Purification of the Type II and Type III isozymes of rat hexokinase, expressed in yeast.

C-terminally His-tagged versions of the Type II and Type III isozymes of rat hexokinase were expressed in Pichia pastoris and Schizosaccharomyces pombe, respectively. Milligram amounts of the homogeneous isozymes were readily obtained in good yield by chromatography on Ni-NTA columns. The specific activities were 133 +/- 4 and 76 +/- 3 u/mg for the purified Type II and Type III isozymes, respectively. The K(m)'s for glucose and ATP were in good agreement with values in the literature for the isozymes isolated from mammalian tissues. The Type III isozyme exhibited substrate inhibition at elevated levels of glucose, as previously observed for this isozyme isolated from mammalian tissue sources.     

Further studies on the role of phospholipids in determining the characteristics of mitochondrial binding sites for type I hexokinase

Previous work has indicated that two types (A and B) of binding sites for hexokinase exist, but in different proportions, on brain mitochondria from various species. Hexokinase is readily solubilized from Type A sites by glucose 6-phosphate (Glc-6-P), while hexokinase bound to Type B sites remains bound even in the presence of Glc-6-P. Type A:Type B ratios are approximately 90:10, 60:40, 40:60, and 20:80 for brain mitochondria from rat, rabbit, bovine and human brain, respectively. The present study has indicated that MgCl2-dependent partitioning of mitochondrially bound hexokinase into a hydrophobic (Triton X-114) phase is generally correlated with the proportion of Type B sites. This partitioning behavior is sensitive to phospholipase C, implying that the factor(s) responsible for conferring hydrophobic character is(are) phospholipid(s). Substantial differences were also seen in the resistance of hexokinase, bound to brain mitochondria from various species, to solubilization by Triton X-100, Triton X-114, or digitonin. This resistance increased with proportion of Type B sites. Enrichment of bovine brain mitochondria in acidic phospholipids (phosphatidylserine or phosphatidylinositol), but not phosphatidylcholine or phosphatidylethanolamine, substantially increased solubilization of the enzyme after incubation at 37 degrees C. Collectively, the results imply that the Type A and Type B sites are located in membrane domains of different lipid composition, the Type A sites being in domains enriched in acidic phospholipids which lead to greater susceptibility to solubilisation by Glc-6-P.     

Molecular forms of red blood cell hexokinase

Summary Mammalian red blood cell hexokinase has been shown to exist in two or more distinct molecular forms, which are separable by ion-exchange chromatography. Of these forms just one corresponds to hexokinase type I from other tissues, while the others differ from any previously reported hexokinase isozyme. Analysis of several molecular properties of the three major forms (la, Ib and Ic in the order of their elution from DE-52 columns) of hexokinase prepared from human red cells and of the two forms purified from rabbit reticulocytes, shows significant differences in the isoelectric point. The kinetic and regulatory characteristics, the molecular weight, the temperature and pH-dependence of the various isozymes were similar.The hexokinase isozymic pattern is largely dependent upon red blood cell age. Among all, hexokinase Ib is the predominant form in rabbit reticulocytes and becomes the minor component in the older cells; a similar situation has also been found in the human erythrocyte. At present the molecular basis of hexokinase heterogeneity remains unknown, however preliminary experimental findings indicate a post-translational modification as a possible mechanism.     

Evidence for multiple genes coding for the isozymes of hexokinase in the highly glycolytic AS-30D rat hepatoma.

We have compared Southern blots of rat hepatoma DNA probed with Types I, II and III hexokinase cDNAs isolated from normal rat tissues. Hybridization patterns show several fragments recognized by both the Type I and II clones while no resemblance is observed between the Type III probe and the other two isozymes. It therefore appears that the Type I-like and Type II-like hepatoma isozymes are coded for by similar yet separate genes, while a dissimilar third gene codes for the Type III-like isozyme. In addition, a loss of heterozygosity was detected at the Type III locus in the AS-30D hepatoma when compared to normal tissue. As only the Type II-like isozyme is highly expressed in highly glycolytic tumors, these data have implications for differential gene regulation between the tumor isozymes.     

Microheterogeneity of human placental lactogen showing different patterns in individuals.

Samples of human placental lactogen, obtained either as a standard pooled preparation or prepared from individual placentas, were shown to migrate as a single band on acrylamide gel electrophoresis. When subjected to isoelectric focusing in polyacrylamide gels, the pooled sample was resolved into bands at pI values 5.0, 5.5, 5.8. 6.0, 6.1 and 6.2. Different batches of the standard pooled sample gave different proportions of each isoprotein species. Isolation and refocusing of individual bands did not alter the pI of each. Treatment with urea or with p-chloromercuribenzoate did not eliminate microheterogeneity seen on isofucising, indicating that the observed heterogeneity is probably not due to conformational differences or to restriction of molecular shape of disulfide bonds. It was shown by immunodiffusion that all the isofocusing reacted similarly against a common antibody to human placental lactogen. When placental lactogen was extracted from individual full term human placentas, the same isoprotein bands were observed but their proportions varied markedly from one placenta to another, and not all bands were present. Thus human placental lactogen displays considerable microheterogeneity which varies with individual placentas.     

Differential inhibition of human placental prostaglandin release in vitro by a GnRH antagonist

Previously, we have demonstrated that the production of prostaglandins by human placental tissue varied with gestational age. In addition, we have shown that placental prostaglandin release was affected by GnRH, and that its response was also dependent on the gestational age of the placenta. Thus, we have studied the effect of a GnRH antagonist ([N-Ac-Pro1,D-p-Cl-Phe2,D-Nal(2)3,6-LHRH, Syntex Research, Palo Alto, CA) on basal prostaglandin release from placentas of 6 to 15 weeks' gestation and found that this antagonist (1 microgram/ml) effects an inhibition of the release of prostaglandin E, prostaglandin F, and 13,14-dihydro-15-keto-prostaglandin from placentas of 13 and 15 weeks of gestation. This effect was not overridden by GnRH at 10 times the antagonist concentration in the 13-week placental cultures, but was totally reversed by GnRH (10 micrograms/ml) in the 15-week placental cultures. These data demonstrate that this GnRH antagonist can affect human placental prostaglandin production at 13 to 15 weeks of gestation and indicate that endogenous placental GnRH-like activity may exert a control over placental prostaglandin release at this gestational stage.     

Effect of experimental diabetes on the activity of hexokinase isoenzymes in tissues of the rat

The effect of experimental diabetes on the activity of hexokinase isoenzymes was studied in a wide range of tissues of the rat. In the tissues known to require insulin for glucose phosphorylation, the activity of hexokinase was markedly decreased; the fall being mainly in the Type IV (Glucokinase) in liver and Type II in other tissues, these tissues also exhibit glucose underutilization in diabetes. In the tissues which are commonly known not to require insulin, the activity of Type I hexokinase was significantly increased, these tissues exhibit aspects of glucose overutilization in diabetes in particular kidney and lens. These changes are discussed in relation to Spiro's hypothesis of glucose under and overutilization in tissues in diabetes.     

Complete amino acid sequence of the type II isozyme of rat hexokinase, deduced from the cloned cDNA: comparison with a hexokinase from novikoff ascites tumor

The 917-residue amino acid sequence of the Type II isozyme of rat hexokinase has been deduced from the nucleotide sequence of cloned cDNA. The sequences of 197 nucleotides in the 5' untranslated region and 687 bases of the 3' untranslated region have also been determined. A region of overlap between two discrete cDNA clones was confirmed by isolation and sequencing of a genomic DNA clone that spanned the region. Within this region, the 634-nucleotide coding sequence was divided into three exons, each of 150-250 nucleotides; these results suggest that the gene encoding Type II hexokinase is likely to be quite complex. There is extensive similarity between the sequences of the N- and C-terminal halves of the Type II isozyme, as previously seen with the Type I and III isozymes; this is consistent with the view that these enzymes evolved by a process of gene duplication and fusion. A cDNA encoding the entire C-terminal half of a hexokinase from Novikoff ascites tumor cells was also isolated and found to be identical to a cDNA encoding the corresponding region of the Type II isozyme of skeletal muscle. Northern analysis indicated that a single mRNA, approx 5200 nucleotides in length, encoded both the skeletal muscle and the tumor enzymes. These results do not support previous speculation that the hexokinase isozymes of normal tissue are distinct from those of tumors, and suggest the possibility that post-translational modifications of a single protein species might account for apparent differences between the isozymes of normal and tumor tissues.     

Differential inhibition of human prostaglandin release in vitro by a GnRH antagonist

Previously, we have demonstrated that the production of prostaglandins by human placental tissue varied with gestational age. In addition, we have shown that placental prostaglandin release was affected by GnRH, and that its response was also dependent on the gestational age of the placenta. Thus, we have studied the effect of a GnRH antagonist ([N-Ac-Pro¹, D-p-Cl-Phe², D-Nal (2)^3,6-LHRH, Syntex Research, Palo Alto, CA) on basal prostaglandin release from placentas of 6 to 15 weeks' gestaton and found that this antagonist (1 μg/ml) effects an inhibition of the release of prostaglandin E, prostaglandin F, and 13, 14-dihydro-15-keto-prostaglandin from placentas of 13 and 15 weeks of gestation. This effect was not overridden by GnRH at 10 times the antagonist concentration in the 13-week placental cultures, but was totally reversed by GnRH (10 μg/ml) in the 15-week placental cultures. These data demonstrate that this GnRH antagonist can affect human placental prostaglandin production at 13 to 15 weeks of gestation and indicate that endogenous placental GnRH-like activity may exert a control over placental prostaglandin release at this gestational stage.     

Inhibition of mitochondrial and soluble hexokinase from various rat tissues by glucose 1,6-bisphosphate

Mitochondrial and soluble Type I and Type II hexokinase from various rat tissues differed in their susceptibility to inhibition by glucose-1,6-bisphosphate (Glc-1,6-P2). In tissues where Type I is the predominant form, the mitochondrial enzyme was less susceptible to inhibition by Glc-1,6-P2 than the soluble enzyme, especially at high Mg2+ concentration. In tissues where Type II is the predominant form, the mitochondrial enzyme was more susceptible to inhibition by Glc-1,6-P2 than the soluble enzyme, especially at low Mg2+ concentration. The results suggest that changes in the intracellular concentrations of Glc-1,6-P2 and Mg2+ under various conditions would affect the activity of the bound and soluble hexokinase from different tissues in a different manner.     

Lipopolysaccharide induces the expression of interleukin-1alpha distinctly in different compartments of term and preterm human placentae

The aim of the study was to investigate the stimulatory effect of lipopolysaccharide (LPS) on IL-lalpha production in different compartments of term and preterm placental tissues. Homogenates from amnion, chorion, and from fetal (subchorionic placental tissues, maternal decidua, and mid-placental tissue before and after perfusion of isolated placental cotyledons of 5 term placentas and 4 placentas obtained after preterm birth (28-34 W of gestation) were examined. Isolated placental cotyledons were dually perfused LPS (100 ng/kg perfused placental tissue) was perfused into the maternal side during 10 hours. Homogenates of the samples were examined by ELISA for IL-1alpha levels, and paraffin sections of the samples were stained by immunohistochemical staining, to characterize the cellular origin of placental IL-1alpha. Paired t test and ANOVA determined statistical significance. In the homogenates, there was a tendency towards higher IL-lalpha levels in all preterm placental compartments as compared to the term compartments before perfusion. A significant increase was observed only in the chorion compartment (p = 0.035). LPS had significantly increased IL-la levels only in the decidua compartment of term placentas as compared to other placental compartments (p = 0.0004), and had decreased IL-1alpha levels in the mid-placenta (p = 0.034). In preterm placentas, addition of LPS did not affect the expression levels of IL-1alpha in either fetal or maternal compartments as determined by ELISA and immunohistochemical staining. IL-la levels in the chorion compartment of preterm placenta were significantly higher as compared to term placenta. LPS affects placental tissues of term and preterm placentas differently. Also, in the term placentas, LPS affected the different compartments differently. Thus, IL-1alpha may have a key role (as a autocrine/paracrine factor) in the regulation of normal and pathological pregnancy and parturition.     

Phospholipase A2 isozymes in pregnancy and parturition

Mammalian cells contain several structurally different phospholipase (PLA2) enzymes that exhibit distinct localisation, function and mechanisms of regulation. PLA2 isozymes have been postulated to play significant roles in the parturition process. Both secretory and cytosolic PLA2 isozymes have been identified in human gestational tissues, and there is differential expression of these PLA2 isozymes in human fetal membranes and placenta obtained at preterm and term. The aims of this commentary are: (1) to review recent data concerning the expression, role and regulation of PLA2 isozymes in human gestational tissues; and (2) to present novel data demonstrating the regulation of PLA2 isozymes in human gestational tissues by nuclear factor-kappa B (NF-kappaB) and peroxisome proliferator-activated receptor (PPAR)-g.     

Aromatase isozyme in goldfish brain and stereochemistry of the aromatization [Poster 40]

Aromatase activity of goldfish brain synaptosomes was not suppressed by a mouse anti-human placental aromatase cytochrome P-450 monoclonal antibody. A rabbit antiserum to human placental aromatase cytochrome P-450 did not show a significant suppression of the goldfish brain activity when compared to the placental aromatase. However, the stereomechanism of 1,2-hydrogen elimination during the brain aromatization was determined to be stereospecific 1ß, 2ß-elimination which was identical to that of human placentas and ovaries.     

Variability of placental expression of cyclin E low molecular weight variants

Cyclin E, a G(1) cyclin serving to activate cyclin-dependent kinase 2, is the only cyclin gene for which alternative splicing leading to structurally different proteins has been described. Different cyclin E proteins are present in tumor tissues but absent from normal (steady) tissues. Cyclin E contributes to the regulation of cell proliferation and ongoing differentiation and aging. Because trophoblast has invasive properties and differentiates into syncytium and placental aging may develop at term, we examined cyclin E protein variants in human placenta. Placental samples were collected from 27 deliveries between 33 and 41 wk and were compared with ovarian cancer (positive control). Both placental and tumor tissues showed seven cyclin E low molecular weight (LMW) bands migrating between 50 and 36 kDa. Placental expression of cyclin E showed certain variability among cases. Lowest cyclin E expression was detected in normal placentas (strong expression of Thy-1 differentiation protein in villous core and low dilatation of villous blood sinusoids). Abnormal placentas (significant depletion of Thy-1 and more or less pronounced dilatation of sinusoids) showed significant increase either of all (early stages of placental aging) or only certain cyclin E proteins (advanced aging). Our studies indicate that a similar spectrum of cyclin E protein variants is expressed in the placental and tumor tissues. Low cyclin E expression in normal placentas suggests a steady state. Overexpression of all cyclin E proteins may indicate an activation of cellular proliferation and differentiation to compensate for developing placental insufficiency. However, an enhanced expression of some cyclin E LMW proteins only might reflect an association of cyclin E isoforms with placental aging or an inefficient placental adaptation.     

Gestational and hormonal regulation of human placental lipoprotein lipase

The fetal demand for FFA increases as gestation proceeds, and LPL represents one potential mechanism for increasing placental lipid transport. We examined LPL activity and protein expression in first trimester and term human placenta. The LPL activity was 3-fold higher in term (n = 7; P < 0.05) compared with first trimester (n = 6) placentas. The LPL expression appeared lower in microvillous membrane from first trimester (n = 2) compared with term (n = 2) placentas. We incubated isolated placental villous fragments with a variety of effectors [GW 1929, estradiol, insulin, cortisol, epinephrine, insulin-like growth factor-1 (IGF-1), and tumor necrosis factor-alpha] for 1, 3, and 24 h to investigate potential regulatory mechanisms. Decreased LPL activity was observed after 24 h of incubation with estradiol (1 micro g/ml), insulin, cortisol, and IGF-1 (n = 12; P < 0.05). We observed an increase in LPL activity after 3 h of incubation with estradiol (20 ng/ml) or hyperglycemic medium plus insulin (n = 7; P < 0.05). To conclude, we suggest that the gestational increase in placental LPL activity represents an important mechanism to enhance placental FFA transport in late pregnancy. Hormonal regulation of placental LPL activity by insulin, cortisol, IGF-1, and estradiol may be involved in gestational changes and in alterations in LPL activity in pregnancies complicated by altered fetal growth.     

Immunoelectron microscopic localization of aromatase in human placenta and ovary using microwave fixation

In this study we investigated the immunohistochemical localization of a unique aromatase, a single protein of 51,000 daltons, in the human placenta and ovary at light and electron microscopic levels. Microwave fixation was adopted for the immunoelectron microscopic study because it is an excellent method for preserving antigenicity and subcellular structures in frozen sections. Tissue samples from four immature human placentas, four full-term human placentas, and two human ovaries fixed in 10% formalin were examined by light microscopy. In addition, tissues from three full-term human placentas and one immature human placenta fixed in 4% paraformaldehyde were examined by electron microscopy. By light microscopy, immunoreactivity for this aromatase was located in the syncytiotrophoblast and a part of the cytotrophoblast of the placenta and in the lutein and granulosa cells of the ovary. Immunoelectron microscopy revealed that the aromatase antigen was localized on the surface of the microvilli, the lateral plasma membrane, and in the endoplasmic reticulum (ER) in the syncytiotrophoblast of the placenta. The positive immunoreactivity in the syncytiotrophoblast ER is consistent with previous results using antibodies for other types of aromatase, whereas the reactivity on the microvilli has not been previously described. The present report describes the fine localization of this unique aromatase in placental and ovarian tissues; its localization on the plasma membranes requires further physiological investigation.     

Infant growth restriction is associated with distinct patterns of DNA methylation in human placentas

The placenta acts not only as a conduit of nutrient and waste exchange between mother and developing fetus, but also functions as a regulator of the intrauterine environment. Recent work has identified changes in the expression of candidate genes, often through epigenetic alteration, which alter the placenta's function and impact fetal growth. In this study, we used the Illumina Infinium HumanMethylation27 BeadChip array to examine genome-wide DNA methylation patterns in 206 term human placentas. Semi-supervised recursively partitioned mixture modeling was implemented to identify specific patterns of placental DNA methylation that could differentially classify intrauterine growth restriction (IUGR) and small for gestational age (SGA) placentas from appropriate for gestational age (AGA) placentas, and these associations were validated in a masked testing series of samples. Our work demonstrates that patterns of DNA methylation in human placenta are reliably and significantly associated with infant growth and serve as a proof of principle that methylation status in the human term placenta can function as a marker for the intrauterine environment, and could potentially play a critical functional role in fetal development.