Postnatal differentiation of sex-specific distribution patterns of G6Pase,G6PDH and ME in the rat liver

Summary The activity of the liver enzymes G6Pase, G6PDH and ME was studied in rats of 2–9 weeks old by histochemical means. In addition, G6PDH and ME activity was quantitatively determined in homogenates. In the 2nd and 3rd week G6Pase is similarly distributed in both sexes: while in the periportal zone high activity is demonstrable, the perivenous zone shows only low activity. After this period a nearly homogeneous distribution pattern becomes evident in all animals. Sex difference occurs after the 6th week: in the livers of male rats the periportal maximum is sometimes combined with a second peak in the perivenous area, in females a steep gradient emerges with high activity in the periportal zone and a low one in the perivenous zone. In the first postnatal weeks G6PDH activity is very low in parenchymal cells, but very prominent in Kupffer cells. Around the 5th week there is an increase, predominantly in the perivenous zone of both sexes. While there is again a further decrease demonstrable in male rats, the G6PDH activity of female rats rises to high adult values. This increase seems to be restricted to the perivenous zone. ME can be demonstrated at first in leucocytes. In the course of the 3rd week there is an increase of activity in both sexes: ME is demonstrable in parenchymal cells of the perivenous area and in scattered hepatocytes of the periportal area. In male rats, the perivenous activity is diminished towards the end of the investigation period, in females, however, a high activity remains in the perivenous zone. The data show that in females the activity of NADP dependent enzymes is high in the perivenous zone, so it may be assumed that a lipogenic area is situated around the terminal efferent vessels. Because of the sex difference this area may be hormone-dependent. The lipogenic area is situated opposite to the gluco(neo)genic area which corresponds to the periportal zone.Parts of this study were presented as an Inaugural Dissertation to the Medical Faculty of the University of Freiburg by H.H.Supported by grants from the Deutsche Forschungsgemeinschaft (Sa 127/7 and the SFB 46 (Molgrudent)     

NADP-dependent dehydrogenases in rat liver parenchyma. II. Comparison of qualitative and quantitative G6PDH distribution patterns with particular reference to sex differences.

Qualitative histochemical G6PDH distribution patterns obtained in the liver acinus of adult male and female rats with an improved method (Rieder et al., 1978) served as a basis for the isolation by microdissection of tissue samples of defined zonal affiliation. G6PDH activity was assayed quantitatively in tissue samples of zones 1 and 3 by a microfluorometric method, using the oil well technique and enzymatic cycling (Burch et al., 1963; Lowry and Passonneau, 1972). With the use of a correlation system further evidence could be presented for the validity of the recently described qualitative distribution patterns. From a total of 50 analyzed tissue samples the following G6PDH activities were calculated: 4.25 +/- 1.56 U/g dry weight in zone 1 and 2.08 +/- 0.46 U/g dry weight in zone 3 of male and 7.21 +/- 1.03 U/g dry weight in zone 1 and 11.10 +/- 2.56 U/g dry weight in zone 3 of female rats. These data were corrected for interference from the G6PDH activity of the Kupffer cells within zone 1 samples (approximately 80 U/g dry weight), so that the actual relative values for the parenchymal activity could be estimated for the first time: 2 U/g dry weight in zones 1 and 3 of male animals, 5 U/g dry weight in zone 1 and 11 U/g dry weight in zone 3 of female animals. In female livers G6PDH activity in zone 1 is therefore 2.5 times higher, and in zone 3 5 times higher than in the male. These zonal as well as sex-differences are clearly indicative of a heterogeneous functional organization of the liver acinus in terms of capacity for NADPH production, mainly in connection with reductive reactions in fatty acid synthesis.     

Quantitative determination of G6Pase activity in histochemically defined zones of the liver acinus

Summary Qualitative histochemical G6Pase distribution patterns obtained with an improved method (Teutsch, 1978) served as the basis for a zonal microdissection of the liver acinus. G6Pase activity was determined quantitatively in tissue samples of zones 1 and 3 by a microfluorometric method (Burch et al., 1978). Using a correlation system it could be demonstrated that the histochemical distribution pattern obtained with the improved method was in better agreement with quantitatively estimated zonal differences of G6Pase activity, both in fed and starved female rats, than with the Wachstein and Meisel medium (1956). From a total of 50 tissue samples analyzed the following average G6Pase activities were calculated: in fed animals 15.36±3.48 U/g dry weight in zone 1, and 9.28±2.15 U/g dry weight in zone 3; in starved female rats 42.50±8.20 U/g dry weight in zone 1, and 29.25±5.68 U/g dry weight in zone 3. The qualitative histochemical as well as quantitative zonal differences of G6Pase activities are taken as further support for the hypothesis of metabolic zonation of liver parencyma.Supported by a grant from the Deutsche Forschungsgemeinschaft     

Quantitative determination of G6Pase activity in histochemically defined zones of the liver acinus.

Qualitative histochemical G6Pase distribution patterns obtained with an improved method (Teutsch, 1978) served as the basis for a zonal microdissection of the liver acinus. G6Pase activity was determined quantitatively in tissue samples of zones 1 and 3 by a microfluorometric method (Burch et al., 1978). Using a correlation system it could be demonstrated that the histochemical distribution pattern obtained with the improved method was in better agreement with quantitatively estimated zonal differences of G6Pase activity, both in fed and starved female rats, than with the Wachstein and Meisel medium (1956). From a total of 50 tissue samples analyzed the following average G6Pase activities were calculated: in fed animals 15.36 +/- 3.48 U/g dry weight in zone 1, and 9.28 +/- 2.15 U/g dry weight in zone 3; in starved female rats 42.50 +/- 8.20 U/g dry weight in zone 1, and 29.25 +/- 5.68 U/g dry weight in zone 3. The qualitative histochemical as well as quantitative zonal differences of G6Pase activities are taken as further support for the hypothesis of metabolic zonation of liver parenchyma.     

Different distribution patterns of the two mannose 6-phosphate receptors in rat liver.

Two mannose 6-phosphate receptors, cation-dependent and -independent receptors (CDMPR and CIMPR), play an important role in the intracellular transport of lysosomal enzymes. To investigate functional differences between the two in vivo, their distribution was examined in the rat liver using immunohistochemical techniques. Positive signals corresponding to CIMPR were detected intensely in hepatocytes and weakly in sinusoidal Kupffer cells and interstitial cells in Glisson's capsule. In the liver acinus, hepatocytes in the perivenous region showed a more intense immunoreactivity than those in the periportal region. On the other hand, positive staining of CDMPR was detected at a high level in Kupffer cells, epithelial cells of interlobular bile ducts, and fibroblast-like cells, but the corresponding signal was rather weak in hepatocytes. In situ hybridization analysis also revealed a high level of expression of CIMPR mRNAs in hepatocytes and of CDMPR mRNA in Kupffer cells. By double immunostaining, OX6-positive antigen-presenting cells in Glisson's capsule were co-labeled with the CDMPR signal but were only faintly stained with anti-CIMPR. These different distribution patterns of the two MPRs suggest distinct functional properties of each receptor in liver tissue.     

Postnatal changes in sublobular distribution of NADPH-cytochrome P-450 reductase in rat liver

Summary Immunohistochemical distribution of NADPH-cytochrome P-450 reductase (NADPH-ferrihaemoprotein reductase; EC 1.6.2.4.) in the liver lobule was examined during development of the rat. From the 19th day of gestation to 4 days after birth, the enzyme was distributed uniformly throughout the lobule. The immunostaining for the enzyme was weak before birth, and became slightly stronger after birth. A slightly uneven distribution of immunoreactivity, stronger in perivenular zones, appeared at 5 days after birth. Then, the staining intensity in perivenular zones became progressively stronger with age, except for a slight increase between 10 and 20 days of age. The intensity in periportal zones also increased gradually, although it remained weaker than that in perivenular zones. Around 30 days of age, the distribution of the immunostaining, stronger in perivenular than in periportal zones, was similar to that seen in the lobules of adult animals. thus, heterogeneity among hepatocytes with respect to the enzyme content is not present in fetal and newborn rats but develops gradually during postnatal development; the postnatal growth of the liver is accompanied by a change in the pattern of the distribution of this enzyme within the lobule.     

The Leishmania donovani complex: genotypes of five metabolic enzymes (ICD, ME, MPI, G6PDH, and FH), new targets for multilocus sequence typing

Flagellates of the Leishmania donovani complex are causative agents of human cutaneous and visceral leishmaniasis. The complex is comprised of L. donovani, Leishmania infantum and Leishmania archibaldi, although the latter is not now considered to be a valid species. Morphological distinction of Leishmania species is impractical, so biochemical, immunological and DNA-based criteria were introduced. Multilocus enzyme electrophoresis (MLEE) is the present gold standard. We have sequenced the genes encoding five metabolic enzymes used for MLEE, both to resolve the DNA diversity underlying isoenzyme mobility differences and to explore the potential of these targets for higher resolution PCR-based multilocus sequence typing. The genes sequenced were isocitrate dehydrogenase, malic enzyme, mannose phosphate isomerase, glucose-6-phosphate dehydrogenase, and fumarate hydratase, for 17 strains of L. infantum, seven strains of L. donovani, and three strains of L. archibaldi. Protein mobilities predicted from amino acid sequences did not always accord precisely with reported MLEE profiles. A high number of heterozygous sites was detected. Heterozygosity was particularly frequent in some strains and indirectly supported the presence of genetic exchange in Leishmania. Phylogenetic analysis of a concatenated alignment based on a total of 263 kb protein-coding sequences showed strong correlation of genotype with geographical origin. Europe and Africa appear to represent independent evolutionary centres.     

Postnatal growth and differentiation in three hindlimb muscles of the rat

Summary The postnatal development, between 0 and 90 days, of three hindlimb muscles and diaphragm of the rat was investigated with respect to fiber types and diameter (histochemistry) and substrate oxidation rates and enzyme activities (biochemistry). The process of muscle fiber differentiation into mature patterns was evaluated by visual classification into 3 or 4 groups having different staining intensities for 3 enzyme-histochemical reactions, enabling 26 fiber types to be distinguished. These exhibited specific sizes and growth rates that varied among the muscles. One of the hindleg muscles (flexor digitorum brevis) remained much more immature than soleus and extensor digitorum longus.The histochemical and biochemical findings correlated well. The capacity for pyruvate and palmitate oxidation, and the activities of cytochrome c oxidase and citrate synthase, increased markedly between 9 and 37 days in soleus and extensor digitorum longus (except citrate synthase in the latter) but not in flexor digitorum brevis. Creatine kinase activity increased in all hindlimb muscles. Both the capacity and the activity of pyruvate oxidation (determined in homogenates and intact isolated muscles, respectively), were in accordance with the fiber type composition. In contrast to oxidation capacity, the activity of pyruvate oxidation decreased after birth until the mature stage, when a value of 18–42% of that of early postnatal muscles was recorded.     

草莓G6PDH基因片段的克隆与序列分析

根据不同植物G6PDH同源基因的保守区设计合成简并引物,采用PCR技术从草莓基因组中分离出一个DNA片段并克隆到pMD18-T载体中。序列测定和分析表明,该片段长717 bp,与拟南芥等植物的G6PDH基因的核酸序列具有83%-87%的同源性。结果表明,克隆的片段为草莓G6PDH基因片段。     

杨树葡萄糖-6-磷酸脱氢酶(G6PDH)基因启动子的克隆与分析

葡萄糖-6-磷酸脱氢酶是磷酸戊糖途径的关键性调控限速酶,其主要功能是为脂肪酸合成、氮还原和谷胱甘肽等生物分子合成提供还原力NADPH,也为核酸合成提供戊糖;此外,还参加非生物逆境胁迫应答反应.因此,G6PDH对植物的生长发育起着非常重要的作用.本文利用甜杨G6PDH基因和毛果杨基因组序列,通过PCR获得了甜杨G6PDH基因上游1 400bp的序列.序列分析结果表明,该序列具有启动子的基本元件TATA-bOX、CAAT-box.此外,还包含多个胁迫诱导元件,如低温诱导元件LTR,盐诱导元件GT-1,抗冻、缺水、脱落酸、抗寒元件MYB和MYC,以及光响应元件L-box、G-box、3AF-1、TC丰富区等.     

Postnatal development of the glycine cleavage system in rat liver

The development of the glycine cleavage system was studied in liver mitochondrial extracts from neonatal and adult rats. The enzyme activity in 2-day-old pups was 29.3% of that measured in the adult and was found to increase in an age-dependent manner. Measurement of hepatic free amino acid concentrations at the neonatal ages showed that glycine levels were highest at 2 days and at 14 days were about 48% of those in the 2-day-old pups. Serine levels did not change between the age of 2 and 14 days. A developmental delay in the glycine cleavage system is responsible for the high levels of glycine in the neonatal rat liver.     

Microquantitative determination of the distribution patterns of alcohol dehydrogenase activity in the liver of rat,guinea-pig and horse

Summary Microquantitative measurements of ADH-activity were carried out on the livers of male and female rats, guinea-pigs and horses (two geldings and a mare). Lyophilized cryostat sections of liver parenchyma were microdissected the whole way along the sinusoidal length from the terminal afferent vessels to the terminal efferent venule. ADH activity in samples of about 50–150 ng was measured in a microbiochemical assay using the oil-well technique without enzymatic cycling, by direct luminometric determination of NADH. On the basis of the single measurements, mean values of total hepatic ADH activity could be calculated and the specific distribution patterns graphically demonstrated.Total activity of ADH in the liver of the female rat is 1.6 times higher than in the male; the male distribution pattern exhibits a relative maximum in the intermediary zone of the acinus while the activity in the liver of female rats increases towards a perivenous maximum. Mean values for total ADH activity in the livers of male and female guinea-pigs are almost equal and there is, moreover, no clear intra-acinar gradient. Mare and castrated male horses show high hepatic ADH activity which is evenly distributed in the liver acinus.Supported by grants from the Schweizerische Stiftung für Alkoholforschung and the Deutsche Forschungsgemeinschaft (Sa 127/8-4)Dedicated to Prof. Dr. W. Graumann on the occasion of his 70th birthday     

Microquantitative determination of the distribution patterns of alcohol dehydrogenase activity in the liver of rat, guinea-pig and horse

Microquantitative measurements of ADH-activity were carried out on the livers of male and female rats, guinea-pigs and horses (two geldings and a mare). Lyophilized cryostat sections of liver parenchyma were microdissected the whole way along the sinusoidal length from the terminal afferent vessels to the terminal efferent venule. ADH activity in samples of about 50-150 ng was measured in a microbiochemical assay using the oil-well technique without enzymatic cycling, by direct luminometric determination of NADH. On the basis of the single measurements, mean values of total hepatic ADH activity could be calculated and the specific distribution patterns graphically demonstrated. Total activity of ADH in the liver of the female rat is 1.6 times higher than in the male; the male distribution pattern exhibits a relative maximum in the intermediary zone of the acinus while the activity in the liver of female rats increases towards a perivenous maximum. Mean values for total ADH activity in the livers of male and female guinea-pigs are almost equal and there is, moreover, no clear intra-acinar gradient. Mare and castrated male horses show high hepatic ADH activity which is evenly distributed in the liver acinus.     

Expression and catalysis of sex-specific cytochrome P450 isozymes in rat liver

Research interest in the study of cytochromes P450 has recently been shifting to the characterization of "constitutively" expressed isozymes from that of the inducible forms. Several "constitutive" cytochrome P450 isozymes have been purified from rat liver including five immunochemically related proteins designated cytochromes P450f, P450g, P450h, P450i, and P450k. These hemoproteins have been identified as distinct isozymes on the basis of spectral, electrophoretic, and catalytic properties and NH2-terminal sequence analysis. Purification and immunoquantitation studies have indicated that these isozymes are expressed in a developmental as well as sex-related manner, and are relatively refractory to induction by xenobiotics. Cytochromes P450h and P450g are male-specific proteins, cytochrome P450i is a female-specific isozyme, while cytochromes P450f and P450k are present in both male and female adult rats. In addition, the expression of cytochrome P450g was shown to segregate into two phenotypes in outbred rats. Genetic studies utilizing inbred strains have indicated that the gene responsible for inheritance of high levels of cytochrome P450g is autosomal. Although considerable progress has been made in understanding the role of gonadal hormones and growth hormone in the hepatic regulation of cytochromes P450g, P450h, and P450i in particular, the physiological significance of the "constitutive" isozymes in the liver remains largely unresolved.     

Postnatal ontogenesis of the circadian clock within the rat liver

In mammals, the circadian oscillator within the suprachiasmatic nuclei (SCN) entrains circadian clocks in numerous peripheral tissues. Central and peripheral clocks share a molecular core clock mechanism governing daily time measurement. In the rat SCN, the molecular clockwork develops gradually during postnatal ontogenesis. The aim of the present work was to elucidate when during ontogenesis the expression of clock genes in the rat liver starts to be rhythmic. Daily profiles of mRNA expression of clock genes Per1, Per2, Cry1, Clock, Rev-Erbalpha, and Bmal1 were analyzed in the liver of fetuses at embryonic day 20 (E20) or pups at postnatal age 2 (P2), P10, P20, P30, and in adults by real-time RT-PCR. At E20, only a high-amplitude rhythm in Rev-Erbalpha and a low-amplitude variation in Cry1 but no clear circadian rhythms in expression of other clock genes were detectable. At P2, a high-amplitude rhythm in Rev-Erbalpha and a low-amplitude variation in Bmal1 but no rhythms in expression of other genes were detected. At P10, significant rhythms only in Per1 and Rev-Erbalpha expression were present. At P20, clear circadian rhythms in the expression of Per1, Per2, Rev-Erbalpha, and Bmal1, but not yet of Cry1 and Clock, were detected. At P30, all clock genes were expressed rhythmically. The phase of the rhythms shifted between all studied developmental periods until the adult stage was achieved. The data indicate that the development of the molecular clockwork in the rat liver proceeds gradually and is roughly completed by 30 days after birth.