Lee, J.C., & Timasheff, S.N. (1977) Biochemistry 16, 1754-1765]. The assembly reaction is characterized by a complex heat uptake pattern comprising both endothermic and exothermic processes.     

Studies on the nephrotoxicity of p-nitrophenylarsonic acid: changes in rat kidney and urinary enzyme activities following the administration of p-nitrophenylarsonic acid.

Histological studies showed that the administration of p-nitrophenylarsonic acid to rats resulted in renal tubular necrosis. The nephrotoxin was administered intraperitoneally and doses greater than 30 mg/kg were found to be fatal. The severity of the renal lesion depended on the amount of the nephrotoxin used. Elevated serum urea levels, urinary protein and volume were recorded over an 8-day period following the injection of the nephrotoxin. These changes were paralleled by an increase in the activity of lactate dehydrogenase, acid and alkaline phosphatase, N-acetyl-beta-glucosaminidase and beta-glucosidase in the urine. beta-Glycosidase activities increased in kidney homogenates, immediately after the injection of the nephrotoxin, but this eventually fell to well below the normal range. Subcellular fractions were prepared from sucrose homogenates by differential centrifugation and beta-glycosidases and cytochrome oxidase were used as enzyme markers. Only minor changes in the activity of cytochrome oxidase activity resulted from the administration of p-nitrophenylarsonic acid. One of the earliest indications of renal damage was a decrease in lysosomal latency. The activities of the lysosomal and soluble enzymes were elevated above normal during the first two days after the injection of p-nitrophenylarsonic acid, but they fell to values, significantly lower than normal, on the third day. The isoenzymic forms of beta-galactosidase, beta-glucosidase and N-acetyl-beta-glucosaminidase in normal and damaged kidneys were studied, using starch gel electrophoresis. The activities of both the lysosomal and the soluble forms of these enzymes decreased following the injection of the nephrotoxin, confirming the results obtained with whole homogenates. The relationship between the changes in renal enzyme activity and urinary enzyme excretion during the nephrotoxic process is discussed.     

The reaction of rabbit muscle creatine kinase with some derivatives of iodoacetamide.

The dimeric enzyme creatine kinase from rabbit muscle was treated with three derivatives of iodoacetamide that are capable of introducing fluorescent groups into the enzyme. All the three reagents (4-iodoacetamidosalicylate (IAS), 5-[N-(iodoacetamidoethyl)amino]-naphthalene-1-sulphonate (IAEDANS) and 6-(4-iodoacetamidophenyl)aminonaphthalene-2-sulphonate (IAANS)) were shown to react at the same single thiol group on each enzyme subunit, leading to complete inactivation of the enzyme. The reaction with IAS was extremely rapid by comparison with the reaction with iodoacetamide or iodoacetate, but various lines of evidence suggest that IAS is not a true affinity label. However, kinetic and binding studies indicate that salicylate itself probably binds at the nucleotide-binding site on the enzyme. As the size of the modifying reagent increased, the first thiol group reacted more rapidly than the second; this trend was more pronounced at 0 degree C than at 25 degree C. With the largest modifying reagent used (IAANS), the pronounced biphasic nature of the modification reaction permitted the preparation of a hybrid enzyme in which only one subunit was modified, but a study of the thiol-group reactivity showed that this hybrid enzyme preparation underwent subunit rearrangement.     

Variability of the inflorescence ofMicroseris laciniata (Compositae: Cichorieae)

Quantitative characters of the flowering head of a garden population ofMicroseris laciniata were scored during the second, third, and fourth season of growth. Number of achenes per head, number of phyllaries per head and the average number of pappus parts per achene in single heads show significant plant to plant variation. Achenes per head and pappus parts per achene were scored in identical plants in two subsequent seasons. The number of pappus parts per achene varies freely between five and ten. This contrasts with annual species ofMicroseris in which either five or ten pappus parts are found, depending on the species. In spite of a clear plant-specific average of pappus parts, both high and low pappus part determination can be demonstrated in all specimens. The number of pappus parts depends on the position of an achene on the receptacle, marginal achenes usually having fewer pappus parts than central ones. This gradient is not closely correlated with the position of an achene on the genetic spiral.