Glucose-6-phosphatase dependent substrate transport in the glycogen storage disease type-1a mouse.

Glycogen storage disease type 1a (GSD-1a) is caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis. A G6Pase knockout mouse which mimics the pathophysiology of human GSD-1a patients was created to understand the pathogenesis of this disorder, to delineate the mechanisms of G6Pase catalysis, and to develop future therapeutic approaches. By examining G6Pase in the liver and kidney, the primary gluconeogenic tissues, we demonstrate that glucose-6-P transport and hydrolysis are performed by separate proteins which are tightly coupled. We propose a modified translocase catalytic unit model for G6Pase catalysis.

Specification of pituitary cell lineages by the LIM homeobox gene Lhx3.

During pituitary organogenesis, the progressive differentiation of distinct pituitary-specific cell lineages from a common primordium involves a series of developmental decisions and inductive interactions. Targeted gene disruption in mice showed that Lhx3, a LIM homeobox gene expressed in the pituitary throughout development, is essential for differentiation and proliferation of pituitary cell lineages. In mice homozygous for the Lhx3 mutation, Rathke's pouch formed but failed to grow and differentiate; such mice lacked both the anterior and intermediate lobes of the pituitary. The determination of all pituitary cell lineages, except the corticotrophs, was affected, suggesting that a distinct, Lhx3-independent ontogenetic pathway exists for the initial specification of this lineage.

Autonomous growth in serum-free medium and production of hepatocellular carcinomas by differentiated hepatocyte lines that overexpress transforming growth factor alpha 1.

Transforming growth factor alpha (TGF-alpha) is a polypeptide closely associated with hepatocyte proliferation in vivo and in vitro. In order to investigate the mechanisms by which TGF-alpha contributes to hepatocyte replication and transformation, we isolated hepatocytes from mice bearing a human TGF-alpha transgene and examined their growth properties and gene expression in defined, serum-free culture. The transgenic hepatocytes continued to overexpress human TGF-alpha mRNA and peptide, and were able to proliferate without exogenous growth factors in primary culture, in contrast to nontransgenic mouse hepatocytes. In short-term culture the transgenic hepatocytes underwent 1 wave of DNA replication at 72-96 h in culture before senescing, similar to nontransgenic hepatocytes supplemented with epidermal growth factor. Constitutive expression of TGF-alpha rendered the transgenic hepatocytes unresponsive to further growth stimulation by exogenous TGF-alpha, as well as other mitogens such as epidermal growth factor and hepatocyte growth factor. However, it did not alter their sensitivity to growth inhibition by TGF beta 1, 2 and 3. The addition of nicotinamide to the culture medium enabled both transgenic and epidermal growth factor-supplemented normal hepatocytes to replicate repeatedly and survive for > or = 2 months in primary culture while maintaining differentiated traits. From these long-term primary cultures of transgenic and nontransgenic hepatocytes, we established immortalized cell lines (designated TAMH and NMH lines, respectively). Both lines continued to express differentiated adult hepatocytic markers such as albumin, alpha-1-antitrypsin, transferrin, and connexin 26 and 32 mRNAs, but also expressed mRNAs for the oncofetal markers alpha-fetoprotein and insulin-like growth factor II. Unlike the near-diploid NMH hepatocyte line, the transgenic TAMH hepatocyte line was quasi-tetraploid, strongly expressed human TGF-alpha mRNA, and was highly tumorigenic in nude mice. Well-differentiated hepatocellular carcinomas developed in nude mice given injections of the TAMH line, and these appeared similar to the primary liver tumors seen in TGF-alpha transgenic mice with regard to histology and strong expression of mouse and human TGF-alpha, insulin-like growth factor II, and alpha-fetoprotein mRNAs. Our data show that TGF-alpha overexpression causes autonomous hepatocyte proliferation and contributes to neoplasia but that additional cellular alterations must occur for carcinogenesis. Inappropriate expression of insulin-like growth factor II may constitute one of these steps. The TGF-alpha transgenic mouse hepatocyte line TAMH appears to undergo transformation in a similar manner to that of hepatocytes overexpressing TGF-alpha in vivo, and should serve as an ideal system in which to study hepatocarcinogenesis.

Higher cholesterol in human LDL is associated with the increase of oxidation susceptibility and the decrease of antioxidant defence: experimental and simulation data.

Increased low-density lipoprotein (LDL) cholesterol is a recognized risk factor for atherosclerosis. There is also strong evidence that oxidatively modified LDL initiates the development of this pathological process and the administration of antioxidants might have a protective effect. However, the appropriate trials did not provide completely consistent results. We found in this study that the oxidation kinetics and also the antioxidant effectiveness are different depending on the cholesterol content in LDL. Higher cholesterol in LDL causes an acceleration of its oxidation as well as an increase of resistance to the antioxidative effect of ascorbic acid. In searching for a theoretical background of this dual impact of cholesterol in LDL, computer simulation of LDL oxidation was used. It was found that the pre-existing level of lipid hydroperoxides together with the total amount of oxidizable lipid substrate associated with the cholesterol level in LDL were satisfactory prerequisites for a best fit to the experimental data. In conclusion, this study provides at least a partial explanation for some failures to arrest, by administration of antioxidants, the progression of atherosclerosis in animal and human hypercholesterolemia.

Copper-induced and photosensitive oxidation of serum low-density lipoprotein. The relation to cholesterol level and inter-species differences.

Hypercholesterolemia is associated with a higher risk for developing atherosclerotic coronary heart disease. During the past few years, evidence has been increasing that modification of lipoproteins, particularly low-density lipoprotein (LDL) oxidation, might be involved in the pathogenesis of atherosclerosis. To compare these factors metal-dependent and -independent photodynamic methods were used for the screening of several indexes of LDL oxidation. Lipid oxidation has been continuously monitored by the increase of conjugated dienes and verified by iodometric and thiobarbituric reaction assay. A close association between LDL cholesterol concentration (and/or serum cholesterol concentration) and LDL maximum diene formation was found using both methods and different sources of sera. With copper-induced oxidation, highly significant correlation coefficient r = 0.86, and with photo-sensitive oxidation r = 0.84 were noted. The data standardized to protein unit showed a reduced but still significant correlation. The extent of LDL oxidation was also closely related to preformed dienes, i.e., to the data obtained before the start of oxidation (r = 0.91). The rate of LDL oxidation was positively linked to LDL cholesterol using both oxidation methods but with photo-sensitive oxidation the rate was much higher. The lag time was inversely related to LDL cholesterol (standardized data) with Cu2+ induced oxidation but it was absent in the photosensitive oxidation. In animals known to be resistant to spontaneous atherosclerosis (rats, guinea pigs) a prolonged lag time, markedly reduced diene formation and lower LDL cholesterol in LDL in parallel was demonstrated. The fact that, using various methods (epidemiology, arteriography, autopsy), the cholesterol level in men was found positively linked to atherosclerosis development on the one hand, and positively associated to oxidation of human LDL on the other, strongly supports the concept on the important role of LDL oxidative modification in this pathological process.

Collapsin response mediator protein-3/unc-33-like protein-4 gene: organization, chromosomal mapping and expression in the developing mouse brain.

CRMPs (collapsin response mediator proteins)/ULIPs (unc-33-like proteins) are a family of intracytoplasmic proteins that are expressed mainly in the brain. The involvement of CRMP/ULIP members in neuronal differentiation, growth cone motility and axonal collapse has been suggested. We recently found that a member of this family, CRMP3/ULIP4, corresponds to POP66 (paraneoplastic oligodendrocyte protein of 66 kDa), a protein which may be associated with auto-immune induced-neuronal degeneration in paraneoplastic neurological syndromes. However, the physiological functions of these proteins remain to be elucidated. Further studies, including the generation of cell lines and of animals with modified/disrupted CRMP/ULIP gene expression, are necessary to explore the functions of this protein. We have cloned and determined the organization and chromosomal localization of the mouse gene encoding CRMP3/ULIP4. The gene is composed of 14 exons and spans more than 20 kb. We assigned the mouse CRMP3/ULIP4 gene to the distal end of chromosome 7. In mouse brain, in situ hybridization showed that CRMP3/ULIP4 mRNA is expressed mainly in the dentate gyrus of hippocampus, in the granular layers of cerebellum and in the inferior olive of the pons, the nucleus which controls movement and posture, and adjusts the major output of descending motor system.

[Determination of paternity using DNA analysis of chorionic villi]. / Urcení paternity analýzou DNA z choriových klku.

Using probes recognizing hypervariable regions in human DNA high precision paternity testing can be provided even in the first trimester of pregnancy. Paternity tests were performed in 14 families using DNA isolated from chorionic villi. The results indicate that by a single probe confirmation or exclusion of paternity from a DNA sample of chorionic villi the 1st trimester of pregnancy can be performed with a probability more than 99.99%.

Reperfusion of the ischaemic myocardium: the role of calcium and perfusion pressure.

1. Our experimental findings do not support the hypothesis that myocardial injury during post-ischaemic reperfusion is caused by a sudden calcium inflow into cardiac cells. This follows from these facts: a) omission of calcium from the reperfusion medium does not prevent, only delays, injury during reperfusion unless the medium has been employed also before and during ischaemia. b) increased calcium concentration in the reperfusion medium does not enhance reperfusion injury unless this increased concentration has been achieved also before and during ischaemia. 2. Manifestation or increase of ischaemic injury during reperfusion can be prevented only with difficulty. One of the important factors which can accelerate or increase reperfusion injury, is perfusion pressure and/or coronary flow rate. The mechanism of this effect is not well understood. Judging by the rate at which it unfolds, one can assume involvement of some physical processes such as explosive cellular expansion, wall "stress", etc.

Heart infarction-like effect induced by natural catecholamines in vitro.

Isolated rat hearts were perfused using retrograde technique under constant perfusion pressure or under constant coronary flow. The addition of L-epinephrine or L-nor-epinephrine (1 microgram per ml) into the perfusion medium for one hour caused visible and irreversible morphological changes. They became apparent usually after 4 hours of perfusion in the form of small, pale, opaque spots of streaks gradually enlarging on the surface or on the transverse section of myocardium. Light microscope and electron microscope examination showed the disintegration process analogous to myocardial infarction but lacking the infiltration with blood elements. The structural changes were preceeded by increased release of lactate dehydrogenase into the effluent, the most characteristic metabolic change a accompanying myocardial injury. Although the underlying mechanism of cardiotoxic action catecholamines remains to be clarified, several factors under consideration could be eliminated like hyperlipidemia, thrombogenic process or reduced total coronary inflow rate.

Human low-density lipoproteins: oxidative modification and its relation to age, gender, menopausal status and cholesterol concentrations.

Recently much evidence has accumulated indicating that oxidative modification of atherogenic lipoproteins plays an important role in atherogenesis. The goal of this study was to ascertain whether any association exists between this and the previously incriminated risk factors of atherosclerotic cardiovascular disease like age, gender and cholesterol concentration. Serum lipid profile, low-density lipoprotein (LDL) composition and indicators of LDL oxidation were examined in a cohort of healthy, predominantly middle aged men and women. LDL oxidation was assessed using the copper catalysis method, and monitored routinely by the increase in conjugated dienes over 4 to 24 hours. A more objective computer-aided technique was used to estimate the oxidative indices based on the sigmoidal fit to data. No marked differences between men and women were found with respect to mean age, total and LDL cholesterol, LDL protein and oxidation of LDL. The post-menopausal as compared to pre-menopausal status was associated with a greater extent of LDL oxidation, as well as with higher total serum cholesterol and its fractions, LDL cholesterol and LDL protein. No such differences were found in the data for men appropriately separated according to age. In a group with high risk LDL cholesterol, the total LDL oxidation was higher, as well as age and total cholesterol. Lag time and half-time of LDL oxidation were significantly shorter, while the oxidation rate of LDL was significantly faster when compared with data in the lower quartile. About six percent of participants had a considerably prolonged initial oxidation phase. These persons also showed low total and LDL cholesterol. High oxidation resistance was reversible and most probably caused by very low pre-existent oxidation products. Multiple regression analysis showed that the closest association among age, gender, lipid profiles and LDL oxidation indices existed between LDL cholesterol and conjugated diene production in both sexes (men: r = 0.93; women: r = 0.81). This association remained high even if adjusted for age. As in earlier epidemiological studies using logistic regression and showing age- and gender-related rising frequency of coronary heart disease, the present paper demonstrated age- and gender-related rising frequency of highly oxidized LDL. In both cases it was closely associated with an increasing LDL cholesterol concentration.