A quantitative analysis of germ cells and the histone variants in the testes of vitamin A-deficient rats and during subsequent repletion with vitamin A

A quantitative analysis of the different types of germ cells present in the seminiferous tubules of vitamin A-deficient-retinoate maintained rats revealed that the number of pachytene spermatocytes and spermatogonia was greatly reduced in the deficient rats. Spermatids were virtually absent in the deficient tubules which contained mostly spermatogonia and preleptotene spermatocytes along with the Sertoli cells. There was no change in the number of Sertoli cells present in the tubules of deficient rats as compared to that of normal rats. Following supplementation of retinyl acetate to vitamin A-deficient-retinoate maintained rats, there was an immediate thinning of the germinal epithelium resulting from the sloughing off of the damaged spermatocytes which were beyond repair. However, after 12 days of vitamin A supplementation fresh batch of pachytene spermatocytes started appearing while by day 16 round spermatids could be seen. Analysis of the acid soluble proteins from nuclei on different types of Polyacrylamide gel electrophoretic systems has revealed that the levels of the testis specific histone variants Hlt, TH2A and TH2B, synthesized predominantly in the pachytene spermatocytes were greatly reduced in the testes of retinoate maintained rats. Following supplementation of retinyl acetate for either 4 days or 8 days the levels of these histone variants further decreased which correlated with the decrease in the number of pachytene spermatocytes. However, by day 12 of supplementation onwards, their levels started increasing and reached near normal levels by day 24 of vitamin A-supplementation     

Synthesis and glycosylation of fibronectin in hepatocytes from vitamin A-deficient rats

Summary Recent work from our laboratory (Kim and Wolf, J Biol Chem 262: 365–371, 1987) has shown increased uptake of labeled amino acids into fibronectin (FN), increased net synthesis of FN and increased levels of FN-mRNA in primary cultures of hepatocytes from vitamin A-deficient rats compared to controls. We now find, surprisingly, decreased uptake of labeled sugars into the oligosaccharide chains of FN from vitamin A-deficient hepatocytes. This decrease could be reversed by added retinoic acid at physiological concentration. At the same time, FN from deficient hepatocytes (–A.FN) was more susceptible to proteolytic degradation. Decreased uptake of the core sugar mannose into –A.FN was similar to that of glucosamine, yet the percent of label in sialic acid was the same as in +A.FN, suggesting a smaller number of oligosaccharide chains per molecule of –A.FN. Upon enzymatic removal of oligosaccharide and labeling with sodium borotritide, it was found that both –A.FN and +A.FN had biantennary oligosaccharide structures. Selective enzymatic removal of sialic acid showed that +A.FN had both sialic acids in an 23 linkage, whereas –A.FN apparently had one 23 and one 26-linked sialic acid. The borotritide experiments allowed us to calculate that +A.FN appeared to have 5 oligosaccharide chains per FN monomer, whereas the –A. FN showed only 4 chains. These results would account for the decreased glycosylation and increased susceptibility to proteolysis of the –A. FN. We conclude that vitamin A controls both the rate of synthesis of the polypeptide chain of FN via its mRNA, as well as the rate of its glycosylation.Abbreviations FN Fibronectin - ELISA Enzyme-linked Immunosorbent Assay - DOC Deoxycholate - TCA Trichloroacetic Acid - PMSF Phenylmethylsulfonyl Fluoride - PBS Phosphate-buffered Saline - BSA Bovine Serum Albumin - AGP Alpha-1 acid Glycoprotein - SDS-PAGE Sodium Dodecylsulfate-Polyacrylamide Gel Electrophoresis     

Characterization of calcium accumulation in the brain of rats administered orally calcium: The significance of energy-dependent mechanism

The characterization of calcium accumulation in the brain of rats administered orally calcium chloride solution was investigated. Rats received a single oral administration of calcium (15–50 mg/100 g body weight), and they were sacrificed by bleeding-between 15 and 120 min after the administration. The administration of calcium (50 mg/100 g) produced a significant increase in serum calcium concentration and a corresponding elevation of brain calcium content, indicating that the transport of calcium into the brain is associated with the elevation of serum calcium levels. The increase in brain calcium content by calcium administration was not appreciably altered by the pretreatment with Ca²⁺ channel blockers (verapamil or diltiazem with the doses of 1.5 and 3.0 mg/100 g). In thyroparathyroidectomized rats, the administration of calcium (50 mg/100 g) caused a significant increase in brain calcium content, indicating that calcium-regulating hormones do not participate in the brain calcium transport. Now, brain calcium content was clearly elevated by fasting (overnight), although serum calcium level was not significantly altered. Calcium administration to fasted rats induced a further elevation of brain calcium content as compared with that of control (fasted) rats. The fasting-induced increase in brain calcium content was appreciably restored by refeeding. This restoration was also seen by the oral administration of glucose (0.4 g/100 g) to fasted rats. The present study demonstrates that serum calcium is transported to brain, and that the increased brain calcium is released promptly. The release of calcium from brain may be involved in energy metabolism, and this release may be weakened by the reduction of glucose supply into brain. The finding suggests a physiological significance of energy-dependent mechanism in the regulation of brain calcium.     

Iron accumulation in tissues of magnesium-deficient rats with dietary iron overload

The mineral imbalances in magnesium-deficient rats with dietary iron overload were studied. Forty-four male Wister rats were divided into six groups and fed six diets, two by three, fully crossed: magnesium adequate or deficient, and iron deficient, adequate, or excess. The concentrations of iron, magnesium, calcium, and phosphorus in tissues of the rats were measured. The results were as follows: (1) The excess iron intake reinforced the iron accumulation in liver and spleen of magnesium deficient rats; (2) The saturation of iron binding capacity was enormously elevated in the magnesium deficient rats fed excess iron; and (3) Dietary iron deprivation diminished the degree of calcium deposition in kidney of magnesium deficient rats. These results suggest that magnesium-deprived-rats have abnormal iron metabolism losing homeostatic regulation of plasma iron, and magnesium deficient rats with dietary iron overload may be used as an experimental hemochromatosis model.     

Alterations in cytokeratin expression precede histological changes in epithelia of vitamin A-deficient rats

Summary Normal epithelial cell differentiation is charactezied by the production of distinct cytokeratin proteins. It is well known that epithelia of several organs show squamous metaplasia in a vitamin A-deficient status. It is not yet known whether these histological changes are concomitant with a change in cytokeratin expression. Therefore, 3-week-old female rats (BN/BiRij) were fed a vitamin A-deficient diet for 8 weeks. The cytokcratin expression in epithelia of various organs was monitored immunohistochemically during the induction of vitamin A deficiency. Therefore, monoclonal antibodies specific for human cytokeratin 4, 5, 5+8, 7, 10, 14, 18 and 19 were used. In a normal vitamin A status, the distributional pattern for the different cytokeratins in rats was similar to that reported for human tissue. No change in cytokeratin expression was seen in trachea, skin, liver and colon at any time point studied. Squamous metaplasia in urinary bladder and salivary glands was observed after six weeks on the vitamin A-deficient diet. This was concomitant with a substitution of cytokeratins 4, 5+8, 7, 18 and 19 by cytokeratin 10. The latter cytokeratin is specific for keratinzed squamous epithelium. A change in cytokeratin expression was observed in bladder, ureter, kidney, salivary glands, uterus and conjunctiva before histological alterations appeared. In conclusion, the changes in cytokeratin expression observed under vitamin A deficiency in epithelia in vivo are in agreement with those described in other studies for epithelial cells in vitro. The changes in cytokeratin expression and the subsequent differentiation into squamous cells occurs in basal cells of the bladder but not in transitional cells. Furthermore, histological alterations are preceded by changes in cytokeratin expression indicating that vitamin A status controls cytokeratin expression in vivo.     

Postnatal development and GABA allosteric modulation of benzodiazepine receptor binding in the vitamin B-6 deficient rat brain

We have measured the postnatal development and GABA modulation of benzodiazepine receptors in neuronal membranes from vitamin B-6 deficient and normal rats. In rats fed vitamin B-6 adequate and deficient diets there were age-dependent changes in [³H]flunitrazepam binding site affinity and in the number of binding sites. Vitamin B-6 deficiency produced a significant reduction in the potency of GABA to enhance [³H]flunitrazepam binding to cortical membranes prepared from 14 day old rats. These results suggests an uncoupling of the GABAa/benzodiazepine receptor at a developmental period when the animals are most susceptible to spontaneous seizures.     

Choline and betaine ameliorate liver lipid accumulation induced by vitamin B6 deficiency in rats

We investigated the efficacy of supplementing the diet with choline or betaine in ameliorating lipid accumulation induced by vitamin B₆ (B₆) deficiency in rat liver. Male Wistar rats were fed a control, B₆-deficient, choline-supplemented (2, 4, or 6 g choline bitartrate/kg diet) B₆-deficient diet or betaine-supplemented (1, 2, or 4 g betaine anhydrous/kg diet) B₆-deficient diet for 35 d; all diets contained 9 g L-methionine (Met)/kg diet. Choline or betaine supplementation attenuated liver lipid deposition and restored plasma lipid profiles to control levels. These treatments restored the disruptions in Met metabolism and the phosphatidylcholine (PC)/phosphatidylethanolamine (PE) ratio induced by B₆ deficiency in liver microsomes. These results suggest that choline and betaine ameliorated liver lipid accumulation induced by B₆ deficiency via recovery of Met metabolism and very low-density lipoprotein secretion by restoring the supply of PC derived from PE.     

Role of mitochondrial calcium uniporter‐mediated Ca2+ and iron accumulation in traumatic brain injury

Previous studies have suggested that the cellular Ca²⁺ and iron homeostasis, which can be regulated by mitochondrial calcium uniporter (MCU), is associated with oxidative stress, apoptosis and many neurological diseases. However, little is known about the role of MCU‐mediated Ca²⁺ and iron accumulation in traumatic brain injury (TBI). Under physiological conditions, MCU can be inhibited by ruthenium red (RR) and activated by spermine (Sper). In the present study, we used RR and Sper to reveal the role of MCU in mouse and neuron TBI models. Our results suggested that the Ca²⁺ and iron concentrations were obviously increased after TBI. In addition, TBI models showed a significant generation of reactive oxygen species (ROS), decrease in adenosine triphosphate (ATP), deformation of mitochondria, up‐regulation of deoxyribonucleic acid (DNA) damage and increase in apoptosis. Blockage of MCU by RR prevented Ca²⁺ and iron accumulation, abated the level of oxidative stress, improved the energy supply, stabilized mitochondria, reduced DNA damage and decreased apoptosis both in vivo and in vitro. Interestingly, Sper did not increase cellular Ca²⁺ and iron concentrations, but suppressed the Ca²⁺ and iron accumulation to benefit the mice in vivo. However, Sper had no significant impact on TBI in vitro. Taken together, our data demonstrated for the first time that blockage of MCU‐mediated Ca²⁺ and iron accumulation was essential for TBI. These findings indicated that MCU could be a novel therapeutic target for treating TBI.     

Effect of iron and/or vitamin A re-supplementation on vitamin A and iron status of rats after a dietary deficiency of both components

Iron and vitamin A deficiency are common nutritional problems in developing countries. From animal experiments and intervention studies, growing evidence is pointing to a possible influence of iron on vitamin A metabolism. We assessed the affects of an oral supplementation of vitamin A and/or iron on the recovery of rats from vitamin A and iron deficiency. Weanling male Wistar rats were kept for four weeks on an iron and vitamin A deficient diet. Thereafter, rats were repleted with iron 35 mg/kg feed, with vitamin A 4500 IU/kg feed both, or with iron 35 mg/kg and vitamin A 4500 IU/kg for five weeks. Retinol and retinyl esters in plasma and tissues were determined by HPLC. Iron was determined by atomic absorption spectrophotometry. The determination of haematological parameters showed that rats developed an anaemia during depletion. This was reversed by the re-supplementation with iron but not vitamin A alone. The simultaneous supplementation of vitamin A was of no additional benefit. When rats were resupplemented with iron alone a substantial further decrease in plasma retinol (P < 0.002) and liver vitamin A (P < 0.05) was observed. A similar but less pronounced decrease in plasma retinol was observed in the rats re-supplemented with vitamin A alone, despite a substantial increase in liver vitamin A (P < 0.002). Despite lower liver vitamin A levels compared to the group re-supplemented with vitamin A lone, the group re-supplemented with iron and vitamin A had substantial higher plasma levels compared to the one supplemented with iron alone (P < 0.002). In conclusion, the study supports an interaction of iron and vitamin A on the level of retinol transport in plasma. Despite a comparable availability of vitamin A as indicated by the comparable liver levels only the re-supplementation of both iron and vitamin A can normalize the retinol level in plasma. This might be of nutritional consequence in developing countries with regard to the supplementation regime of both nutrients iron and vitamin A to prevent a functional deficiency of vitamin A despite sufficient dietary availability.     

Relationship between vitamin D status and deposition of bound calcium in skeletal muscle of the rat

Summary— The effects of vitamin D on the intramuscular distribution of total and bound calcium, phosphate and on available cytosolic calcium, were investigated in skeletal muscle. Total calcium and phosphorus were measured on ashed subcellular fractions of muscles from vitamin D-repleted and vitamin D-deprived rats. The variations in available calcium were followed by determining the activities of calcium-sensitive enzymes in isolated cytosol. Bound-calcium was revealed ultra-microscopically by pyroantimonate. In vitamin D-repleted muscles, the pyroantimonate method revealed specific areas of intense bound-calcium deposition: the myofibrils, where they formed pronounced lines parallel to the Z-bands. In vitamin D-deficient muscles, the calcium-pyroantimonate deposits appeared clearly reduced. This loss was accompanied by a marked reduction in total calcium and phosphorus in all the subcellular fractions, as compared to vitamin D-repleted muscles. Unexpectedly, the activity of the Ca²⁺-activated isocitrate-dehydrogenase was increased in the cytosol, while that of the Ca²⁺-inhibited pyruvate-kinase decreased. Prolonged vitamin D-administration to vitamin D-repleted rats led to an intensification of calcium-pyroantimonate deposits and a general increase in total calcium and phosphorus, but no change in the cytosolic Ca²⁺-sensitive enzyme activities. Cessation of vitamin D-administration to vitamin D-repleted rats produced a regression of calcium-pyroantimonate deposits, a general decrease of total calcium and phosphate levels, and stimulation of the Ca²⁺-activated isocitrate-dehydrogenase accompanied by lowering of the Ca²⁺-inhibited pyruvate-kinase. The results clearly indicate a correlation between vitamin D-repletion and the total and bound calcium content of skeletal muscle. In addition, they demonstrate an apparent contradiction between the decrease of total and bound calcium, and the activities of cytosolic Ca²⁺ sensitive enzymes during vitamin D-deprivation, which can only be explained by an increase in available calcium. It is suggested that vitamin D stimulates intramuscular mechanisms tending to lower available calcium by inactivating the cation via the formation of calcium chelates.     

Progesterone and vitamin D: Improvement after traumatic brain injury in middle-aged rats

Progesterone (PROG) and vitamin D hormone (VDH) have both shown promise in treating traumatic brain injury (TBI). Both modulate apoptosis, inflammation, oxidative stress, and excitotoxicity. We investigated whether 21 days of VDH deficiency would alter cognitive behavior after TBI and whether combined PROG and VDH would improve behavioral and morphological outcomes more than either hormone alone in VDH-deficient middle-aged rats given bilateral contusions of the medial frontal cortex. PROG (16 mg/kg) and VDH (5 μg/kg) were injected intraperitoneally 1 h post-injury. Eight additional doses of PROG were injected subcutaneously over 7 days post-injury. VDH deficiency itself did not significantly reduce baseline behavioral functions or aggravate impaired cognitive outcomes. Combination therapy showed moderate improvement in preserving spatial and reference memory but was not significantly better than PROG monotherapy. However, combination therapy significantly reduced neuronal loss and the proliferation of reactive astrocytes, and showed better efficacy compared to VDH or PROG alone in preventing MAP-2 degradation. VDH + PROG combination therapy may attenuate some of the potential long-term, subtle, pathophysiological consequences of brain injury in older subjects.     

Influence of vitamin E on the levels of fatty acids and MDA in some tissues of diabetic rats

This study was performed to determine whether vitamin E supplementation in streptozotocin-induced diabetic rats treated with insulin could affect the levels of fatty acid composition and malondialdehyde (MDA) of brain, liver and muscle tissues. Thirty Wistar albino rats were used during the experiments. They were randomly divided into three groups, each consisting of six individuals. The first group was diabetic, the second was control, and the third was diabetic but fed vitamin E. The level of stearic acid in brain tissues decreased (p<0.05) in the second and the third groups as compared to the first group. The percentage of arachidonic and polyunsaturated fatty acids slightly decreased (p<0.05) in the diabetic group in comparison to the second and third groups. The proportion of docosahexaenoic acid significantly increased (p<0.01) in the second and third groups in contrast to the first group. The level of docosatrienoic was slightly higher (p<0.05) in the third group than in other groups. In the liver tissues, the proportion of stearic, oleic and total monounsaturated fatty acids was slightly higher (p<0.05) in the first group than in the other groups. The level of arachidonic, docosahexaenoic, unsaturated and total polyunsaturated fatty acid slightly increased (p<0.05) in the second and third groups as compared to the first group. The level of myristic and stearic acids in muscle tissue slightly increased (p<0.05) in the first group as compared to the second and third groups. The proportion of arachidonic, docosahexaenoic and unsaturated fatty acids slightly increased (p<0.05) in the second and third groups relative to the first group. The amount of MDA was slightly higher in the diabetic group than in the other groups in all tissues. The results indicate that vitamin E supplementation, in experimental diabetes could play a role in controlling the oxidative status and altered fatty acid metabolism in tissues, thereby maintaining favourable fatty acid distribution in the tissues affected by diabetic complications.     

Alterations in surface carbohydrates and in some functional properties of liver lysosomal membrane in vitamin A-deficient rat

A significant decrease in total carbohydrates and particularly in mannose, galactose and sialic acid has been observed in vitamin A-deficient rat liver lysosomal membrane. These alterations adversely affect the membrane permeability and structure-linked latency of the lysosomal enzymes.Significant reduction in the pH-dependent in vitro binding of the lysosomal arylsulfatase B to the highly purified membrane has been observed in vitamin A deficiency. This is attributed to the decrease in electro-negativity, mainly due to the observed reduction in negatively-charged sialic acid residues on the outer side of the membrane.Similar reduction in sialic acid content on the inner side of the membrane affects the microenvironment in the lysosomes. Intralysosomal pH, measured by computing the proteolytic activity of lysed lysosomes and of phagolysosomes, endocytosed with denatured ¹³¹I-labelled human serum albumin, is slightly but consistently higher in vitamin A-deficient groups compared to that in control one. This is reflected in the low rate of degradation of the entrapped proteins in vitamin A deficiency.The possible physiological significance of the observations is discussed with special reference to the loss of surface carbohydrates, particularly sialic acid, in vitamin A-deficient rats.     

雌激素改善维生素D受体基因敲除雌性小鼠的骨、钙代谢

以维生素 D 受体基因敲除雌性小鼠为模型,研究雌激素对骨、钙代谢的调节作用。外源性给予雌二醇一个月后,观察小鼠血钙水平的变化,同时测定小鼠骨密度,并利用胫骨非脱钙 von Kossa 染色观察钙化的骨小梁和未钙化的类骨质面积的变化。结果显示,外源性给予雌二醇一个月后,维生素 D 受体基因敲除小鼠的血钙水平,从(2.10±0.37) mmol/L 上升到(2.80±0.41) mmol/L (P<0.05); 骨密度从(0.037±0.006) g/cm2增高到(0.048±0.007) g/cm2,显著改善(P<0.05) ;钙化骨小梁面积显著增加,未钙化的类骨质面积显著缩小。结果提示,外源性雌二醇对骨、钙代谢具有非依赖于维生素 D 的正向调节作用。     

钙信号调节小鼠前体脂肪细胞分化和脂质蓄积的机制

本试验用醋酸钙、p38丝裂原激活蛋白激酶(p38MAPK)抑制剂SB203580及钙通道阻滞剂和激动剂刺激小鼠前体脂肪细胞。通过实时定量PCR技术检测前体脂肪细胞分化标志基因和钙信号相关受体基因表达水平,用油红O染色提取法和Fura-2/AM荧光法测定胞内脂质蓄积情况及胞浆游离Ca2+浓度([Ca2+]i)变化,以探讨钙信号调节前体脂肪细胞分化的潜在机制。结果表明:钙通道阻滞剂和激动剂显著改变了脂蛋白脂酶(LPL),过氧化物增殖激活受体γ(PPARγ)、脂肪酸合成酶(FAS)的表达水平,且影响细胞内的脂质蓄积。与降低外钙摄入相比,降低内钙释放能促进前体脂肪细胞分化(P<0.01),而提高外钙摄入与提高内钙释放相比,提高外钙摄入显著抑制前体脂肪细胞分化(P<0.01)。SB203580可降低胞浆[Ca2+]i浓度,促进前体细胞分化和脂质蓄积(P<0.01)。但钙信号并未影响维生素D受体(VDR)和细胞外钙敏感受体(CaSR)的表达水平。提示钙信号可能通过p38MAPK通路影响前体脂肪细胞分化和脂质蓄积。     

Differences in calcium accumulation between human plantar and palmar aponeuroses

To elucidate the characteristics of calcium accumulation of human plantar and palmar aponeuroses, the authors determined the calcium content of human plantar and palmar aponeuroses by atomic absorption flame emission spectrophotometry. The subjects consisted of 9 men and 14 women, ranging in age from 61 to 93 yr. In the plantar aponeurosis, the calcium content was significantly higher in the anterior and posterior parts than in the middle part. It is known that pressure distribution under the sole of a foot is higher in the anterior and posterior parts than in the middle part. The present study suggests that the accumulation of calcium in the plantar aponeurosis is related with the pressure distribution under the sole of a foot. The calcium content increased progressively with aging in the anterior part of the plantar aponeurosis, but not in the middle and posterior parts. Regarding the palmar aponeurosis, the calcium content was significantly higher in the anterior and posterior parts in comparison with the middle part. It was found that the calcium content increased progressively with aging in the posterior part of the palmar aponeurosis, whereas it did not increase significantly with aging in the anterior and middle parts. Regarding the relationship between the calcium content of the aponeuroses and the bone mineral density, a significant correlation was found between the calcium content in the anterior part of the palmar aponeurosis and the bone mineral density of the scaphoid bone.     

The action of excessive,inorganic silicon (Si) on the mineral metabolism of calcium (Ca) and magnesium (Mg)

The influence of silicon treatment on the levels of calcium and magnesium in blood serum and tissues was studied in rats. The concentrations of both elements were estimated in samples of sera and tissues of rats receiving per os a soluble, inorganic silicon compound—sodium metasilicate nonahydrate (Na₂SiO₃·9H₂O (REACHIM, USSR)), dissolved in the animals' drinking water. A decrease of magnesium concentration in serum was observed with accompanying elevation of registered calcemia. Moreover, a reduction of tissue calcium levels was found with a simultaneous increase of magnesium tissue pool. The results provide evidence for silicon involvement in mineral metabolism. It could result in a modification of pathological processes concerning bone tissue.     

Alterations in binding characteristics of peripheral benzodiazepine receptors in testes by vitamin A deficiency in guinea pigs

The correlation of vitamin A with the binding characteristics of peripheral benzodiazepine receptors (PBRs) in testes have been implicated on the basis of findings of involvement of vitamin A in testicular physiology and the abundance of PBRs in testicular tissue. Both vitamin A and PBRs are involved in the control of cell proliferation and differentiation but no data exists regarding the relationship between them. In the present study, we have examined the effects of vitamin A deficiency on the affinity and density of PBRs in testes of guinea pigs. Weanling guinea pigs were divided into three groups: control, pair-fed control and vitamin A deficient. They were fed a complete semipurified diet. The vitamin A deficient diet was similar to the control diet except vitamin A palmitate was omitted. Vitamin A deficiency status was achieved after 90 days of feeding. Binding of [³H]Ro 5-4864, a specific ligand for peripheral benzodiazepine receptors was determined in whole homogenate of testicular tissue. There was a 77% decrease in the receptor density (B_max) in vitamin A deficient group compared to control. The B_maxvalues for control, pair-fed control and vitamin A deficient groups were: 12.4 ± 0.4, 8.8 ± 0.2 and 3.0 ± 0.6 pmol/g, respectively. The equilibrium dissociation constant (K_D) values were also 86% decreased in the vitamin A deficient group compared to the other groups. The K_D values for control, pair-fed control and vitamin A deficient groups were: 3.4 ± 0.7, 2.8 ± 0.5 and 0.5 ± 0.01, respectively. The decrease in the binding characteristics of PBRs in testes due to vitamin A deficiency was accompanied with a corresponding decrease in the levels of testosterone in plasma. These results suggest a close functional relationship of vitamin A with PBRs in testes.