Variant type of sialyl Lewis X antigen expressed on adult T cell leukemia cells is associated with skin involvement

Expression of a variant type of sialyl Le(x) antigen defined by 2F3 monoclonal antibody on leukemia cells was studied in 15 adult T cell leukemia (ATL) patients. The expression of 2F3-defined sialyl Le(x) antigen on CD4+CD45+ cells, which is an ATL cell-rich population, was higher in patients with skin involvement (50.1 +/- 23.1% were positive) than in patients without skin involvement (18.1 +/- 12.5%) (P < 0.01). The other surface markers including classical sialyl Le(x) antigen defined by SNH3 or FH6 and LFA-1, VLA-4, CD4, CD25, ICAM-1, Leu8, and HLA-DR did not show a significant difference regardless of skin involvement. In the skin lesion of four patients that we could examine, infiltrating leukemia cells strongly expressed 2F3-defined sialyl Le(x) antigen. In one patient, we could also examine the expression of classical sialyl Le(x) antigen defined by SNH-3 and CSLEX-1, but this was almost negligible. Both skin and lymph node biopsy specimens were examined in two patients. Leukemia cells in the skin strongly expressed 2F3-defined sialyl Le(x) antigen, while its expression was almost negligible on the leukemia cells in the lymph node. These findings suggest that the expression of 2F3-defined sialyl Le(x) antigen on ATL cells is associated with skin involvement of ATL.     

Developmental modulation of mouse hypoglossal nerve inspiratory output in vitro by noradrenergic receptor agonists

The ontogeny of the noradrenergic receptor subtypes modulating hypoglossal (XII) nerve inspiratory output was characterized. Noradrenergic agents were locally applied over the XII nucleus of rhythmically active medullary slice preparations isolated from mice between zero and 13 days of age (P0-P13) and the effects on XII inspiratory burst amplitude quantified. The alpha1 receptor agonist phenylephrine (PE, 0.1-10 microM) produced a dose-dependent, prazosin-sensitive (0.1-10 microM) increase in XII nerve inspiratory burst amplitude. The magnitude of this potentiation increased steadily from a maximum of 15+/-8% in P0 mice to 134+/-4% in P12-P13 mice. The beta receptor agonist isoproterenol (0.01-1.0 mM) produced a prazosin-insensitive, propranolol-sensitive potentiation of XII nerve burst amplitude. The isoproterenol-mediated potentiation increased with development from 27+/-5% in P0-P1 slices, to 37+/-3% in P3 slices and 45+/-4% in P9-P10 slices. The alpha2 receptor agonist clonidine (1 mM) reduced XII nerve inspiratory burst amplitude in P0-P3 slices by 29+/-5%, but had no effect on output from P12-P13 slices. An alpha2 receptor-mediated inhibition of inspiratory activity in neonates (P0-P3) was further supported by a 19+/-3% reduction in XII nerve burst amplitude when norepinephrine (NE, 100 microM) was applied in the presence of prazosin (10 microM) and propranolol (100 microM). Results indicate that developmental increases in potentiating alpha1 and, to a lesser extent, beta receptor mechanisms combine with a developmentally decreasing inhibitory mechanism, most likely mediated by alpha2 receptors, to determine the ontogenetic time course by which NE modulates XII MN inspiratory activity.     

Ontogeny of vesicular monoamine transporter mRNAs VMAT1 and VMAT2. I. The developing rat central nervous system

We used in situ hybridization histochemistry to study the expression of the mRNA of the two vesicular monoamine transporters (VMAT1 and VMAT2) during embryonic and postnatal development of the central nervous system (CNS) in the rat. In the adult rat, VMAT2 mRNA is present exclusively in monoaminergic cell groups of the CNS and VMAT1 mRNA was reported to be present in the adrenal medulla and certain intestinal epithelial cells. In contrast to the above, the expression of VMAT1 mRNA has previously never been detected in the central nervous system. This study shows the first evidence that both transporter molecules are expressed in CNS during ontogenesis. We here demonstrate four main expression patterns detected during development: 1. VMAT2 mRNA expression in monoaminergic neurons of the brainstem beginning as early as embryonic day E13. 2. Expression of VMAT2 mRNA in all major sensory relay nuclei of central nervous system. 3. Co-expression of VMAT1 and VMAT2 mRNA in most limbic structures, basal ganglia, as well as in some hypothalamic nuclei. 4. Exclusive expression of VMAT1 mRNA in the neocortical subventricular zone, in the amygdala at early (E15-18) and late (P1-P28) timepoints, the granular cell layer of cerebellum, and in several brainstem motor nuclei. Based on their distribution during development we suggest that monoamines, released in a controlled fashion, might affect wiring of sensory and also motor circuits. VMAT1 mRNA expression may reflect a specific effect of monoamines in glial differentiation and cerebellar granule cell migration and/or differentiation.     

AMPA receptor protein in developing rat brain: glutamate receptor-1 expression and localization change at regional, cellular, and subcellular levels with maturation

We tested the hypothesis that the regional, cellular, and synaptic localizations of the glutamate receptor 1 (GluR 1) subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor are regulated developmentally in rat brain. By immunoblotting, GluR1 was first detected in whole brain at embryonic day E15.5, and levels increased progressively during late embryonic (E20) and early postnatal (P2-P11) days. Regionally, GluR1 increased in cerebral cortex but decreased in striatum with postnatal maturation. These changes occurred in the presence of increased presynaptic maturation, as determined by synaptophysin detection. By immunocytochemistry, distinct cellular populations showed different temporal profiles of GluR1 expression during postnatal maturation. The neocortex and hippocampus showed a progressive maturation-related enrichment of GluR1, whereas the striatum showed a gradual reduction in GluR1 during maturation. In cerebellum, GluR1 protein was expressed transiently at restricted times postnatally by granule cells (P0-P11) and Purkinje cells (P13-P19), but by P21 and thereafter these neurons had sparse GluR1 immunoreactivity. By immunoelectron microscopy. GluR1 was found in neurites, specifically in both dendritic and axon terminal components of developing synapses. GluR1 was clustered at the plasma membrane of apparent growth cone appositions, neuronal cell bodies, and dendrites of developing neurons. The presence of GluR1 at presynaptic sites dissipated with synaptic maturation, as GluR1 became confined to the somatodendritic compartment as maturation progressed. We conclude that the regional expression as well as the cellular and synaptic localizations of the GluR1 are developmentally regulated and are different in immature and mature brain. Differences in glutamate receptor expression and synaptic localization in immature and mature brain may be relevant to the phenomenon that the perinatal and adult brain differ in their regional vulnerability to hypoxia-ischemia and excitotoxicity.     

Tissue plasminogen activator mRNA expression in granule neurons coincides with their migration in the developing cerebellum

Tissue plasminogen activator activity in the developing cerebellum, as quantified by zymography of cerebellar homogenates from embryonic day (E) 17 to adult mice, shows a peak of activity at postnatal day (P) 7, followed by a steady 75% decrease into adulthood. Northern blot analysis reveals a similar pattern for tissue plasminogen activator mRNA levels, which are low at E17 but increase dramatically, reaching their highest levels of specific mRNA/micrograms RNA in P1-P7 mice and declining about threefold in the adult mouse. In situ hybridization of whole mouse brain sections with a tissue plasminogen activator antisense cRNA probe shows pronounce reactivity in the cerebellum. Although some binding is associated with the cerebellar meninges, the external granule layer is devoid of tissue plasminogen activator mRNA at all ages. However, highly labeled elongated cells, which also bind antibody to neuronal nuclear antigen and are adjacent to Bergmann glial fibers (i.e., migrating granule neurons), are readily visible throughout the molecular and Purkinje layers at P7 and P14. In the adult mouse cerebellum, tissue plasminogen activator mRNA labeling is restricted to cells in the Purkinje/internal granule layers. Thus, tissue plasminogen activator gene expression is induced as granule neurons leave the external granule layer and begin their inward migration.     

Immunohistochemical study on the expression of carbohydrate antigens in normal squamous epithelia and squamous cell carcinomas of the uterine cervix

The expression of 4 kinds of carbohydrate antigens, CA50, CA19-9, sialyl SSEA-1, and DU-PAN-2 was studied immunohistochemically in 15 normal cervical squamous epithelia and 49 cervical carcinomas. (1) In normal epithelia, CA50 and CA19-9 were expressed in all cell layers and all cell layers except for the basal layer, respectively, and a gradual decrease in the intensity of staining was observed in the upper layer. Sialyl SSEA-1 was expressed only in the superficial layer, but DU-PAN-2 was not found in any normal epithelia. (2) In cervical carcinomas, CA50, CA19-9, sialyl SSEA-1 and DU-PAN-2 were observed in 51.0%, 49.0%, 55.1% and 26.7%, respectively. (3) The number of Ki67 positive cells tended to be lower in the area with sialyl SSEA-1 immunostaining. (4) In the cases in which sialyl SSEA-1 positive cells were distributed diffusely in cancer nests, the incidence of lymph node metastasis was significantly higher. These result suggest that the expression of CA50, CA19-9 and sialyl SSEA-1 is related to the differentiation of normal squamous epithelial cells, and sialyl SSEA-1 may be related to cell differentiation also in cervical carcinomas, and the features of its expression may be useful in predicting the biologic properties of cervical carcinomas.     

Transient kinetics of formation and reaction of the uridylyl-enzyme form of galactose-1-P uridylyltransferase and its Q168R-variant: insight into the molecular basis of galactosemia

Galactose-1-phosphate uridylyltransferase catalyzes the reaction of UDP-glucose with galactose 1-phosphate (Gal-1-P) to form UDP-galactose and glucose 1-phosphate (Glc-1-P) through a double displacement mechanism, with the intermediate formation of a covalent uridylyl-enzyme (UMP enzyme). Gln 168 in E. coli uridylyltransferase engages in hydrogen bonding with the phosphoryl oxygens of the UMP moiety, which is bonded to His 166 in the intermediate [Wedekind, J. E., Frey, P. A., and Rayment, I. (1996) Biochemistry 35, 11560-11569]. In humans, the point variant Q188R accounts for 60% of galactosemia cases. The corresponding E. coli variant Q168R has been overexpressed and purified. In preparation for kinetic correlation of Q168R and wild-type uridylyltransferases, we tested the kinetic competence of the wild-type UMP-enzyme. At 4 degreesC, the first-order rate constant for uridylylation by UDP-glucose is 281 +/- 18 s-1, and for deuridylylation it is 226 +/- 10 s-1 with Glc-1-P and 166 +/- 10 s-1 with Gal-1-P. Inasmuch as the overall turnover number at 4 degreesC is 62 s-1, the covalent intermediate is kinetically competent. The variant Q168R is uridylylated by UDP-glucose to the extent of about 65% of the potential active sites. Uridylylation reactions of Q168R with UDP-glucose proceed with maximum first-order rate constants of 2.2 x 10(-)4 s-1 and 4.2 x 10(-)4 s-1 at 4 and 27 degreesC, respectively. In experiments with uridylyl-Q168R and glucose-1-P, the mutant enzyme undergoes deuridylylation with maximum first-order rate constants of 4.8 x 10(-)4 s-1 and 1.68 x 10(-)3 s-1 at 4 and 27 degreesC, respectively. The value of Km for uridylylation of Q168R is slightly higher than for the wild-type enzyme, and for deuridylylation it is similar to the wild-type value. The wild-type enzyme undergoes uridylylation and deuridylyation about 10(6) times faster than Q168R. The wild-type activity in the overall reaction is 1.8 x 10(6) times that of Q168R. The wild-type enzyme contains 1.9 mol of Zn+Fe per mole of subunits, whereas the Q168R-variant contains 1.36 mol of Zn+Fe per mole of subunits. The mutation stabilizes the uridylyl-enzyme by 1.2 kcal mol-1 in comparison to the wild-type enzyme. These results show that the low activity of Q168R is not due to overstabilization of the intermediate or to the absence of structural metal ions. Instead, the main defect is very slow uridylylation and deuridylation.     

Regulation of skeletal muscle hexokinase II by insulin in nondiabetic and NIDDM subjects

Impaired muscle glucose phosphorylation to glucose-6-phosphate by hexokinases (HKs)-I and -II may contribute to insulin resistance in NIDDM and obesity. HK-II expression is regulated by insulin. We tested the hypothesis that basal and insulin-stimulated expression of HK-II is decreased in NIDDM and obese subjects. Skeletal muscle HK-I and HK-II activities were measured in seven lean and six obese normal subjects and eight patients with NIDDM before and at 3 and 5 h of a hyperinsulinemic (80 mU x m(-2) x min(-1)) euglycemic clamp. To assess whether changes in HK-II expression seen during a glucose clamp are likely to be physiologically relevant, we also measured HK-I and HK-II activity in 10 lean normal subjects before and after a high-carbohydrate meal. After an overnight fast, total HK, HK-I, and HK-II activities were similar in lean and obese control subjects; but HK-II was lower in NIDDM patients than in lean subjects (1.42 +/- 0.16 [SE] vs. 2.33 +/- 0.24 nmol x min(-1) x mg(-1) molecular weight, P < 0.05) and accounted for a lower proportion of total HK (33 +/- 3 vs. 47 +/- 3%, P < 0.025). HK-II (but not HK-I) activity increased during the clamp in lean and obese subjects by 34 and 36% after 3 h and by 14 and 22% after 5 h of hyperinsulinemia; no increase was found in the NIDDM patients. In the lean subjects, muscle HK-II activity also increased by 15% 4 h after the meal, from 2.47 +/- 0.19 basally to 2.86 +/- 0.28 nmol x min(-1) x mg(-1) protein (P < 0.05). During the clamps, muscle HK-II activity correlated with muscle citrate synthase activity in the normal subjects (r = 0.58, P < 0.05) but not in the NIDDM patients. A weak relationship was noted between muscle HK-II activity and glucose disposal rate at the end of the clamp when all three groups were combined (r = 0.49, P < 0.05). In summary, NIDDM patients have lower muscle HK-II activity basally and do not increase the activity of this enzyme in response to a 5-h insulin stimulus. This defect may contribute to their insulin resistance. In nondiabetic obese subjects, muscle HK-II expression and its regulation by insulin are normal.     

Mechanism of enhanced Na-K-ATPase activity in cortical collecting duct from rats with nephrotic syndrome

The maximal hydrolytic activity of Na-K-ATPase is specifically increased in the cortical collecting duct (CCD) of rats with puromycin-induced nephrotic syndrome (NS). This stimulation is independent of aldosterone and of endogenous ouabain-like substance. To investigate the mechanism responsible for this change, we compared the maximal Na-K-ATPase hydrolytic activity, the ouabain sensitive 86Rb influx, the specific [3H]ouabain binding, and the sensitivity of Na-K-ATPase to ouabain in the CCD of control rats and of rats given an intraperitoneal injection of puromycin 7 d before study. Both Na-K-ATPase activity and ouabain-sensitive 86Rb influx increased two-fold in rats with NS (ATPase activity: 34.1 +/- 2.1 vs. 18.0 +/- 0.7 pmol.mm-1 x min-1 +/- SE, n = 6, P < 0.001; Rb influx: 14.4 +/- 0.7 vs. 7.4 +/- 0.4 peq.min-1 +/- SE, n = 6, P < 0.001) whereas specific [3H]ouabain binding decreased in rats with NS (6.9 +/- 0.7 vs. 9.0 +/- 0.6 fmol.mm-1 +/- SE, n = 6, P < 0.005). Therefore, the maximal turnover rate of Na-K-ATPase increased over twofold in rats with NS (5,053 +/- 361 vs. 2,043 +/- 124 cycles.min-1 +/- SE, n = 6, P < 0.001). Analysis of the curves of inhibition of Na-K-ATPase by ouabain showed the presence of two Na-K-ATPase populations in both control and NS rats: a highly sensitive population (apparent Ki: 1.4 x 10(-6) M and 0.9 x 10(-6) M) and a less sensitive moiety (apparent Ki: 2.6 x 10(-4) M and 1.1 x 10(-4) M). The enhancement of Na-K-ATPase activity observed in the CCD of rats with NS was entirely due to the stimulation of the population of Na-K-ATPase with low ouabain sensitivity. These results suggest that a dysregulation of this subclass of Na-K-ATPase might be the primary cause of sodium retention in this model of nephrotic syndrome.     

Localization and developmental changes of the expression of two inward rectifying K(+)-channel proteins in the rat brain

We have raised affinity-purified polyclonal antibodies specific for the inward rectifying K+ channel (IRK1/Kir2.1) and the G protein-activated inward rectifying K+ channel (GIRK1/Kir3.1) examined their distributions in the rat brain immunohistochemically. The regional expression pattern of the IRK1 and GIRK1 proteins were similar to those of mRNA of the previous in situ hybridization study. The subcellular distribution was studied in the cerebellum; cerebral cortex and hippocampus. In the cerebellum, the IRK1 protein was clearly detected in the somata and proximal dendrites of Purkinje cells, while the GIRK1 protein was present in the somata and clustered dendrites of granule cells. In the cerebral cortex and hippocampus, both IRK1- and GIRK1-immunoreactivities were detected in the somata and apical dendrites of the pyramidal cells. The presence of IRK1 or GIRK1 proteins in the axons could not proved by the present study. The developmental changes of the expression pattern of the GIRK1 protein were also investigated in the hippocampus and in the cerebellum of postnatal day (P) 7 to P17 rats. The GIRK1 protein was detected neither in the subgranular zone of the dentate gyrus nor in the proliferative zone of the external granule cell layer of the cerebellum, in which granule cell precursors are reported to proliferate, while it was clearly detected in the adjacent layer in which postmitotic but immature cells exist. These results imply that the expression of the GIRK1 protein starts just after the neuronal precursors finished the last mitotic cell division.     

Evolution of anisotropic diffusion in the developing rat corpus callosum

Changes in the ability of substances to diffuse in the intersticial space of the brain are important factors in the pathophysiology of cerebrovascular diseases. Extracellular space (ECS) volume fraction alpha (alpha = ECS volume/ total tissue volume), tortuosity lambda (lambda 2 = free diffusion coefficient/apparent diffusion coefficient), and nonspecific uptake (k')-three diffusion parameters of brain tissue were studied in cortex and subcortical white matter (WM) of the developing rat during anoxia. Changes were compared with the rise in extracellular potassium concentration ([K+]e), extracellular pH (pHe) shifts, and anoxic depolarization (AD). Diffusion parameters were determined from extracellular concentration-time profiles of tetramethylammonium (TMA+) or tetraethylammonium (TEA+), TMA+, TEA+, K+, and pH changes were measured using ion-selective microelectrodes. In the cortex and WM of animals at 4-12 postnatal days (P4-P12), the volume fraction, alpha, is larger than that of animals at > or = P21. Anoxia evoked by cardiac arrest brought about a typical rise in [K+]e to approximately 60-70 mM, AD of 25-30 mV, decrease in alpha, increase in lambda, and increase in k'. At P4-P6, alpha decreased from approximately 0.43 to 0.05 in cortical layer V and from approximately 0.45 to 0.5 in WM. Tortuosity, lambda, increased in the cortex from 1.50 to 2.12 and in WM from approximately 1.48 to 2.08. At P10-P12 and at P21-P23, when alpha in normoxic rats is lower than at P4-P6 by approximately 25 and 50%, respectively, the final changes in values of alpha and lambda evoked by anoxia were not significantly different from those in P4-P6. However, the younger the animal, the longer the time course of the changes. On P4-P6 final changes in alpha, lambda and k' in cortex and WM were reached after 37 +/- 3 min and 54 +/- 2 min; on P10-P12, after 24 +/- 2 and 27 +/- 3 min; and on P21-P23 at 15 +/- 1 and 17 +/- 3 min, respectively (mean +/- SE, n = 6). The time course of the changes was longer in WM than in gray matter (GM), particularly during the first postnatal week, i.e., in the period during which WM is largely unmyelinated. Changes in diffusion parameters occurred in three phases. The first slow and second fast changes occurred simultaneously with the rise in [K+]e and AD. Peaks in [K+]e and AD were reached simultaneously; the younger the animal, the longer the time course of the changes. The third phase outlasted the rise in [K+]e and AD by 10-15 min and correlated with the acid shift in pHe. Linear regression analysis revealed a positive correlation between the normoxic size of the ECS volume and the time course of the changes. Slower changes in ECS volume fraction and tortuosity in nervous tissue during development can contribute to slower impairment of signal transmission, e.g., due to lower accumulation of ions and neuroactive substances released from cells and their better diffusion from the hypoxic area in uncompacted ECS.     

Caffeine counteracts the ergogenic action of muscle creatine loading

This study aimed to compare the effects of oral creatine (Cr) supplementation with creatine supplementation in combination with caffeine (Cr+C) on muscle phosphocreatine (PCr) level and performance in healthy male volunteers (n = 9). Before and after 6 days of placebo, Cr (0.5 g x kg-1 x day-1), or Cr (0.5 g x kg-1 x day-1) + C (5 mg x kg-1 x day-1) supplementation, 31P-nuclear magnetic resonance spectroscopy of the gastrocnemius muscle and a maximal intermittent exercise fatigue test of the knee extensors on an isokinetic dynamometer were performed. The exercise consisted of three consecutive maximal isometric contractions and three interval series of 90, 80, and 50 maximal voluntary contractions performed with a rest interval of 2 min between the series. Muscle ATP concentration remained constant over the three experimental conditions. Cr and Cr+C increased (P < 0.05) muscle PCr concentration by 4-6%. Dynamic torque production, however, was increased by 10-23% (P < 0.05) by Cr but was not changed by Cr+C. Torque improvement during Cr was most prominent immediately after the 2-min rest between the exercise bouts. The data show that Cr supplementation elevates muscle PCr concentration and markedly improves performance during intense intermittent exercise. This ergogenic effect, however, is completely eliminated by caffeine intake.     

Cerebellar granule cell differentiation in mutant and X-irradiated rodents revealed by the neural adhesion molecule TAG-1

In the external granular layer of the cerebellum, the granule cell precursors express the transient axonal glycoprotein TAG-1, a molecule involved in adhesion and neurite outgrowth. Granule cells express TAG-1 transiently, just as they extend neurites before migrating over the radial glia. The present study aims to investigate whether the expression pattern of TAG-1 is altered when granule cells develop abnormally. We studied in vivo models in which Purkinje and/or granule cell defects occur during postnatal development. These include the cerebellar mutant mice staggerer and lurcher as well as rats irradiated during postnatal development. Neither alterations in Purkinje cell differentiation nor the related granule cell loss in the mouse mutants impairs the ability of the surviving granule cell precursors to express TAG-1. Also, early granule cell loss in the X-irradiated rats do not disturb the TAG-1 expression phase in the patches of surviving granule cell precursors. Ectopic granule cells found in the adult cerebellum of X-irradiated rats do not bear the molecule, although they are located in the most superficial part of the molecular layer, occupied by the immunopositive cells a few days earlier. Thus, TAG-1 marks a very precise stage of granule cell differentiation, and the inward migration process itself is not required for the cessation of the expression. We postulate that TAG-1 may be involved in local differentiation steps restricted to the deep external granular layer such as parallel migratory routes or synchrony of axonal growth.