Quantitative agglutination of specific populations of sea urchin embryo cells with concanavalin A

It has been demonstrated that specific changes in carbohydrate-containing cell surface lectin receptor sites occur with differentiation and maturation of sea urchin embryo cells. In this study, evidence is presented, using a quantitative electronic particle counter assay to measure agglutination, which indicates that concanavalin A (Con A) mediated agglutination of dissociated 32/64 cell sea urchin embryos differs dramatically with respect to specific cell populations. The migratory cell type, the micromere, is significantly more agglutinable with Con A than the other cell types and colchicine treatment markedly increases sea urchin embryo cell agglutinability. The results indicate that like many malignant cells which display extensive migratory behavior, specific migratory populations of embryonic cells are agglutinable with Con A. The results are discussed with respect to the possible nature of lectin receptor sites on specific populations of embryonic cells and the possible role of colchicine-sensitive structures in controlling the display patterns of these sites.     

Cell density-dependent stimulation of glutamine synthetase activity in cultured mouse teratoma cells

A density dependent stimulation of glutamine synthetase (GS) activity has been observed in cultures of mouse teratoma cells. GS specific activity increased as cultures approached confluency to a level greater than 2-fold over the basal level found in sparse cultures. After confluency the GS specific activity returned to the basal level found in sparse cultures. The enzyme increase could not be attributed to age of cultures, medium or glutamine depletion, cell leakage of GS, or change in the amount of cellular protein. Dibutyryl cyclic AMP (db-cAMP) plus theophylline lowered GS specific activity both in cultured teratoma and in teratoma obtained from ascites grown tumors. The enzyme increase observed in cultured teratoma cells could be prevented by cycloheximide, and enhanced by hydrocortisone or actinomycin D.     

Two-dimensional 1H NMR of three spin-labeled derivatives of bovine pancreatic trypsin inhibitor

Three nitroxide spin-labeled monoderivatives of bovine pancreatic trypsin inhibitor were prepared with the amino-specific reagent succinimidyl 1-oxy-2,2,5,5-tetramethyl-3-pyrroline-3-carboxylate. The monoderivatives were purified by ion-exchange and affinity chromatography. Thin-layer maps of tryptic peptides of the monoderivatives showed that the spin-label was incorporated at either the alpha-amino group, Lys-15, or Lys-26. Two-dimensional J-correlated 1H NMR spectra of the monoderivatives were recorded. Spectra were also recorded after reduction by ascorbic acid of the nitroxide label to hydroxylamine. With the nitroxide label present, significant line-broadening effects on many of the cross peaks in the spectra were observed. The extent of line broadening for the C alpha H-NH cross peaks was qualitatively correlated with the distance between the labeled amino group and the average C alpha H-NH position in the crystal structure. The spin-label affects cross peaks of protons within approximately 15 A. This study suggests that it is feasible to accumulate sufficient intramolecular distances in order to determine protein solution structures with the aid of distance geometry algorithms.     

A myb gene required for leaf trichome differentiation in Arabidopsis is expressed in stipules

The GL1 gene is required for the initiation of differentiation of hair cells (trichomes) on the crucifer, Arabidopsis thaliana. This gene has been localized to a 4.5 kb DNA fragment by molecular complementation of gl1 mutants. DNA sequence analysis has shown that the protein encoded by GL1 contains a Myb DNA-binding motif. Southern analysis and subsequence analysis of isolated lambda clones has established that GL1 is a member of an extensive myb gene family in Arabidopsis. The putative GL1 promoter directs the expression of the GUS reporter gene in non-trichome-bearing structures that appear to be stipules. This pattern of expression suggests that GL1 may control the synthesis of a diffusible signal that activates the developmental pathway for trichome differentiation.     

Identification of rat S14 protein and comparison of its regulation with that of mRNA S14 employing synthetic peptide antisera

The rat S14 gene encodes a protein of unknown function and has an amino acid sequence unrelated to any published sequences. Expression of mRNA S14 and lipogenesis in liver, fat, and mammary gland are regulated coordinately by dietary and hormonal stimuli, suggesting that the S14 protein may be associated with lipogenesis. Antisera to synthetic peptides corresponding to portions of the deduced amino acid sequence of the protein were used to identify the protein and to compare its regulation with that of mRNA S14. Antisera specifically recognized the in vitro translation product of mRNA S14 as defined by its migration on two-dimensional gel electrophoresis. A product of identical Mr was identified on Western blots of liver homogenates from hyperthyroid, carbohydrate-fed rats. Subcellular fractionation showed that S14 protein is primarily cytosolic. The protein was detectable in tissues with abundant S14 gene expression, including hyperthyroid liver and epididymal fat and hypothyroid brown adipose tissue, whereas it was undetectable in hypothyroid liver and euthyroid kidney, testis, and spleen. Diurnal variation in hepatic mRNA S14 correlated with comparable changes in levels of the protein. Surprisingly, no S14 protein was observed in the livers of chronically (3 week) hypothyroid rats treated with triiodothyronine (T3) until 12 h had elapsed, despite attainment of maximal levels of mRNA S14 within 4 h. Rapid appearance of protein after T3 treatment was observed in both euthyroid and short term (4 day) hypothyroid rats, suggesting that long-term hypothyroidism is associated with a defect in the translational efficiency of mRNA S14.     

Thyroid hormone and circadian regulation of the binding activity of a liver-specific protein associated with the 5'-flanking region of the S14 gene

Recent studies have described a DNase I hypersensitive site in the 5'-flanking region of the rat hepatic S14 gene that is closely associated with its expression. A 111-base pair subfragment (-389 to -279) of this region interacts specifically in a gel shift assay with a protein present in hepatic nuclear protein extracts. This protein, designated P1, was not present in extracts of other tissues, even those in which the gene is expressed and hormonally regulated. The binding activity of P1 is exceedingly low in extracts from hypothyroid rats and is markedly increased by administration of thyroid hormone. However, the slow accumulation of P1 after thyroid hormone administration indicates that increased levels of P1 are not necessary for the acute hormonal induction of S14 gene expression. The level of P1 binding activity increases in the evening, synchronous with circadian variation of hepatic mRNA S14. Since neither P1 binding activity nor circadian variation in mRNA-S14 levels are observed in the other tissues expressing the S14 gene, P1 may function to modulate the circadian rhythm observed in hepatic S14 gene expression. DNase I footprinting analysis revealed that P1 binds to a defined nucleotide sequence, 5'-AAAAGAGCTATTGATTGCCTGCA-3', located between -310 and -288 in the S14 gene.     

Growth hormone acts at a pretranslational level in hepatocyte cultures

We have examined the effects of ovine growth hormone and recombinant DNA synthesized human growth hormone on hepatocytes maintained in serum free cultures. Both growth hormone preparations augmented or attenuated 3 specific mRNA sequences as revealed by two-dimensional gel electrophoresis of [35S] methionine labeled products synthesized in vitro in an mRNA dependent rabbit reticulocyte lysate system. The results clearly indicate that growth hormone, free of potential pituitary contaminants, acts directly on hepatocytes at a pretranslational level.     

Thyroid hormone-carbohydrate interaction at the hepatic nuclear level

We have studied the interaction between triiodothyronine (T3) and carbohydrate (CHO) in the induction of hepatic lipogenic enzymes under both in vivo and in vitro conditions. Our studies demonstrate a synergistic relationship between T3 administration and CHO feeding in the induction of these enzymes. Likewise, in states characterized by CHO deprivation such as starvation and diabetes, the response to T3 is also inhibited. Studies in the aging animal have documented a diminished response both to CHO and to T3. Our studies suggest that T3 multiplies a primary CHO-generated signal by a constant factor, and that this signal declines with age. Additional studies with primary hepatocyte cultures provide evidence that glucose is the main factor responsible for the induction of hepatic malate dehydrogenase: decarboxylating (EC 1.1.1.40) (ME). Glucose induces ME in the absence of changes in extrahepatic hormones or metabolites and in the complete absence of T3. In the cultured hepatocyte system, T3 also acts as a constant multiplier of the primary glucose-derived signal. Our results provide further support for the thesis that the primary action of T3 at the molecular level is a multiplication of other nuclear signals. The complexity of response pattern to both T3 and CHO administration, however, is illustrated by recent studies in which we have analyzed the translated products of total poly(A+) RNA extracted from livers of rats subjected to various physiological stimuli.     

Colorimetric device for measurement of transvascular fluid flux in blood-perfused organs

The aim of this study was to develop a device capable of measuring transvascular fluid flux in blood-perfused organs. For any given blood flow through the organ (QT), transvascular flux (QF) can be considered as the fraction of QT exchange. Presumably, QF would change the background concentration of an impermeable tracer residing in the perfusate. Thus QF could be calculated from the relative changes in tracer concentration for any given QT. We have used Blue Dextran (1 g/l of blood) as the reference tracer. Because the minimum molecular weight of Blue Dextran is 2 X 10(6), we anticipated it to behave as an impermeable tracer in most organs. QF was simulated with continuous infusions of plasma, normal saline solution, and a 50% mixture of both. Changes in Blue Dextran concentration were continuously followed colorimetrically by changes in transmission of specific light at a wavelength of 632 nm. Because 632-nm light is affected by hematocrit and O2 saturation changes, two additional wavelengths were used: 815-nm, which is not affected by saturation or Blue Dextran concentration changes, was used to account for changes in hematocrit, and 887-nm specific light, which is not affected by Blue Dextran, served to correct for saturation changes. Red cells could not be used as the reference tracer because of the possibility of hematocrit changes independent of fluid flux (Fahraeus effect). The device so constructed proved capable of measuring rates of fluid infusion in the order of 0.1% of QT with a variability of 10% around the mean.