Affect Protein Levels Research Articles

Tracheal occlusion stimulates cell cycle progression and type I cell differentiation in lungs of fetal rats.

Fetal tracheal occlusion (TO) has been reported to stimulate lung growth but decreases number and maturation of type II cells, effects that vary with gestational age and duration of TO. We examined effects of a novel method of TO (unipolar microcautery to seal the trachea) produced at 19.5-20 days (d) of gestation in fetal rats; fetuses were delivered at term, 22 d. Controls were sham operated and unoperated littermates. TO increased wet lung weight but not dry lung weight or lung DNA and protein. To evaluate further the effects of TO, we examined the cell cycle regulators, cyclins D1 and A, in fetal lungs. Cyclin D1 increased with TO (P < 0.005). TO also increased expression of the type I epithelial cell marker RTI40 (mRNA and protein). TO decreased mRNA for surfactant proteins (SP)-A and -C but did not affect protein levels of SP-A and -B and of RTII70, a type II epithelial cell marker. We conclude that TO by microcautery, even of short duration, has diverse pulmonary effects including stimulating increased levels of cyclin D1 with probable cell cycle progression, type I cell differentiation, and possibly inhibiting type II cell function.

Social recognition in mice: Behavioral disruption with ketamine and blockade with antipsychotic drug

Withdrawal anxiety following chronic ethanol exposure is often associated with relapse in recovering alcoholics. It is likely that brain regions regulating anxiety-like behaviors adapt during chronic ethanol exposure to ultimately regulate such behaviors. The central amygdala contains numerous neurotransmitter systems that have been implicated in the regulation of anxiety-like behavior, including corticotropin releasing factor (CRF) and NMDA-type glutamate receptors. Chronic ethanol exposure causes functional adaptations in both CRF and NMDA receptors that are likely to regulate anxiety-like behaviors expressed during withdrawal. However, the molecular mechanisms governing these adaptations remain unexplored. We therefore evaluated these neurotransmitter systems in Sprague–Dawley rats during chronic ingestion of an ethanol-containing liquid diet. Quantitative real-time reverse transcription–PCR demonstrated that preproCRF mRNA was significantly upregulated by chronic ethanol exposure, whereas mRNA expression of CRF binding protein did not change. There were also no significant changes observed in any of the NMDA subunit mRNAs, although there was a trend toward greater NR2A mRNA expression during chronic ethanol exposure. Using Western blotting analysis we measured NMDA receptor subunit protein expression. Chronic ethanol exposure did not affect protein levels of the NR1 and NR2B subunits. Like the mRNA measures, chronic ethanol exposure did influence NR2A protein levels but the effects were modest. Our results demonstrate that NMDA receptor subunit mRNA and protein expressions are not strongly influenced by exposure to chronic ethanol. This suggests that the functional NMDA receptor adaptations identified in previous studies [Roberto, M., Schweitzer, P., Madamba, S. G., Stouffer, D. G., Parsons, L. H., & Siggins, G. R. (2004). Acute and chronic ethanol exposure alter glutamatergic transmission in rat central amygdala: an in vitro and in vivo analysis. J Neurosci 24, 1594–1603] are likely to be mediated by post-translational events. In contrast, enhanced levels of CRF during/after chronic ethanol exposure are likely to be mediated by increased levels of prepro CRF mRNA. Together, our findings suggest that adaptations to chronic ethanol exposure by proanxiety factors expressed in the central nucleus appear to be mediated by distinct cellular and molecular mechanisms.

Inhibition of stearoyl-CoA desaturase activity by the cis-9, trans-11 isomer and the trans-10, cis-12 isomer of conjugated linoleic acid in MDA-MB-231 and MCF-7 human breast cancer cells

Conjugated linoleic acid (CLA) is a collective term for a group of positional and geometric conjugated dienoic isomers of linoleic acid. CLA has been shown to have strong inhibitory effects on mammary carcinogenesis both in vitro and in vivo. In this study, we investigated the regulation of human stearoyl-CoA desaturase (SCD, EC 1.14.99.5) expression by CLA in human breast cancer cell lines, MDA-MB-231 and MCF-7. Treatment of the cells with the cis-9, trans-11 and trans-10, cis-12 CLA isomers (45 μM) did not repress SCD mRNA in both MDA-MB-231 and MCF-7 cells. However, the cis-9, trans-11 and trans-10, cis-12 CLA isomers significantly decreased SCD protein levels and SCD activity in MDA-MB-231 cells. In MCF-7 cells, both isomers did not affect protein levels, but they inhibited SCD activity. These results suggest that in MDA-MB-231 cells the cis-9, trans-11 and trans-10, cis-12 CLA isomers regulate human SCD by reducing SCD protein levels, while in MCF-7 cells both isomers have a direct inhibitory effect on SCD enzyme activity.

Regulation of fibronectin fibrillogenesis by protein kinases in cultured rat osteoblasts.

Fibronectin (Fn) plays an important role in the regulation of adhesion, migration, and maturation of osteoblasts. Fn fibrillogenesis is involved in the process of bone mineralization. To elucidate the regulatory role of protein kinases in the formation of fibrillar Fn matrix, Fn synthesis and assembly were examined in cultured osteoblasts. Osteoblasts assembled the endogenously released soluble Fn into immobilized form on the substratum in a time-dependent manner. Both 12-O-tetradecanoylphorbol-13 acetate (TPA) and forskolin increased the synthesis of Fn. However, the extracellular assembly of Fn fibril from both endogenously released and exogenously applied soluble Fn was increased by TPA but decreased by forskolin. Protein kinase C (PKC) inhibitors, such as H7, Ro 318220, and Gö 6976, inhibited Fn fibrillogenesis. These results suggest that the dynamic of Fn fibrillogenesis is differentially regulated by the activation of PKC and protein kinase A (PKA). Both classic and novel isoforms of PKC are involved in the action of TPA in osteoblasts. It has been reported that alpha5beta1 integrin is related to Fn fibrillogenesis. Immunocytochemistry and flow cytometry showed that TPA and forskolin increased and inhibited, respectively, the clustering and surface expression of alpha5 integrins. TPA and forskolin did not affect protein levels of alpha5 integrins. The Western blot and reverse transcriptase-polymerase chain reaction showed that protein and mRNA levels of beta1 integrins also were not affected by TPA and forskolin. These results suggest that TPA and forskolin may affect the surface expression of alpha5beta1 integrins. cAMP response element-binding protein phosphorylation is involved in the action of forskolin but not that of TPA. Our results suggest that PKC activation enhanced Fn fibrillogenesis, whereas PKA activation inhibited extracellular Fn fibrillogenesis in primary cultured osteoblasts. Cytosolic Fn synthesis and extracellular Fn assembly may be differentially regulated by the activation of PKA.

Genetic and phenotypic polymorphisms of the A subunit of Coagulation factor XIII in Japanese population.

To clarify whether genetic polymorphisms in exon 14 of Coagulation factor XIII A-subunit gene (FXIIIA) affect phenotype expressions, we studied genetic polymorphisms in exon 14 of FXIIIA in a Japanese population and the relationship between the genetic polymorphisms and phenotype expression. Genetic polymorphisms in exon 14 of FXIIIA of 144 unrelated Japanese were analyzed by single-strand conformation polymorphism. Plasma FXIIIA antigen concentrations, FXIII activities, and phenotype were also determined by two-dimensional electrophoresis. The frequencies of the three genotypes, the homozygote (AD), the homozygote (BC) and the heterozygote (AD/BC), were 77.1, 0.7, and 22.2%, respectively. The gene frequencies of AD and BC were 0.88 and 0.12. We detected AD (GTT x GAG) and BC (ATT x CAG) at codon 650 and 651 of exon 14. There were no significant differences of FXIIIA antigen concentrations and FXIII activities between these genotypes. We detected three pl differences among them as being pls of 5.3, 5.6, 5.8 in the homozygote (AD) and the heterozygote (AD/BC), and a pl of 5.8 in the homozygote (BC). These polymorphisms affected isoelectric mobility, but did not affect protein levels, enzyme activities, or the molecular weight of FXIII.

Expression and redistribution of cellular Bad, Bax, and Bcl-X(L) protein is associated with VCD-induced ovotoxicity in rats.

Previous studies have shown that 4-vinylcyclohexene diepoxide (VCD)-induced ovotoxicity in rats is likely caused by acceleration of the normal rate of atresia (apoptosis). VCD-induced ovotoxicity is specific for small preantral follicles and is associated with increased activity of caspase cascades. The present study was designed to investigate the alteration of expression and distribution of several Bcl-2 family member proteins induced by dosing of VCD in rat small ovarian follicles. Female F344 rats were given a single dose of VCD (80 mg/kg, i.p., 1 day; a time when ovotoxicity is not initiated), or dosed daily for 15 days (80 mg/kg, i.p., 15 days; a time when significant ovotoxicity is underway). Four hours following the final dose, livers and ovaries were collected. Ovarian small (25-100 microm) and large (100-250 microm) preantral follicles were isolated, and subcellular fractions (cytosolic and mitochondrial) were prepared. Compared with controls, levels of the proapoptotic protein, Bad, were greater in both cytosolic and mitochondrial fractions of small preantral follicles collected from 15-day VCD-treated rats (cytosol, 1.97 +/- 0.16; mitochondria, 2.20 +/- 0.24, VCD/control, P < 0.05). After 15 days of daily VCD dosing, total cellular antiapoptotic Bcl-x(L) protein levels were unaffected in small preantral follicles, but its distribution in mitochondrial and cytosolic components was altered (mitochondria, 0.635 +/- 0.08; cytosol, 1.39 +/- 0.14, VCD/control, P < 0.05). Likewise, VCD did not affect protein levels of proapoptotic Bax in small follicles on Day 15. However, consistent with a Bax-mediated mechanism of apoptosis, the relative ratio of Bax/Bcl-x(L) in the mitochondrial fraction of small preantral follicles was significantly increased by VCD dosing (1.62 +/- 0.21, VCD/control, P < 0.05). Immunofluorescence staining intensity evaluated by confocal microscopy visualized cytochrome c protein in the cytosolic compartment in granulosa cells of preantral follicles in various stages of development. Relative to controls, within the population of small preantral follicles, staining intensity was less (P < 0.05) and presumably more diffuse, specifically in stage 1 primary follicles from VCD-treated animals (15 days). VCD caused none of these effects in large preantral follicles or liver (not targeted by VCD). These data provide evidence that the apoptosis induced by VCD in ovarian small preantral follicles of rats is associated with increased expression of Bad protein, redistribution of Bcl-x(L) protein and cytochrome c from the mitochondria to the cytosolic compartment, and an increase in the Bax/Bcl-x(L) ratio in the mitochondria. These observations are consistent with the involvement of Bcl-2 gene family members in VCD-induced acceleration of atresia.

Mechanisms of inhibition of the Ras-MAP kinase signaling pathway in 30.7b Ras 12 cells by tea polyphenols (-)-epigallocatechin-3-gallate and theaflavin-3,3'-digallate.

Our previous study showed that tea polyphenols inhibited MAP kinase and AP-1 activities in mouse epidermal JB6 cells and the corresponding H-ras-transformed cell line 30.7b Ras 12. The present study investigated the mechanisms of this inhibition. The cells were incubated with (-)-epigallocatechin-3-gallate (EGCG) or theaflavin-3,3'-digallate (TFdiG) (20 mM) for different times, and the cell lysate was analyzed by immunoblotting. EGCG treatment decreased the levels of phospho-Erk1/2 and -MEK1/2 time-dependently (by 60% at 60 min). TFdiG lowered their levels by 38%-50% at 15 min. TFdiG effectively decreased total Raf-1 protein levels, most likely through lysosomal degradation. EGCG did not affect protein levels or the activity of Raf-1 significantly but decreased its association with MEK1 as determined by co-immunoprecipitation. In addition, EGCG and TFdiG (10 mM) inhibited the phosphorylation of Elk-1 by isolated phospho-Erk1/2 in vitro. This inhibition of Erk1/2 activity is Elk-1 concentration-dependent and ATP concentration-independent, which suggests that EGCG and TFdiG interfere with the binding of the protein substrate to the kinase. The presently demonstrated specific mechanisms of inhibition of MAP kinases by EGCG and TFdiG may help us to understand the effects of tea consumption on cancer, inflammatory diseases, and cardiovascular diseases.

Immunochemical studies on the putative plasmalemmal receptor for 1,25-dihydroxyvitamin D 3. III. Vitamin D status

The effect of vitamin D status on levels of the putative 1,25(OH) 2D 3 membrane receptor (pmVDR) was studied in chick intestine, kidney, and brain. Western analyses and assays for specific [ 3H]1,25(OH) 2D 3 binding indicated that, in intestine, pmVDR levels were greatest in −D chicks relative to +1,25D and +D animals ( P < 0.05). In kidney, protein levels and specific binding followed the order +D > +1,25D, −D. In brain, vitamin D status did not affect protein levels or specific binding levels. In tissue from normal chicks, both protein and specific binding followed the order of intestine > kidney > brain membranes. Intestinal cells were further evaluated for the effect of 1,25(OH) 2D 3 on selected “rapid responses.” Extrusion of 45Ca in response to 130 pM 1,25(OH) 2D 3 in vitro was greater in cells from −D chicks than from +1,25D or normal birds. Analyses of signal transduction events revealed diminished hormone-induced intracellular calcium oscillations (as assessed by fura-2 fluorescence), and lack of steroid-enhanced protein kinase (PK) A activity in intestinal epithelial cells from −D chicks relative to +D chicks. PK C activation by 130 pM 1,25(OH) 2D 3 was approximately twofold in cells from +D or −D chicks. The combined results indicate that vitamin D status differentially affects the pmVDR in intestine, kidney, and brain. In intestine, vitamin D deficiency differentially affects 45Ca handling, intracellular calcium oscillations, PK A and PK C activities in response to 1,25(OH) 2D 3.

Inhibition of insulin-like growth factor I receptor signaling by the vitamin D analogue EB1089 in MCF-7 breast cancer cells: A role for insulin-like growth factor binding proteins.

Insulin-like growth factors I and II (IGF-I and IGF-II) are potent mitogens involved in growth regulation of breast epithelial cells and are implicated in the pathophysiology of breast cancer. Their bioactivity is enhanced or inhibited by specific IGF-binding proteins (IGFBPs). Vitamin D-related compounds (VDRCs) have been shown to inhibit proliferation and induce apoptosis of MCF-7 breast carcinoma cells. We have previously demonstrated that VDRCs antagonize the growth-promoting activity of IGF-I by stimulating autocrine production of IGFBP-5 in MCF-7 cells, but the effect of VDRCs on IGF-I receptor (IGF-IR) intracellular signaling has not been elucidated. We report here that the vitamin D analogue EB1089 interferes with the IGF-IR signaling pathway by attenuating IGF-I-induced tyrosine phosphorylation of IRS-1, and to a lesser extent, IRS-2. It does not affect protein levels of IRS-1, IRS-2 or IGF-IR. However, EB1089 does not inhibit tyrosine phosphorylation of IRS-1 induced by des(1-3) IGF-I, an IGF-I analogue with greatly reduced affinity for IGFBPs. Furthermore, we demonstrate that an antisense IGFBP-5 oligodeoxynucleotide attenuates EB1089-induced inhibition of IGF-I-stimulated tyrosine phosphorylation of IRS-1 and EB1089-induced IGFBP-5 accumulation. These data strongly suggest that IGFBP-5 plays a functional role in the interfering action of EB1089 with the IGF-IR signal transduction pathway.

New collaborations make pharmacogenomics a SNP

Single nucleotide polymorphisms (SNPs) are variations of one nucleotide between the DNA sequence of individuals. The variation can be a substitution, an insertion or a deletion. The occurrence of SNPs is approximately one every 800 bp in the human genome. They are found in genes that encode proteins (sometimes called cSNPs) and in non-coding regions. These polymorphisms could be the basis for most human diseases because of their potential functional importance. If the SNP occurs in the protein-coding region of a gene, it could be in the ‘wobble’ position of a codon and so not affect the amino acid sequence of the protein (i.e. it is ‘silent’); or it could be in a conserved position and could potentially alter the function of the protein. If the SNP results in the formation of a stop codon, the protein will be truncated and might or might not be functional. cSNPs that are insertions or deletions can cause shifts in the reading frame of the gene, resulting in the wrong amino acid sequence. Additionally, if the SNP occurs in the control region of the gene, it could affect protein levels through effects on transcription and/or translation.

Analysis of an even-skipped rescue transgene reveals both composite and discrete neuronal and early blastoderm enhancers, and multi-stripe positioning by gap gene repressor gradients.

The entire functional even-skipped locus of Drosophila melanogaster is contained within a 16 kilobase region. As a transgene, this region is capable of rescuing even-skipped mutant flies to fertile adulthood. Detailed analysis of the 7.7 kb of regulatory DNA 3' of the transcription unit revealed ten novel, independently regulated patterns. Most of these patterns are driven by non-overlapping regulatory elements, including ones for syncytial blastoderm stage stripes 1 and 5, while a single element specifies both stripes 4 and 6. Expression analysis in gap gene mutants showed that stripe 5 is restricted anteriorly by Krüppel and posteriorly by giant, the same repressors that regulate stripe 2. Consistent with the coregulation of stripes 4 and 6 by a single cis-element, both the anterior border of stripe 4 and the posterior border of stripe 6 are set by zygotic hunchback, and the region between the two stripes is 'carved out' by knirps. Thus the boundaries of stripes 4 and 6 are set through negative regulation by the same gap gene domains that regulate stripes 3 and 7 (Small, S., Blair, A. and Levine, M. (1996) Dev. Biol. 175, 314-24), but at different concentrations. The 3' region also contains a single element for neurogenic expression in ganglion mother cells 4-2a and 1-1a, and neurons derived from them (RP2, a/pCC), suggesting common regulators in these lineages. In contrast, separable elements were found for expression in EL neurons, U/CQ neurons and the mesoderm. The even-skipped 3' untranslated region is required to maintain late stage protein expression in RP2 and a/pCC neurons, and appears to affect protein levels rather than mRNA levels. Additionally, a strong pairing-sensitive repression element was localized to the 3' end of the locus, but was not found to contribute to efficient functional rescue.

Choroid plexus ion transporter expression and cerebrospinal fluid secretion.

The Cl-/HCO3- exchanger (AE2 isoform) and the Na+/K(+)-ATPase at the choroid plexus are both thought to be involved in CSF secretion. However, both transport mechanisms are also postulated to have a role in CSF ion homeostasis raising questions as to which parameters control the expression of these transporters? Northern blots have been used to assess AE2 mRNA levels in rats subjected to alterations in blood pH or blood osmolality (a factor affecting CSF secretion). Six hours of alkalosis induced a 40% increase in AE2 mRNA (p < 0.01), suggesting that alterations in the expression of this transporter play a role in CSF pH homeostasis. In contrast, changes in osmolality did not affect AE2 mRNA. Western blots of Na+/K(+)-ATPase subunits were also examined to determine whether hypo and hyperkalemia affect protein levels of this transporter. There was a positive correlation between the plasma K+ concentration and both alpha 1- and beta 1 subunit protein levels suggesting a role for this transporter in CSF K+ homeostasis. As changes in plasma K+ and pH affect choroid plexus ion transporters but do not appear to alter CSF production, these results suggest the presence of compensatory mechanisms. Understanding of such mechanisms may facilitate therapeutic control of CSF production.

Acute depletion of serotonin down-regulates serotonin transporter mRNA in raphe neurons.

Serotonin transporter (5-HTT) mRNA and 5-HTT sites were measured 3 days after treatment with p-chlorophenylalanine, a tryptophan hydroxylase inhibitor. While 5-HTT mRNA levels decreased (P < 0.001) in the dorsal raphe nucleus, 5-HTT binding sites remained unchanged, suggesting that an acute depletion of 5-HT may induce an increase in the turnover of 5-HTT mRNA without affecting protein levels at 3 days.

Influence of nitrogen availability on the biochemical composition of three unicellular marine algae of commercial importance

Chaetoceros calcitrans (Paulsen) Takano, Tetraselmis suecica (Kylin) Butch. and Isochrysis galbana Parke were grown in artificial seawater containing either normal (9·8 mg atoms litre −1) or reduced (0·613 mg atoms litre −1) nitrogen. When grown in the nitrogen-deficient medium protein decreased and carbohydrate increased in all three species; lipid, however, increased in Chaetoceros and Isochrysis but decreased in Tetraselmis. The biochemical composition of Tetraselmis and Isochrysis sampled during the post-exponential phase of growth from normal medium was similar to that of cells growing exponentially in nitrogen-deficient medium. Salinities of 10–35‰ did not affect protein levels in Tetraselmis or Isochrysis but carbohydrate increased and lipid decreased in Isochrysis over the salinity range. Temperatures of 18–25 °C had little effect on the cell composition of either species. The energy potentially available from a unit volume of Chaetoceros and Isochrysis cells from normal medium was similar to that from cells grown in nitrogen-deficient conditions in both species. Tetraselmis had 34% more energy available when grown in normal compared to nitrogen-deficient medium.

(−)- threo-chlorocitric acid: A novel anorectic agent

A new class of peripherally acting anorectics is described in these studies. The four stereoisomers of chlorocitric acid which are structurally similar to the known anorectic, (−)- threo-hydroxycitric acid, all suppressed food intake when administered at high doses to rats. Only one of these isomers, (−)- threo-chlorocitric acid, retained its anorectic activity at lower doses. The anorectic potency of (−)- threo-chlorocitric acid was approximately 40-fold greater in dogs than in lean and obese rats. The decreased food intake in rats resulted in a significant reduction of body lipid, without affecting protein levels. In contrast to the tolerance which was observed with the continued administration of mazindol or diethylpropion, tolerance to the anorectic effect of (−)- threo-chlorocitric acid did not develop. These studies suggest that (−)- threo-chlorocitric acid might be useful as an antiobesity agent.

Physiological and metabolic variations in Carcinus mediterraneus (Crustacea: Decapoda) parasitized by Thelohania maenadis (Microspora: Microsporida): An ecophysiopathological approach

This paper examines the influence of Thelohania maenadis (Protozoa: Microsporida), a muscle microsporidian, on certain features of the metabolism of Carcinus mediterraneus (Crustacea: Decapoda). Comparison of biochemical parameters reveals that only hemolymph total protein and glucose levels are reduced significantly by this parasitic infection. Muscular effort results in a diminution of muscular glycogen and hemolymph glucose levels, while hepatopancreatic glycogen remains unaffected; lactate accumulates in the muscle tissue. Experimental variations in the environmental water temperature and salinity affect protein levels and metabolic indicators of carbohydrate metabolism. Proteinemia varies with temperature and salinity, but parasitic infection accentuates these variations. Glucose and lactate concentrations in the hemolymph are increased in healthy specimens after muscular effort, under conditions of increased temperature or hypoosmotic shock; microsporidiosis provokes the reverse of these tendencies, on a less pronounced scale. Hyperosmotic shock at high temperature leads to a slight reduction in glucose and lactate concentrations. The paper concludes with a discussion of the physiopathological significance of the results obtained as an expression of the effects of parasitism infestation.

NAA and Sevin on Composition, Development, and Abscission of Apple Fruit1

Abstract Naphthaleneacetic acid (NAA) applied as a spray to apple trees on May 7 reduced fruit set, number of seed per fruit, percent and amount of reducing sugar in flesh of young fruits but did not affect protein level or fruit size. Fruit from trees sprayed with NAA on May 18 had more seed and less reducing sugar than the checks. The late NAA spray also reduced fruit size and set but did not affect the protein level of flesh or seed. Sprays of 1-naphthyl methylcarbamate (Sevin) applied on the same dates reduced fruit set but did not affect other factors measured. We propose that the reduced metabolite supply (reducing sugar) in the young fruit is the primary reason for reduced fruit set on trees sprayed with NAA and that effect on seed number is not related to this change in composition.

Hepatic drug-metabolising enzyme activity and duration of hexobarbitone anaesthesia in barbitone-dependent and withdrawn rats

The activity of several hepatic drug-metabolising enzymes and the duration of hexobarbitone anaesthesia were determined in barbitone-dependent and withdrawn rats. Rats were made dependent on barbitone sodium by the administration of increasing amounts in the drinking water over a 32-day period. Abrupt withdrawal was accompanied by a characteristic withdrawal syndrome. The body weight, liver weight and body weight/liver weight ratio were found to be increased during barbitone treatment and to fall after withdrawal. The liver protein and glycogen content did not change during barbitone treatment. Withdrawal produced a transient reduction in hepatic glycogen content but did not affect protein levels. The capacity of hepatic microsomal preparations to metabolise hexobarbitone, aminopyrine and prontosil and to conjugate o-aminophenol was increased by barbitone treatment. Following withdrawal, the enzyme activity fell below that of control animals Four months after withdrawal the liver enzyme activity was still subnormal. The acute administration of phenobarbitone to 4-month withdrawn animals was found to restimulate hepatic microsomal drug-metabolising enzymes but the level attained was not as high as that found in phenobarbitone-treated control rats. The same degree of tolerance to injected hexobarbitone, as indicated by reduction in sleeping time, was found at all stages of barbitone treatment. A hypersensitivity to hexobarbitone was produced by withdrawal. It is concluded that both the tolerance in barbitone-dependent rats and the hypersensitivity of withdrawn animals can be adequately explained by altered hepatic drug-metabolising enzyme activity.