Administration Of Puromycin Research Articles

Puromycin-induced kidney injury and subsequent regeneration in adult zebrafish

ABSTRACT Puromycin treatment can cause glomerular injury to the kidney, leading to proteinuria. However, the pathogenesis of acute kidney injury and subsequent regeneration after puromycin administration in animal models remain unclear. In this work, we examined the characteristics of kidney injury and subsequent regeneration following puromycin treatment in adult zebrafish. We intraperitoneally injected 100 μg of puromycin into zebrafish; sacrificed them at 1, 3, 5, 7, or 14 days post-injection (dpi); and examined the morphological, functional, and molecular changes in the kidney. Puromycin-treated zebrafish presented more rapid clearance of rhodamine dextran than control animals. Morphological changes were observed immediately after the puromycin injection (1–7 dpi) and had recovered by 14 dpi. The mRNA production of lhx1a, a renal progenitor marker, increased during recovery from kidney injury. Levels of NFκB, TNFα, Nampt, and p-ERK increased significantly during nephron injury and regeneration, and Sirt1, FOXO1, pax2, and wt1b showed an increasing tendency. However, TGF-β1 and smad5 production did not show any changes after puromycin treatment. This study provides evidence that puromycin-induced injury in adult zebrafish kidneys is a potential tool for evaluating the mechanism of nephron injury and subsequent regeneration.

Purification of human iPSC-derived cells at large scale using microRNA switch and magnetic-activated cell sorting.

SummaryFor regenerative cell therapies using pluripotent stem cell (PSC)-derived cells, large quantities of purified cells are required. Magnetic-activated cell sorting (MACS) is a powerful approach to collect target antigen-positive cells; however, it remains a challenge to purify various cell types efficiently at large scale without using antibodies specific to the desired cell type. Here we develop a technology that combines microRNA (miRNA)-responsive mRNA switch (miR-switch) with MACS (miR-switch-MACS) to purify large amounts of PSC-derived cells rapidly and effectively. We designed miR-switches that detect specific miRNAs expressed in target cells and controlled the translation of a CD4-coding transgene as a selection marker for MACS. For the large-scale purification of induced PSC-derived cardiomyocytes (iPSC-CMs), we transferred miR-208a-CD4 switch-MACS and obtained purified iPSC-CMs efficiently. Moreover, miR-375-CD4 switch-MACS highly purified pancreatic insulin-producing cells and their progenitors expressing Chromogranin A. Overall, the miR-switch-MACS method can efficiently purify target PSC-derived cells for cell replacement therapy.

Mesenchymal Stromal Cells Induce Podocyte Protection in the Puromycin Injury Model

Podocytes are specialized cells with a limited capacity for cell division that do not regenerate in response to injury and loss. Insults that compromise the integrity of podocytes promote proteinuria and progressive renal disease. The aim of this study was to evaluate the potential renoprotective and regenerative effects of mesenchymal stromal cells (mSC) in a severe form of the podocyte injury model induced by intraperitoneal administration of puromycin, aggravated by unilateral nephrectomy. Bone derived mSC were isolated and characterized according to flow cytometry analyses and to their capacity to differentiate into mesenchymal lineages. Wistar rats were divided into three groups: Control, PAN, and PAN+ mSC, consisting of PAN rats treated with 2 × 105 mSC. PAN rats developed heavy proteinuria, hypertension, glomerulosclerosis and significant effacement of the foot process. After 60 days, PAN rats treated with mSC presented a significant amelioration of all these abnormalities. In addition, mSC treatment recovered WT1 expression, improved nephrin, podocin, synaptopodin, podocalyxin, and VEGF expression, and downregulated proinflammatory Th1 cytokines in the kidney with a shift towards regulatory Th2 cytokines. In conclusion, mSC administration induced protection of podocytes in this experimental PAN model, providing new perspectives for the treatment of renal diseases associated with podocyte damage.

Multispectral Imaging Application in Chronic Kidney Disease Drug Discovery

Chronic kidney disease (CKD) is characterized by gradual loss of kidney functions over time. Several markers such as fibrosis, kidney injury marker-1 (KIM-1) and infiltrating macrophages (F4/80) have been shown to closely associate with CKD stages and treatment efficacy. Immunohistochemistry (IHC) provides detailed antigen expression patterns at sub-mm resolution. Manual IHC evaluation by pathologists is accurate but time consuming and prone to error when the number of sections for review is numerous. The Vectra high-throughput system utilizes multispectral imaging technology and machine learning algorithm to automatically identify and quantify antigen expression. We compared the automated Vectra platform with manual analysis procedures to quantitate kidney antigen expression following unilateral ureteral obstruction (UUO) in rodents. The results showed excellent correlations (fibrosis score r2=0.82, p=0.0008; KIM-1,r2=0.98, p<0.0001; F4/80, r2=0.804, p=0.002). Puromycin aminonucleoside-induced nephrosis (PAN) model induces podocyte damage and proteinuria, a primary prognostic marker of podocyte injury in rats. Using the automated Vectra platform to evaluate several podocyte related markers after puromycin administration we found GLEPP1(as an indicator of podocyte function) correlated significantly to proteinuria (r2=0.53, p<0.0001 with outlier; r2=0.90, p<0.0001 without outliers). In addition results from Vectra are highly reproducible among users. Our tests show that the Vectra imaging platform provides deeper analysis, highly reproducible among users and is a fast and reliable tool for monitoring CKD progression and preclinical drug development.

Estrogen directly induces expression of retinoic acid biosynthetic enzymes, compartmentalized between the epithelium and underlying stromal cells in rat uterus.

Estrogen (E2) has been shown to induce the biosynthesis of retinoic acid (RA) in rat uterus. Here we examined whether E2 could directly induce the enzymes involved in this process by using the ovariectomized rat. A retinol dehydrogenase that we have previously described, eRolDH, and the retinal dehydrogenase, RalDH II, were found to have markedly increased uterine mRNA levels within 4 h of E2 administration, independent of the prior administration of puromycin. eRolDH and RalDH II and their mRNAs were also increased in uteri of rats during estrus. This indicated that RA biosynthesis in rat uterus is directly controlled by E2 and provides a direct link between the action of a steroid hormone and retinoid action. We also examined the cell-specific localization of RalDH II by immunohistochemistry. The enzyme was observed in the stromal compartment, particularly in cells close to the uterine lumenal epithelium. eRolDH was observed only in the lining epithelial cells. Taken together with the previous observations of cellular retinol-binding protein and cellular retinoic acid-binding protein, type two also being expressed in the lumenal epithelium, we propose that RA production is compartmentalized, with retinol oxidation occurring in the lumenal epithelium and subsequent oxidation of retinal to RA occurring in the underlying stromal cells.

Cellular Retinoic Acid-binding Protein II Gene Expression Is Directly Induced by Estrogen, but Not Retinoic Acid, in Rat Uterus

It has been suggested that cellular retinoic acid-binding protein (II) (CRABP(II)) may have a role in the movement of retinoic acid (RA) to its nuclear receptors, thereby enhancing the action of RA in the cells in which it is expressed. RA has also been shown to increase expression of CRABP(II). Previous work from our laboratory has shown that 17 beta-estradiol (E2) administration to prepubertal female rats leads to acquisition of the ability of the lining epithelium to synthesize RA as well as to express CRABP(II). To determine whether this appearance of CRABP(II) was dependent on the production of RA, both E2 and RA were administered to ovariectomized rats. E2 administration induced expression of the CRABP(II) gene in the uterus within 4 h, and this induction was not inhibited by prior administration of puromycin, indicating that the induction was direct. In contrast, RA caused no change in CRABP(II) message level, even at times as late as 48 h after administration. Isolation and analysis of 4.5 kb of the 5'-flanking region of the gene revealed no apparent E2-response element. Using this portion of the gene to drive expression of the luciferase gene in transfected cells allowed identification of a region containing an imperfect estrogen-response element and estrogen-response element half-site, necessary for E2-driven induction. A possible Sp1 binding site in the 5'-flanking region of the CRABP(II) gene was also required for this induction. The ability of E2 to induce expression of CRABP(II) suggests that it can enhance the activity of RA, directly affecting expression of retinoid-responsive genes.

Renal structural abnormalities following recovery from acute puromycin nephrosis

Rats that recover from acute puromycin nephrosis later develop widespread glomerular and tubulointerstitial injury. The current study sought to identify structural changes present in the recovery phase that could precipitate progressive renal disease. Stereologic studies were performed 10 weeks after administration of puromycin (PAN) or saline (Cont). Serial sections were examined to assess glomerular structure. Rats receiving puromycin developed heavy proteinuria that returned nearly to control levels at 10 weeks. Kidneys in these animals were moderately enlarged and exhibited expansion of the interstitium (PAN, 254 +/- 47 mm3; Cont, 152 +/- 23 mm3; P < 0.05). The average glomerular volume was not different from control (PAN, 1.90 +/- 0.38 x 10(6) microm3; Cont, 2.07 +/- 0.47 x 10(6) microm3), but a subpopulation of glomeruli of about half normal size was found in PAN rats. Serial sections revealed that most of these glomeruli were not connected to normal tubule segments. Serial sections also revealed that more than 90% of glomeruli in rats recovering from nephrosis had synechias joining the tuft to Bowman's capsule. Synechias occupied an average of 8 +/- 11% of the Bowman's capsule surface in PAN animals versus less than 1% of the surface in controls. The appearance of synechias was not associated with a reduction in the mean number of visceral or parietal epithelial cells per glomerulus. Acute puromycin nephrosis does not cause a notable reduction in visceral epithelial cell number. However, widespread glomerular injury characterized by synechia between the tuft and Bowman's capsule is present following remission of proteinuria. Progression of this residual glomerular injury could contribute to the late development of glomerular segmental sclerosis following recovery from acute nephrosis.

Down-regulation of hepatic lipase expression in experimental nephrotic syndrome

Hepatic lipase (HL) plays an important role in catabolism of chylomicron remnants, conversion of intermediate density lipoprotein (IDL) to low-density lipoprotein (LDL) and reverse transport of cholesterol to the liver. Several features of the nephrotic dyslipidemia point to the possible presence of HL deficiency. In an attempt to address this possibility, gene expression of HL was studied in rats with puromycin-induced nephrotic syndrome (NS). The results were compared with those obtained in a group of placebo-treated control animals. The NS group showed marked proteinuria, hypoalbuminemia, hypercholesterolemia, hypertriglyceridemia, normal creatinine clearance and normal hepatic tissue cholesterol concentration. HL activity of the liver tissue was reduced by approximately 60% in the NS group as compared to that found in the normal control group. The reduction of HL activity in the NS group was accompanied by a reduction of HL mRNA of virtually similar magnitude. HL activity of the liver tissue was inversely related to urinary protein excretion, serum cholesterol and serum triglyceride concentrations. In contrast, HL activity was directly related to serum albumin concentration and HL mRNA. No significant difference was observed in HL activity between the control group and the pre-nephrotic animals studied at days 1 and 5 following puromycin administration. This observation excludes an acute effect of puromycin as a possible cause of HL deficiency in the NS animals. Thus, NS in this model results in a marked down-regulation of HL expression which may, in part, contribute to the nephrotic dyslipidemia.

Insulin-like growth factor and growth hormone receptor in nephrotic rats.

In an attempt to elucidate the mechanism of growth retardation in the nephrotic syndrome, specific serum and hepatic growth factors were measured in Sprague-Dawley rats in which nephrotic syndrome was produced by administration of puromycin (1.5 mg.100 g body wt-1.day-1) for 12 days. On the 13th day, the results of these nephrotic animals were compared with those of an equal number of pair-fed and control animals: the mean dietary intake of the nephrotic group was 71% that of the control group (P < 0.001). Serum insulin-like growth factor (IGF) binding protein-3 was significantly reduced (P < 0.005) in the nephrotic rats, compared with the pair-fed and the control groups. Hepatic IGF-I mRNA in the nephrotic rats averaged 36% that of control (P < 0.001) and 46% that of the pair-fed animals (P < 0.001). Hepatic growth hormone receptor (GHr) mRNA in the nephrotic rats averaged 19% of that of the control (P < 0.001) and 27% of that of the pair-fed rats (P < 0.001). These results indicate that the growth retardation of the nephrotic rats may be associated with the significant decrease in IGF-I mRNA and reduction in GHr mRNA.

Alterations in the morphology of nuages in spermatogonia of the fish, Oryzias latipes, treated with puromycin or actinomycin D.

A germ-cell specific organelle, nuage, in Oryzias latipes is polymorphic. Nuages with a strand-like structure and amorphous fibrous bodies can be discriminated from each other; furthermore 2 types of nuages of intermediate morphology are also present. After the administration of puromycin or actinomycin D, nuage morphology in spermatogonia was examined by electron microscopy. Puromycin as well as actinomycin D caused a significant increase in the incidence of nuages with a strand-like structure. These observations indicate that the loss of supply of some materials can induce morphological changes in nuages, suggesting that the polymorphism in nuages of O latipes possibly results from the turnover of nuage materials.

Regulation of intestinal apo A-IV mRNA abundance in rat pups during fasting and refeeding

The amount of intestinal apolipoprotein (apo) A-IV mRNA was examined in rat pups during fasting and refeeding. When 14-day old pups were fasted for 15 h, apo A-IV mRNA levels in the whole intestine decreased to 20% of the prefasting level. Refeeding casein and lactose, and the artificial milk composed of Intralipid, casein and lactose, caused an elevation of the apo A-IV mRNA after 3 h, without accompanying an elevation of serum triacylglycerols and apo A-IV (fat-independent elevation of apo A-IV mRNA). Refeeding Intralipid alone simultaneously elevated the apo A-IV mRNA, and serum triacylglycerols and apo A-IV after 3 h (fat-dependent elevation of apo A-IV mRNA). Administration of physiological saline during fasting partly suppressed the reduction of the apo A-IV mRNA (40% of the prefasting level), and the dietary fat-independent elevation of the message disappeared. Refeeding dam's milk to the pups, fasted without water administration, increased the apo A-IV mRNA after 3 and 15 h, although the elevation of serum triacylglycerols and apo A-IV occurred only after 15 h. Refeeding the milk increased the apo A-IV mRNA after 3 h and 15 h. Refeeding dam's milk to the pups fasted with saline administration accelerated the fat-dependent elevation of the apo A-IV mRNA. Simultaneously refeeding Intralipid and Pluronic L-81, an inhibitor of lymphatic fat transport, delayed the elevation of the apo A-IV mRNA and serum triacylglycerols and apo A-IV. Transcription rates of the apo A-IV mRNA, determined by nuclear run/on assay, were similar before and after fasting and refeeding Intralipid. During fasting, administration of puromycin, as compared with actinomycin D, enhanced the disappearance rate of the apo A-IV message. Intestinal mRNA for apo B, but not for apo A-I and β-actin, similarly changed to the apo A-IV message. Thus, it can be concluded that: (1) dietary fat-dependent and -independent factors are involved in the elevation of the intestinal apo A-IV message; (2) the elevation of the message is not mediated by lipid uptake in the enterocytes but rather stimulated by the events leading to secretion of chylomicrons; and, (3) dietary fat-dependent elevation of the message appears to be due to the stabilization of the message.

Tunicamycin, puromycin and brefeldin A influence the subcellular distribution of neuropeptides in hypothalamic magnocellular neurones of rat.

Magnocellular neurones in the supraoptic nuclei of normal Long Evans and homozygous Brattleboro rats were examined electron-microscopically after intracisternal injections of tunicamycin, puromycin, or brefeldin A. Moderate (50 micrograms) or high (200 micrograms) doses of tunicamycin caused the formation of electron-dense filamentous accretions in the endoplasmic reticulum (ER) cisterns of vasopressin neurones, but only the high dose of tunicamycin also caused accretions to form in the ER of some oxytocin neurones. Immunogold labelling of ultrathin sections from tunicamycin-treated rats revealed that, in about 5% of vasopressin neurones, the accretions could be immunogold-labelled for vasopressin and its associated neurophysin. However, in the majority of vasopressin neurones, the sections required trypsinisation before immunolabelling of the accretions could be detected. Small accretions in the ER of oxytocin neurones did not label for oxytocin or its neurophysin without prior trypsinisation, whereas larger accretions in other oxytocin cells could be labelled without prior trypsin treatment. Administration of puromycin resulted in the formation of small ER accretions in both vasopressin and oxytocin neurones. These accretions were immunolabelled with antisera, respectively, to vasopressin and oxytocin, but neurophysin-immunoreactivity was in most cases absent and was not revealed by treatment with trypsin, suggesting that neurophysin-immunoreactive epitopes were absent from truncated peptides forming the accretions. Brefeldin A caused dilatation of ER cisterns and disruption of the Golgi apparatus in both oxytocin and vasopressin neurones, but did not cause accretions to form in the ER.

Inhibition of protein synthesis does not block physiological responses to odors in the frog over the short term

1. 1. Frog olfactory mucosae were stimulated with an odorant every 60 sec and electro-physiological responses (electroolfactograms or EOGs) were monitored continuously—before, during, and after treatment with puromycin, a protein synthesis inhibitor. 2. 2. Repeated administration of puromycin at 4 hr intervals over a 36 hr period failed to suppress EOG responses. 3. 3. In an alternative approach, olfactory responses were inhibited with ethyl bromoacetate, a vaporous alkylating agent. EOG responses failed to reappear over a 72 hr period. 4. 4. We conclude that receptor turnover is not readily influenced by stimulation, and that the turnover time of frog olfactory receptor proteins is of the order of days or longer.

Effect of clofibrate treatment on aconitase activity in rat liver and other tissues

1. 1. The activity of both mitochondrial and cytosolic aconitases was significantly increased in the livers of male rats following treatment with the hypolipidemic drug clofibrate. 2. 2. Cycloheximide or puromycin administration to rats inhibited the inducing effect of clofibrate on the enzyme activity. 3. 3. Aconitase activity in small intestine homogenate was also increased by clofibrate. The drug did not affect the enzyme activity in rat kidney, heart and brain.

The preparation of puromycin antibody and its use in enzyme immunoassay for the quantification using beta-D-galactosidase as a label.

An antibody specific for puromycin (PU) was prepared by immunization of rabbits with a PU conjugate of bovine serum albumin, which was newly synthesized by coupling PU to mercaptosuccinylated bovine serum albumin via a cross-linker, N-(gamma-maleimidobutyryloxy)succinimide. Enzyme labeling of PU was performed using beta-D-galactosidase [EC 3.2.1.23] via N-(m-maleimidobenzoyloxy) succinimide. An ultrasensitive and specific enzyme immunoassay for PU was developed utilizing these reagents by a double antibody technique. The standard curve of the assay was linear in the range of 2 pg to 100 pg, and the lower limit of detection was 28.2 pm (2 pg/tube); so the enzyme immunoassay was found to be approximately 326,000 times more sensitive than a microbiological assay. Further, the enzyme immunoassay is free from interference by any purine or pyrimidine analogs, or by other drugs commonly used for the inhibition of protein synthesis. Using this assay, drug levels were easily determined in rat tissue following PU administration. Since PU is extensively available as an inhibitor of protein synthesis, the enzyme immunoassay should provide useful tool for developing biochemical and toxicity studies of PU.

Inducing effect of clofibrate on alkaline phosphatase and histidine-glyoxylate aminotransferase in rat liver.

The activity of plasma membrane alkaline phosphatase and both mitochondrial and peroxisomal histidine-glyoxylate aminotransferase was significantly increased in the livers of male rats following treatment with the hypolipidemic drug clofibrate. Cycloheximide or puromycin administration to rats inhibited the effects of clofibrate.

Acute effects of selected hepatotoxic agents on polyribosomes and protein synthesis in the livers of rats fed purified diets containing hepatocarcinogens

This investigation was concerned with the effects of certain hepatocarcinogens ingested for long- or short-term intervals upon selected responses of rat liver to specific hepatotoxic stimuli. Rats fed ad libitum for long (3 to 45 weeks) periods or force-fed for short (3 days) periods purified diets containing a single hepatocarcinogen, ethionine, N-2-fluorenylacetamide, 3′-methyl-4-dimethylaminoazobenzene, or thioacetamide, were subjected to the acute administration of a single hepatotoxic agent, such as puromycin, actinomycin D, sparsomycin, hypertonic NaCl or CCl 4, and hepatic protein synthesis ( in vitro) and polyribosomal aggregation were evaluated. Relating to in vitro protein synthesis, in long-term experiments, puromycin induced less inhibition in the experimental groups than in the control (basal diet) group; and actinomycin D inhibited all groups similarly; while in the short-term experiments, puromycin induced less inhibition in the experimental groups than in the control group; actinomycin D inhibited he basal and ethionine groups more than the other groups; sparsomycin induced variable degrees of inhibition, with the ethionine group showing the most and the N-2-fluorenylacetamide and thioacetamide groups showing the least; hypertonic NaCl markedly inhibited all groups similarly; and CCl 4 induced somewhat variable degrees of inhibition, with the thioacetamide group showing less than the other groups. In a few long-term experiments where hepatomas were evaluated in relation to surrounding liver tissue, puromycin administration induced less inhibition in the hepatomas than in the surrounding liver while actinomycin D administration induced similar inhibition in both groups. In general, in all experiments the degree of polyribosomal disagregation was parallel to that of inhibition of protein synthesis ( in vitro). The results reveal that the livers of rats fed purified diets containing selected hepatocarcinogens responded in a variable manner in regard to protein synthesis ( in vitro) and status of polyribosomal aggregation to the acute administration of selected hepatotoxic agents.

Differential transport of cholesterol and oleic acid in lymph lipoproteins: sex differences in puromycin sensitivity

Adult rats of both sexes were prepared with indwelling drainage catheters in the left thoracic lymphatic duct, and with duodenal infusion catheters. Control and puromycin-treated animals were administered an aqueous test emulsion containing [7alpha-(3)H]cholesterol and [1-(14)C]oleic acid, followed two hours later, by a tracer dose of [1-(14)C]-leucine. Successive 2-hr lymph samples were subjected to ultracentrifugal separations of the major lipoprotein classes. These were specifically extracted for lipids, and for DNA- and lipid-free protein. In both sexes, oleic acid absorption was largely associated with the d < 1.006 g/ml chylomicron fraction throughout the 6-hr experimental period. Small but consistent levels of labeled fatty acid appeared in the 1.006 < d < 1.019 g/ml VLDL fraction. However, with both sexes 25-35% of the absorbed cholesterol appearing in lymph was recovered in the VLDL fraction. Furthermore, there were statistically greater levels of cholesterol in this lymph fraction in females than in males. Cumulative protein levels and leucine incorporation into chylomicron proteins was comparable in both sexes. However, VLDL protein in the female was significantly greater than in the male and this difference was mimicked by the greater incorporation of leucine into VLDL proteins in the female. In males, there were no significant effects of puromycin on cholesterol or oleic acid absorption, despite a marked inhibition in chylomicron protein levels and leucine incorporation into this fraction. There was also no effect of the inhibitor on VLDL protein levels or on leucine incorporation into VLDL peptides. Cholesterol but not oleic acid absorption in females was significantly depressed by administration of puromycin, and this was largely attributed to a decrease in VLDL transport of the sterol. Also, unlike males, leucine incorporation into VLDL peptides was inhibited by 75% by puromycin administration. These results emphasize the importance of non-chylomicron transport of cholesterol during absorption and suggest a hormonal influence on intestinal VLDL synthesis in female rats.-Vahouny, G. V., E. M. Blendermann, L. L. Gallo, and C. R. Treadwell. Differential transport of cholesterol and oleic acid in lymph lipoproteins: sex differences in puromycin sensitivity.

Effect of puromycin administration on presynaptic bouton regeneration after hemisection of rat spinal cord

The effect of puromycin on synaptic renewal on spinal neurons 1 to 1.5 mm rostral to the site of a T1-T2 interface after hemisection was studied. Ninety-five Long-Evans rats were utilized (five animals per group) 10, 20, 30, 45, 60, and 90 days posthemisection. The groups consisted of normal animals, those with spinal cord hemisection alone, those with Gelfoam sponge saturated with 0.9% saline placed in the lesion, or those with a Gelfoam sponge saturated with 1.0 m m puromycin dihydrochloride in 0.9% saline placed in the lesion (at surgery). The spinal cords were processed for the light microscopic impregnation of presynaptic boutons. Counts of boutons (lamina IV and IX motoneurons) were made from coded material and analyzed statistically. After spinal cord hemisection alone, there was a cyclic significant renewal of presynaptic boutons on the somata of lamina IV and IX neurons with decreases ( P < 0.05) at 10 to 20 days, increases ( P < 0.05) at 30 days, and spontaneous loss ( P < 0.05) at 45 days. This small number of boutons was retained at 60 days postoperative (DPO) and remains at this value at 90 days on lamina IV neurons or increases on lamina IX neurons. Saline-puromycin-Gelfoam implantation resulted in a significant bouton decrease ( P < 0.05) on lamina IX neurons at only 45 days postoperative and no differences from normal on lamina IV neurons. These data show a “protective” effect that stabilized presynaptic boutons on lamina IV neurons for 90 days and on lamina IX neurons for at least 30 days.

Hepatic lipid metabolism. Age-related changes in triglyceride metabolism.

Age-related changes in hepatic triglyceride formation have been described in developing rats. Triglyceride formation was measured in vitro in the presence of [14C]glycerol-3-phosphate, palmitate, ATP, CoA, and Mg2+ by using liver homogenates and microsomal fractions derived from various age groups of animals. Triglyceride formation was most active in one-day-old rats and then decrease with age. The increase in triglyceride formation following birth was prevented by the administration of puromycin or by denying suckling. In addition, changes in plasma and hepatic triglyceride concentrations, were also determined as functions of age. These studies suggest that the age of the animal significantly influences triglyceride metabolism.