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Biomedical subjects

J Ilan

Publications and source records attributed to J Ilan.

At least 55 records · Page 3Linked to original sources
PubMed1986

Effects of forelimb amputation and denervation on protein synthesis in spinal cord ganglia of the newt.

Analysis of the effects of forelimb amputation and denervation on [35S]methionine incorporation into the protein of newt brachial plexus nerve ganglia showed that amputation increases the level of protein synthesis about 4-fold as compared with that of control (nonamputated) animals. Denervation without amputation nearly doubles the level of protein synthesis as compared with controls. Analysis of labeled protein by means of two-dimensional gel electrophoresis and radioautogram revealed incorporation patterns that are similar to controls; this observation suggests that amputation affects nerve cells quantitatively rather than qualitatively, in influencing the production of putative neurotrophic agents. A group of basic proteins ranging in Mr from 15,000 to 31,000 are prominently expressed in these radioautograms from experimental ganglia and may be important in promoting forelimb regeneration in the newt.

Amputation, Surgical↗
PubMed1986

Expression of insulin-like growth factor II in human placentas from normal and diabetic pregnancies.

The growth and development of the placenta is critical to fetal growth and development; however, little is known regarding the mechanisms controlling placental growth and development. Human placental membranes are known to possess receptors for insulin-like growth factors I and II (IGF-I and IGF-II) from early gestation, and increasing evidence supports a major role for IGF-I and/or IGF-II in fetal growth and development. Therefore, the IGFs may also play a significant role in regulating placental growth and development. We report here that an adult human liver IGF-II cDNA hybridizes to poly(A)+ RNAs of human placentas from different gestational ages. There are four placental poly(A)+ RNA species that hybridize to IGF-II cDNA, the major one of which is about 6000 bases. The sizes of the hybridized transcripts are the same for placentas of different gestational ages. Furthermore, the IGF-II sequences expressed in the human placenta were quantitated by dot blot hybridization. The second trimester placenta expresses more IGF-II mRNA sequences than placenta of first trimester and term. Interestingly, the term placentas from diabetic pregnancies also express more of these sequences than those from normal pregnancies. These results suggest that there are developmental changes in the expression of the IGF-II gene in the placenta and that IGF-II may promote placental growth by way of an autocrine and/or paracrine mechanism. Moreover, fetuses developing in diabetic pregnancies receive a large influx of glucose, which in turn may stimulate the expression of IGF-II sequences in placenta, resulting in higher utilization of glucose and overgrowth of placenta. This may explain the macrosomia and high incidence of malformations and stillbirths known to result from pregnancies in diabetics.

Female↗
PubMed1985

Comparisons of human placental lactogen mRNA levels from placentas of diabetics and normal term.

Overt diabetes and gestational diabetes (1-2.5% of all pregnancies) has been related to perinatal mortality, increased macrosomia and increased frequency of other pregnancy complications. Human placental lactogen (hPL), a hormone similar to growth hormone, is produced by the placenta and is a potent antagonist to insulin action. While hPL's presence in maternal circulation induces a sparing effect on nutrients including glucose, it exacerbates diabetes during pregnancy and may well relate to other clinical complications. To explore possible regulation of hPL in diabetic pregnancy and specifically to examine gestational diabetes, we have evaluated the levels of placental mRNA coding for hPL synthesis as well as other parameters from diabetic and normal term patients. By in vitro translation assays using nuclease-treated reticulocyte lysate, no substantial differences in translatable hPL-mRNA were observed when comparing normal term (3.5% of total synthesis), gestational diabetic (3.4%) and Type C diabetic (3.5%). However, translatable hPL-mRNA in Type R diabetes which was 2.7% of total synthesis was slightly reduced in comparison to normal term. To determine more directly hPL-mRNA levels in gestational diabetic placentas and normal term placentas, total RNA preparations were evaluated qualitatively by northern blot and quantitatively by dot blot of RNA and cDNA hybridization to a nick-translated hPL-pMB9 plasmid. The northern blot revealed no major size differences of the mRNA and the dot blot hybridization was quantitatively similar for both gestational diabetics and normal terms per unit of total RNA. By direct analysis of DNA per g tissue we found the DNA content of placentas from gestational diabetics and normal term to be statistically the same.(ABSTRACT TRUNCATED AT 250 WORDS)

Female↗
PubMed1985

Insulin-related genes expressed in human placenta from normal and diabetic pregnancies.

Rapid growth of human fetal tissues requires insulin or insulin-like growth factors. A high rate of human fetal growth occurs between implantation and about 14 weeks of gestation. Fetal pancreatic insulin secretion begins much later. Since maternal insulin does not cross the blood/placental barrier, other sources of insulin or insulin-like growth factors may be provided for fetal development. We report here that placental polyadenylylated RNAs from the first and third trimester of normal pregnancy as well as from term pregnancies of diabetic mothers hybridize to a 32P-labeled cloned cDNA of an insulin-related sequence expressed in fetal pancreas. Moreover, placentas from diabetic women express much more of these sequences. These results suggest that insulin-related genes are expressed in placental tissue during fetal development and may be a source of growth-promoting hormones for the human fetus. Fetuses developing in diabetic women receive a large influx of glucose. This in turn may stimulate the expression of insulin-related sequences, which may result in higher utilization of glucose, thus bringing about the macrosomia and high incidence of malformation and still-births known to result from pregnancies in diabetics.

DNA↗
PubMed1985

Hybrid selection of messenger ribonucleoprotein for serum albumin: analysis of specific message-bound proteins.

The messenger RNA for chicken serum albumin together with its specific binding proteins was purified by hybrid selection using filter-bound cloned albumin cDNA. Under conditions where hybridization of the protein-mRNA complex was specific for the immobilized cDNA sequence, no dissociation of the complex into its protein and RNA components was apparent. Electrophoretic analysis of albumin mRNA-specific binding proteins showed three major bands. Two of these were prominent in total poly(A) messenger ribonucleoprotein. The third band was of much greater relative intensity in the albumin mRNA-specific proteins than in total poly(A) messenger ribonucleoprotein. The results suggest that the proteins bound to albumin mRNA represent only a subset of the total population of poly(A) mRNA-associated protein.

Animals↗
PubMed1984

Phosphorylation of tyrosine in cultured human placenta.

The [32P]phosphoamino acids in proteins of first-trimester and term-cultured human placentas have been separated and their relative amounts have been measured. Significant phosphorylation of tyrosine residues could be detected in the cultured placental tissue at different stages of gestation. The phosphotyrosine accounts for 2-4% of the total acid-stable phosphate in the phosphoamino acids after partial acid hydrolysis. The difference in the extent of [32P]tyrosine in various placentas seems to be a function of biological variation of the individual placentas, rather than a function of placental age and stage of gestation. In contrast, a significant difference in the phosphorylation ratio of serine and threonine could be measured between first-trimester and term placentas. As more evidence is accumulating that protein phosphorylation of tyrosine is involved in the processes of cellular growth and proliferation, our findings of the relatively high tyrosine phosphorylation in human placenta strongly suggest that this type of protein phosphorylation may play an important role in the placental growth and development. Furthermore, these findings may correlate with the existence of the endogenous RNA virus-like particles found in normal human placenta.

Culture Techniques↗
PubMed1984

Evidence for major differences in ribosomal subunit proteins from Plasmodium berghei and rat liver.

Purified polysomes were isolated in high yield from the erythrocytic stages of the rodent malaria parasite, Plasmodium berghei, and from rat liver. Proteins extracted from the ribosomal subunits derived from these polysomes were fractionated and their number and molecular weights were estimated by two-dimensional polyacrylamide gel electrophoresis. Plasmodial small ribosomal subunits contained 30 proteins ranging in apparent molecular size from 11.7 to 40.7 kDa, while large subunits contained 35-36 proteins ranging from 12.1 to 42.6 kDa. None of these parasite proteins was shared by the two subunits nor altered in electrophoretic mobility by radioiodination. Rat liver 40 S ribosomal subunit proteins numbered 30 and ranged from 9.2 to 37.5 kDa, while liver 60 S subunits contained 41-43 proteins with apparent molecular sizes of 10.3-45.2 kDa. Coelectrophoresis of trace amounts of radioiodinated P. berghei ribosomal subunit proteins and stainable quantities of liver proteins demonstrated that most of these 139 parasite and host ribosomal proteins possessed different two-dimensional electrophoretic mobilities under the conditions of this study. Based upon a comparative analysis of P. berghei and rodent ribosomal RNA and these data, it was concluded that parasite and host ribosomes contain distinct ribosomal RNAs and ribosomal proteins.

Animals↗
PubMed1984

Increased rates of polypeptide chain elongation in placental explants from human diabetics.

Average rates of polypeptide chain elongation were determined in placental explants of first trimester and term placentas from both normal and diabetic human pregnancies. Average ribosome half-transit times were determined by measuring the kinetics of transfer of labeled polypeptides from polysomal-bound to released polypeptides. The average half-transit time decreases from 75 sec per ribosome in first trimester explants to 56 sec per ribosome in term placentas. The average polypeptide molecular weights synthesized by explants from first trimester and in term are 49,300 and 49,600, respectively, which are not significantly different. The average elongation rates for first trimester and term placental explants are 172 and 231 amino acids per minute per ribosome, respectively, which are significantly different. Moreover, the average polypeptide molecular weight synthesized by term placentas from diabetic pregnancies is 48,200, while the average ribosome half-transit time is 40 sec. Thus, ribosomes from explants of term placenta from diabetics move along the average message at a much higher speed than do ribosomes in normal term tissue. The assembly rate of amino acid into polypeptide in explant of placenta of diabetic mothers is 314 amino acids per minute, which is significantly faster than 231 amino acids per minute in normal term tissue. These findings indicate that during placental development and in diabetic pregnancy there is a large change in the actual rates at which amino acids are added to the nascent polypeptide chain--i.e., the rates in polypeptide chain elongation. Therefore, translation-level regulation of protein synthesis in placenta plays a significant part in the magnitude of the response to developmental and other physiological stimulations.

Chorion↗
PubMed1983

Phosphorylation of tyrosine in cultured human placenta.

The [32P]phosphoamino acids in proteins of first trimester and term-cultured human placentas have been separated and their relative amounts were measured. A significant phosphorylation of tyrosine residues could be detected in the cultured placental tissue at different stages of gestation. The phosphotyrosine accounts for 2-4% of the total acid-stable phosphate in the phosphoamino acids after partial acid hydrolysis. The difference in the extent of [32P]tyrosine in various placentas seems to be a function of biological variation of the individual placentas, rather than a function of placental age and stage of gestation. In contrast, a significant difference in the phosphorylation ratio of serine and threonine could be measured between first trimester and term placentas. As more evidence is accumulating that protein phosphorylation of tyrosine is involved in the processes of cellular growth and proliferation, our findings of the relatively high tyrosine phosphorylation in human placenta strongly suggest that this type of protein phosphorylation may play an important role in the placental growth and development. Furthermore, these findings may correlate with the existence of the endogenous RNA virus-like particles found in normal human placenta.

Female↗
PubMed1983

Large-scale isolation of plasmid DNA and purification of lambda phage DNA using hydroxylapatite chromatography.

A rapid and relatively simple procedure for purifying large quantities of plasmid DNA is described. Plasmid thus purified contains no detectable chromosomal DNA and little RNA or protein. The procedure combines alkaline denaturation and hydroxylapatite chromatography and utilizes an improved method of separating DNA from RNA. It was observed that the phosphate concentrations at which previously bound DNA as well as RNA elute from hydroxylapatite changed markedly as a function of urea concentration. In the presence of urea concentrations higher than 4 M, the ranges of phosphate concentration over which DNA and RNA elute show no overlap. This permits efficient washing of hydroxylapatite-bound DNA under conditions which should remove all bound RNA. lambda Phage DNA is also easily eluted from hydroxylapatite under the conditions used.

Adsorption↗
PubMed1983

Effect of transfer RNA from various sources on placental messenger RNA translation.

Poly(A+)-containing mRNA from human term placenta was used to direct protein synthesis in a nuclease-treated rabbit reticulocyte lysate, which is dependent on mRNA and tRNA for maximal activity. The major protein product was human pre-placental lactogen (hPL). Addition of tRNA from rabbit liver, rabbit reticulocyte, human first trimester and term placenta, human liver and yeast resulted in 2-5-fold stimulation of [35S]methionine incorporation into total protein. Although all mammalian tRNA increased hPL synthesis, the relative synthesis as compared to endogenous globin was markedly different and most efficient with tRNA from term placenta. Addition of yeast tRNA increased total incorporation 3-fold but decreased incorporation of [35S]methionine into pre-hPL. These results suggest that the population of isoacceptor tRNAs may influence the expression of hPL in term placenta. Results are discussed by showing codon bias and usage of mRNA coding for hPL, alpha- and beta-hCG, rabbit globin and yeast alcohol dehydrogenase I.

Female↗
PubMed1983

Preferential utilization of exogenously supplied leucine for protein synthesis in estradiol-induced and uninduced cockerel liver explants.

A cockerel liver explant system has been used to study protein synthesis and ribosome transit times. After a 2-hr preincubation of explant tissue in the presence of a large concentration of nonradioactive leucine, a small quantity of [3H]leucine was added and the kinetics of uptake of [3H]leucine into the intracellular acid-soluble leucine pool was compared to the incorporation of [3H]leucine into protein. Incorporation of [3H]leucine into protein reaches a linear rate almost immediately after addition of label, whereas the acid-soluble pool does not reach constant specific activity until much later. The length of time needed to reach a linear rate of incorporation of [3H]leucine into protein is approximately equal to the length of time needed to equilibrate nascent polypeptide chains with labeled precursor--that is, one average ribosome transit time. Therefore, it seems that the immediate precursor pool for protein synthesis reaches constant specific activity almost instantly after addition of [3H]leucine. The results indicate that at least part of the supply of leucine for protein synthesis is derived directly from the exogenous incubation medium and not from the intracellular acid-soluble amino acid pool.

Amino Acids↗
PubMed1982

RNA synthesis in cultured human placenta.

The in vitro synthesis of RNA in the human placental tissue, incubated in organ culture, was investigated. We followed the synthesis of the poly A(-) and poly A(+) RNA fractions, and investigated the distribution of the newly synthesized RNA among the subcellular fractions isolated from first and third trimester placentas. The poly A(-) RNA was the major fraction of the RNA synthesized in vitro. The incorporation of [3H]uridine into the poly A(+) RNA fraction was very low. As protein synthesis occurred during the entire incubation period, we suggest the presence of a pool of mRNA molecules in the form of mRNP particles.

DNA↗
PubMed1982

The synthesis and secretion of human placental lactogen (hPL) in cultured term placenta.

We studied the in vitro synthesis and secretion of hPL by human term placental tissue incubated in organ culture. Placental tissue maintains a constant pool of hPL. The synthesis of hPL may be the driving force for its secretion. The de novo synthesis and secretion rates of hPL were also investigated. The higher specific radioactivities of the secreted hPL than those found in the tissue may suggest that newly synthesized hPL is preferentially released. The intracellular distribution of hPL was also compared.

Female↗
PubMed1982

Proteins associated with poly(A)+RNA of cockerel liver: effects of estradiol stimulation.

The protein population of poly(A)-containing messenger ribonucleoprotein (mRNP) from cockerel liver was analyzed before and after estradiol induction. Polysomal and free mRNP were isolated by thermal elution from oligo(dT)-cellulose. The proteins were separated by two-dimensional electrophoresis (of which the first dimension was a nonequilibrium pH gradient) and were visualized by silver staining. In order to determine similarities and differences between proteins of various mRNP fractions, trace amounts of 125I-labeled protein from one fraction were coelectrophoresed with stainable amounts of protein from another fraction on the same gel. Of 27 proteins analyzed, 15 were common to polysomal and free mRNP, 7 were specific to polysomal mRNP, and 5 were specific to free mRNP. Moreover, estradiol strongly influenced 11 proteins. Six proteins changed either in relative intensity or in distribution between polysomal and free mRNP fractions. Three major proteins appeared in both fractions, and two additional proteins disappeared from the free mRNP fraction after estradiol treatment. The results suggest that the protein population in polysomal mRNP is quite complex and that the profound influence of estradiol on protein synthesis in cockerel liver may be connected to changes in the protein composition of mRNP.

Animals↗
PubMed1981

An analysis of rates of polypeptide chain elongation in avian liver explants following in vivo estrogen treatment. I. Determination of average rates of polypeptide chain elongation.

Average rates of polypeptide chain elongation have been determined in cockerel liver explants on 4 successive days following an in vivo injection of 17 beta-estradiol. Incorporation of [3H]leucine by the explants is linear for at least 24 h, and the rate of protein synthesis increases significantly after estrogen injection. The explants synthesize and secrete serum albumin. B-apolipoprotein, and the phosphoprotein vitellogenin at relative rates which are similar to those reported for liver in vivo. Using this system, changes in the average rates of polypeptide chain elongation have been analyzed as a temporal sequence following a single injection of 17 beta-estradiol into cockerels. For this, average ribosome half-transit times were determined by measuring the kinetics of transfer of labeled polypeptides from polysomal-bound to released polypeptides. The data revealed a dramatic effect of estradiol on the average ribosome half-transit time with a maximum increase of 4.6-fold; however, the average size of polypeptides synthesized by explants at the peak of induction increased only 15% when compared to uninduced liver explants. These findings indicate that injection of estradiol results in large changes in the actual rates at which amino acids are added to the growing nascent polypeptide chain; that is, rates of polypeptide chain elongation. Therefore, translation-level regulation of protein synthesis in cockerel livers plays a significant part in determining the magnitude of the response to hormone stimulation.

Animals↗