The malonyl-CoA-sensitive form of carnitine palmitoyltransferase is not localized exclusively in the outer membrane of rat liver mitochondria

The data used to support the idea that malonyl-coenzyme A (CoA)-sensitive carnitine palmitoyltransferase (CPT-I) is localized on the outer mitochondrial membrane are based on harsh techniques that disrupt mitochondrial physiology. We have turned to the use of the French press, which produces a shearing force that denudes mitochondria of their outer membrane without the physiologically disruptive effects characteristic of phosphate swelling. Our results indicate that the mitoplasts contain just 15-19% of the outer membrane marker enzyme activity while retaining 85% of the total CPT activity and 50% of both CPT-I, as well as long-chain acyl-CoA synthase activity, the latter two supposed outer membrane enzymes. These mitoplasts were shown by electron microscopy to have the configuration of mitochondria that merely have been divested of their outer membranes. Carnitine-dependent fatty acid oxidation was retained in the mitoplasts, showing that they were physiologically intact. Moreover, protein immunoblotting analysis showed that CPT-I, as well as the inner CPT-II, was localized in the mitoplast fraction. The outer membrane fraction, which consisted of membrane "ghosts," contained most (50-60%) of marker enzyme activity, monoamine oxidase-B and porin proteins, but only about 27-29% CPT-I activity. Because CPT-I and long-chain acyl-CoA synthetase appear to be associated with both inner and outer membranes, we postulate that these enzymes reside in contact sites, which represent a melding of both limiting membranes.     

2nd-trimester maternal serum marker results are not altered by delayed analysis

OBJECTIVE: The goal of the study was to evaluate the significance of delayed laboratory analysis of maternal serum alpha-fetoprotein, beta-subunit of human chorionic gonadotropin, and unconjugated estriol for prenatal screening. METHODS: Biochemical analysis of 30 consecutive biochemical screening specimens of maternal serum alpha-fetoprotein, beta-subunit of human chorionic gonadotropin, and unconjugated estriol was performed immediately upon arrival to the laboratory, 7 days later, and again 14 days after maternal blood was drawn. Differences among the results of the three sets of biochemical studies were evaluated by one-way analysis of variance for repeated measures. RESULTS: No significant differences were found among the results of immediate assays as compared with those at a 7- or a 14-day delay for all three biochemical markers. CONCLUSIONS: Our data suggest that up to a 14-day delay in the performance of the 2nd-trimester maternal serum biochemical screening assays will not alter the results significantly. The results of maternal serum screening are, thus, clinically valid even if the laboratory assays were performed several days after maternal blood was drawn.     

The reinforcing effects of ethanol are altered by the endogenous neurosteroid, allopregnanolone

We examined the effect of systemic administration of the endogenously occurring progesterone metabolite, allopregnanolone, on oral self-administration of ethanol by male rats. Rats were trained to perform an operant response for presentation of 0.1 ml of a solution of 10% ethanol in water using the sucrose fading technique. After acquisition of stable lever-press responding on a fixed-ratio 4 schedule, subjects received subcutaneous injections of 1, 3, or 10 mg/kg of allopregnanolone, or vehicle, 20 min prior to the self-administration session. Pretreatment with 3 mg/kg, but not 1 or 10 mg/kg, increased the mean total number of lever press responses made to obtain ethanol, and therefore increased the mean total number of ethanol presentations. The number of responses and response rate were examined as a function of the number of "runs" within the 30-min session; a "run" was defined as a series of consecutive responses with an interresponse interval of <1 min. The increase in total responses after 3 mg/kg was due in part to an increased number of responses for the first run of the session, with no effect on response rates. However, the higher dose of 10 mg/kg decreased response rates within the first run. Thus, allopregnanolone alters ethanol-reinforced responding at concentrations lower than those that depress rates of responding. The effects of administration of the benzodiazepene, diazepam, were determined for comparison with those of the neurosteroid. The subcutaneous injection of 0.3, 1.0, or 3.0 mg/kg of diazepam did not produce any clear dose-dependent changes in measures of ethanol-reinforced operant responding, supporting the suggestion of differences in the contribution of the benzodiazepene and neurosteroid binding sites to GABA(A) receptor function. The results indicate that exogenous administration of allopregnanolone dose-dependently alters ethanol-reinforced operant responding, and suggest that this endogenously occurring neurosteroid could mediate some of the reinforcing effects of ethanol.     

Zinc levels in the rat fetal liver are not determined by transport across the placental microvillar membrane or the fetal liver plasma membrane

During pregnancy, zinc (Zn) levels in the rat fetal liver increase markedly. Why they do so or how the increase is regulated is unknown. We firstly investigated whether the increase occurs as a result of increased Zn transfer across the placenta and then tested whether the regulation occurred at the level of the microvillar membrane of the placenta or the fetal liver plasma membrane. Rats at different stages of gestation were injected with 7.5 microCi 65Zn in 100 microliters rat serum and killed after 1 h. 65Zn levels in the fetus remained constant at equivalent to the amount in 50 microliters maternal plasma per fetus until Day 18; at this time they increased to equivalent to 1.33 ml and then continued to increase until term. We isolated placental microvillar vesicles from placentas at each stage of gestation, characterized them, and measured Zn uptake. Zn uptake rates did not change during pregnancy. Similarly, we isolated vesicles from fetal liver plasma membrane and measured Zn uptake. Again, the uptake properties did not change during pregnancy. The data suggest that some other step in the transport process is rate limiting and that the increase in Zn levels in the fetal liver that occurs during pregnancy is possibly a result, rather than a cause, of metallothionein induction.     

In vitro inhibition of carnitine acyltransferase activity in mitochondria from rat and mouse liver by a diethylhexylphthalate metabolite

The effects of mono(2-ethyl-5-oxohexyl)phthalate [ME(O)HP], a di(2-ethylhexyl)phthalate (DEHP) metabolite and a potent peroxisomal inducer, on the mitochondrial beta-oxidation were investigated. In isolated rat hepatocytes, ME(O)HP inhibited long chain fatty acid oxidation and had no effect on the ketogenesis of short chain fatty acids, suggesting that the inhibition occurred at the site of carnitine-dependent transport across the mitochondrial inner membrane. In rat liver mitochondria, ME(O)HP inhibited carnitine acyltransferase I (CAT I; EC 2.3.1.21) competitively with the substrates palmitoyl-CoA and octanoyl-CoA. An analogous treatment of mouse mitochondria produced a similar competitive inhibition of palmitoyl-CoA transport whereas ME(O)HP exposure with guinea pig and human liver mitochondria revealed little or no effect. The addition of clofibric acid, nafenopin or methylclofenopate revealed no direct effects upon CAT I activity. Inhibition of transferase activity by ME(O)HP was reversed in mitochondria which had been solubilized with octyl glucoside to expose the latent form of carnitine acyltransferase (CAT II), suggesting that the inhibition was specific for CAT I. Our results demonstrate that in vitro ME(O)HP inhibits fatty acid oxidation in rat liver at the site of transport across the mitochondrial inner membrane with a marked species difference and support the idea that induction of peroxisome proliferation could be due to an initial biochemical lesion of the fatty acid metabolism.     

Mature carnitine palmitoyltransferase I retains the N-terminus of the nascent protein in rat liver

Carnitine palmitoyltransferase I was isolated from octylglucoside extracts of rat liver mitochondrial outer membranes. This native enzyme was digested proteolytically with V8 protease. Five major peptides were obtained all of which were found in the amino acid sequence predicted from the full-length cDNA sequence of the protein. One peptide was found to correspond to the extreme N-terminus of the deduced amino acid sequence. Therefore, the mature protein retains the N-terminus of the nascent protein after import into the mitochondrial membrane. Knowledge of the identity of the N-terminus of the mature protein allows a reappraisal of the role of the two main. N-terminal hydrophobic domains of the protein and of the possible topology of the protein within the membrane.     

The effects of a high fat diet on leptin mRNA, serum leptin and the response to leptin are not altered in a rat strain susceptible to high fat diet-induced obesity

Osborne-Mendel (OM) and S5B/Pl rats differ in their sensitivity to develop obesity when fed a high fat (HF) diet; OM rats become obese, whereas S5B/Pl rats remain thin. We have investigated the possibilities that either an impaired leptin response or resistance to leptin action underlies the sensitivity to this form of obesity in OM rats. In Experiment 1, OM and S5B/Pl rats fed a nonpurified diet were killed at d 0 or were fed either a HF (56% fat energy) or a low fat (LF, 10% fat energy) diet for 2 or 7 d. The HF diet increased serum leptin significantly by d 2 to levels that were similar in both rat strains. At 7 d, leptin levels were lower than at d 2 but remained higher than levels in the d 0 control groups. The leptin mRNA:18S RNA ratio in epididymal adipose tissue increased to higher levels in HF-fed OM rats than in S5B/Pl rats fed that diet. However, although the LF diet had no effect in S5B/Pl rats, it increased leptin mRNA levels in epididymal adipose tissue of OM rats compared with the controls fed the nonpurified diet. In Experiment 2, OM and S5B/Pl rats were fed HF or LF diets for 5 wk. At that time, their feeding response to a range of leptin doses (0, 1, 5 or 10 microgram) given intracerebroventricularly was tested after overnight food deprivation. There was a similar dose-dependent reduction in energy intake in response to leptin in both OM and S5B/Pl rats. These responses were independent of the diet. The data suggest that the susceptibility of OM rats to HF diet-induced obesity is not related to either a loss of central sensitivity to leptin or a failure to enhance leptin production acutely, although the failure to maintain chronically increased levels of serum leptin could contribute to the obesity.     

Oleate uptake kinetics in the perfused rat liver are consistent with pseudofacilitation by albumin

We measured uptake of a representative free fatty acid, oleate, by the single-pass perfused rat liver at oleate:albumin molar ratios of 0.01 to 2:1. For each ratio, uptake was studied at albumin concentrations from 50 to 600 microM. When uptake velocity was plotted as a function of the albumin concentration, the data at each ratio exhibited a pseudosaturation pattern as previously observed in isolated cells (J Clin Invest 84: 1325). At a physiologic albumin concentration of 600 microM, a plot of uptake vs. unbound oleate concentrations was best fitted by the Michaelis-Menten equation (Vmax = 235 +/- 8.8 nmol.min-1.g.liver-1; Km = 130 +/- 12 nM). As the albumin concentration was increased from 50 to 250 microM, the unbound oleate clearance, calculated by either the undistributed sinusoidal or venous equilibrium models, increased progressively, in violation of conventional pharmacokinetic theory, indicating an enhancing effect of albumin on ligand uptake at low albumin concentrations. In contrast, there was no significant difference between measures of unbound clearance at albumin concentrations of 350 and 600 microM. To explain this phenomenon, the clearance data were examined for evidence of facilitation (accelerated dissociation of ligand:albumin complexes) by the clearance ratio test ("square root rule"). All deviations from the predictions of conventional theory were entirely attributable to pseudofacilitation. No data required explanation by a true facilitation model.     

Cleavage planes in frog eggs are altered by strong magnetic fields

Early cleavages of Xenopus embryos were oriented in strong, static magnetic fields. Third-cleavage planes, normally horizontal, were seen to orient to a vertical plane parallel with a vertical magnetic field. Second cleavages, normally vertical, could also be oriented by applying a horizontal magnetic field. We argue that these changes in cleavage-furrow geometries result from changes in the orientation of the mitotic apparatus. We hypothesize that the magnetic field acts directly on the microtubules of the mitotic apparatus. Considerations of the length of the astral microtubules, their diamagnetic anisotropy, and flexural rigidity predict the required field strength for an effect that agrees with the data. This observation provides a clear example of a static magnetic-field effect on a fundamental cellular process, cell division.     

Accentuated antagonism in canine subendocardium is not altered by chronic exercise

Acetylcholine often affects cardiac action potential repolarization only during augmented adrenergic tone, i.e., the phenomenon of accentuated antagonism. Since chronic exercise involves repeated changes in autonomic outflow, we determined whether it also influenced adrenergic/cholinergic interactions in isolated canine cardiac tissue. Using standard micro-electrode techniques in thin ventricular subendocardial slices isolated from exercised (EX: 8-10 wk daily exercise) and sedentary (SED): 8-10 wk cage rest) dogs, we examined transmembrane potential responses to isoproterenol (ISO: 10(-8), 10(-7), 10(-6) M) and to ISO in the presence of ACH (10(-5) M). Control transmembrane characteristics at BCL = 500 ms were similar for EX (N = 8 dogs) and SED (N = 9 dogs). ISO (10(-6) M) decreased action potential duration at 50% repolarization (APD50): EX = -29 +/- 15 ms; SED = 11 ms and at 90% repolarization (APD90): EX = -37 +/- 17 ms; and SED = -24 +/- 14 ms (P > 0.05, EX vs SED). ACH alone did not alter APD. With ACH (10(-5) M), delta APD50 with ISO (10(-6) M) was -5 +/- ms and 0 +/- 5 ms for EX and SED, respectively; delta APD90 was -8 +/- 4 ms and -8 +/- 7 ms for EX and SED, respectively (P > 0.05, EX vs SED). Thus, ACH antagonized ISO-mediated acceleration of repolarization equally in both groups. Chronic daily exercise does not influence adrenergic/cholinergic interactions at the cellular level.     

Glucose transport correlates with GLUT2 abundance in rat liver during altered thyroid status

Glucose transport activity ([3H]D-glucose uptake) in liver sinusoidal membrane vesicles (SMVs) from hyperthyroid rats was significantly higher than that from euthyroid controls (2.1-times increase in V(max) with K(m) unchanged at approximately 18 mM), associated with increased GLUT2 expression. In contrast, glucose transport V(max) into SMVs from hypothyroid rats was reduced to 0.75-times that of euthyroid controls, associated with a reduced GLUT2 abundance. GLUT1 expression in SMVs was unaffected by changes in thyroid status. GLUT2, but not GLUT1 abundance on the blood-facing membrane of liver cells is sensitive to changes in thyroid status and these changes in transporter expression directly correlate (r = 0.96) with altered glucose transport activity.     

Vitamin K requirement and the concentration of vitamin K in rat liver

The concentration of vitamin K was determined in the liver of different strains of rats, and in male and female warfarin-resistant rats by feeding 3H-vitamin K in a purified diet. In each case, the level of vitamin K in the liver correlated approximately with the amount of vitamin K fed. The results indicate that differences in the requirement for vitamin K between the sexes and between strains of rats are due principally to different required concentrations of vitamin K in liver and not to differences in absorption or turnover of the vitamin. The results of the determination of vitamin K epoxide levels in male and female warfarin-resistant rats, and other data, suggest that the amount of vitamin K required in liver may be in part due to differences in the activity of the enzyme, vitamin K epoxide reductase.     

Word frequency effects in oral reading are not merely age-of-acquisition effects in disguise.

Four experiments examined the effects of the rated age of acquisition (AoA) and word frequency on oral reading latencies and word pronunciation durations. In Experiment 1, both AoA and frequency had independent (and noninteracting) effects on naming latencies. Experiment 2 found no effect of either AoA or frequency on delayed naming, indicating that prepared articulation time was not a factor contributing to the naming latencies observed in Experiment 1. Experiment 3 reported 2 replications of the study by C. M. Morrison and A. W. Ellis (1995). Both replications found reliable effects of frequency and AoA, whereas Morrison and Ellis found an effect of AoA but no effect of frequency. Experiment 4 found a strong AoA effect on pronunciation durations, with a smaller and less reliable effect of frequency. It was concluded that frequency affects the visual recognition of words and that AoA affects the production of lexical phonology. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Bacterial overexpression, purification, and reconstitution of the carnitine/acylcarnitine carrier from rat liver mitochondria

Seventy-seven patients with locally advanced breast cancer were treated with multimodality therapy comprising of six pulses of neo-adjuvant chemotherapy (doxorubicin, cyclophosphamide, vincristine and prednisolone) at 21-day intervals, followed by surgery (breast conservation or mastectomy) with appropriate axillary surgery, radiotherapy and adjuvant tamoxifen. The serum concentrations of acute phase proteins, C-reactive protein (CRP), á-1-anti-trypsin, albumin and transferrin were measured in serum taken prior to commencement of treatment. Patients were followed up for a median of 31 months and their clinical and histological responses and overall survival recorded. Univariate analyses revealed that tumour stage (p=0.01), clinical lymph node status (p=0. 02) and pre-treatment levels of serum albumin (p=0.002) and á-1-anti-trypsin (p=0.06) predicted overall survival. Using the Cox proportional hazards model reduced pre-treatment levels of serum albumin (p<0.00001), progressive lymph node involvement with tumour (p<0.005), and advancing tumour stage (p<0.01) were independent prognostic indicators for a poorer survival in patients with locally advanced breast cancer receiving neo-adjuvant chemotherapy.     

Recombinant rat liver S-adenosyl-L-methionine synthetase tetramers and dimers are in equilibrium

Wrens, Troglodytes troglodytesare highly polygynous passerines. Males build cock nests that are displayed to females and function as a mate-choice cue. Males with large numbers of nests are more likely to attract females than males with fewer nests. To be useful to females a cue used in mate choice must contain information. One way to evaluate the information content of such a cue may be to examine the factors that influence its size. The number of nests was predicted on territories with known building rates (immigration) and nest destruction rates (death) using an immigration-death function. This analysis suggested that if only building rate and destruction rates were considered then the number of nests on territories was consistently overestimated. In 1994 an experiment was conducted in which nest-site availability was manipulated. Males provided with additional nest sites built more nests and continued to build later in the year resulting in longer nest-building periods than controls. The converse was found when nest sites were experimentally removed. This suggested that the number of nests that could be constructed on a territory was limited by habitat structure. None of the morphological characters considered here significantly influenced the number of nests built by males. Therefore, the information content of this mate-choice cue will be primarily about characteristics of the habitat on the territory rather than characteristics of the male.1997The Association for the Study of Animal Behaviour     

Glycogenolysis stimulation by non-steroidal anti-inflammatories in the perfused rat liver is not accompanied by Ca2+ release

To better understand the characteristics of amyloid deposition in the choroid plexus, we examined autopsied brain by routine histology, immunohistochemistry, and electron microscopy in three group of patients: primary systemic amyloidosis (n = 7), cerebral amyloid angiopathy (CAA, n = 6), and controls (n = 3). Three of the CAA patients had Alzheimer's disease. Congophilic, birefringent amyloid deposits of the choroid plexus were seen in six of the seven cases of systemic light chain amyloidosis. Immunohistochemistry revealed that the deposited amyloids had reactivity for immunoglobulin light chain and amyloid P component. Accumulation of macrophages labeled with monoclonal antibodies against CD 68 and major histocompatibility complex class II antigens were observed around the massive amyloid deposits. The presence of approximately 10 nm amyloid fibrils along the epithelial basement membrane as well as in the vascular walls was ascertained by electron microscopy. In CAA, Congo red-positive amyloid deposits were consistently present in meningeal blood vessels and were often found in senile plaques of the cerebral parenchyma; congophilic amyloid deposits were absent in the choroid plexus. Choroid plexus epithelial cells exhibited immunostaining for beta amyloid precursor protein (APP) with N-terminal- and C-terminal-specific antibodies; in particular, consistent staining was obtained for the latter antibody. Immunoreactivity for amyloid beta protein (A beta) with monoclonal antibodies (6E10, 4G8) was often found in choroid plexus epithelial cells. These findings suggest that amyloid deposition of the choroid plexus depends on the major component protein in amyloidosis, and that the choroid plexus may produce APP and A beta protein although A beta amyloidosis is not evident in the choroid plexus.     

Early events in the development of neuronal polarity in vitro are altered by ethanol

Among the neuropathological effects of prenatal exposure to ethanol is the disruption of neuromorphogenesis. The effects of ethanol on early events in the development of axons and dendrites were studied using cultured embryonic rat hippocampal neurons, which develop in vitro in a stereotypical sequence of events that mimics their development in vivo. During the first 24 hr in culture, hippocampal neurons attach to the substrate and develop into one of three stages identified by phase-contrast microscopy: (i) neurons having lamellipodia and no processes (stage 1); (ii) neurons developing minor processes (<40 microm) that subsequently become the cell's axon or dendrites (stage 2); or (iii) polarized neurons with at least one axon (process with length > or =40 microm) (stage 3). Exposure to ethanol (300 mg/dl or 800 mg/dl) in the culture medium resulted in an increase in both the number of minor processes per neuron and the number of stage 3 neurons having more than the typical single axon. In addition, ethanol exposure significantly altered the proportion of neurons in the three early stages of development at 18 to 24 hr in vitro, without affecting overall neuron survival. With ethanol, there was a smaller proportion of neurons in the first stage of development, and a greater proportion of polarized stage 3 neurons. These findings suggest that ethanol alters the normal establishment of neuronal polarity, disrupting mechanisms that ensure the formation of the appropriate number of processes and that regulate the timing of process outgrowth.