Caveolin-1 and mitochondrial alterations in regenerating rat liver

The liver has a remarkable ability to regenerate after partial hepatectomy (PH), although the factors governing such ability are still poorly understood. During the prereplicative phase of the regeneration, ultrastructural alterations of periportal hepatocytes were seen, including mitochondrial swelling, abnormal accumulation of lipids, and myelin figures which could lead to the formation of lipid droplets. As it has been hypothesized that caveolin-1 is involved in lipidogenesis and in mitochondrial homeostasis, we aimed to study the subcellular distribution of caveolin-1 in hepatocytes at an early stage following PH. Liver samples were processed for light and electron microscopy at 0 h, 24 h, and 96 h after PH. The expression and subcellular distribution of caveolin-1 was assessed by immunohistochemical and immunocytochemical techniques. Following PH, at 24 h, membranes of altered mitochondria of periportal hepatocytes exhibited significant decrease of caveolin-1 expression compared with control. Myelin figures showing high expression of caveolin-1 were also seen. At 96 h, hepatocytes became ultrastructurally similar to the control liver, and the expression of caveolin-1 on mitochondria showed a moderate increase compared with 24 h after PH. Decrease of expression of caveolin-1 in the altered liver mitochondrial membranes at 24 h following PH, and the high expression of caveolin-1 observed on myelin figures, suggests involvement of caveolin-1 is in both mitochondrial homeostasis and lipidogenesis. Addressing the role played by caveolin-1 during liver regeneration might disclose additional features of mitochondrial homeostasis and lipidogenesis during frequent metabolic liver diseases.     

Insight into 5-aminolevulinic acid-induced modulation of cellular antioxidant metabolism to confer salinity and drought tolerance in maize

The current study investigated the comparative oxidative damage in two maize seedlings induced by saline, drought,and combined stress and the ameliorative role of two different doses (20 and 80 µM) of 5-aminolevulinic acid (ALA) againstthe above-mentioned stresses. Hydroponically grown 10-day-old maize (Zea mays, var. BARI Hybrid Maize-7 (BHM-7) andBARI Hybrid Maize-9 (BHM-9)) seedlings were exposed to 12 dS/m of saline solution, 200 mM mannitol-induced droughtstress alone and their combined stress for 7 days. Result revealed that individual stresses retard the plant growth to somedegrees; however, their combined stress has more detrimental effects, which might be correlated with lipid peroxidation(MDA)-induced oxidative stress in seedlings, enhanced Na⁺/K⁺ ratio, and augmented generation of superoxide (O₂^•−) andhydrogen peroxide (H₂O₂). In contrast, exogenous ALA supplementation at 20 µM concentration markedly recoveredfrom chlorosis and growth inhibition, substantially scavenged reactive oxygen species (ROS) and MDA by preserving ionhomeostasis and relaxing oxidative stress; also, by boosting catalase (CAT) and glutathione S-transferase (GST), andexclusively via depressing the activity of lipoxygenase (LOX) antioxidant enzyme. On the contrary, 80 µM ALA madethings worse; nevertheless, higher activities shown by other antioxidant enzymes; like, superoxide dismutase (SOD),ascorbate peroxidase (APX), peroxidase (POD), and glutathione peroxidase (GPX), which were related to lessen theoxidative damage by highly produced O₂^•− and H₂O₂ under combined stress. Non-denaturing gel electrophoresiswas done for further confirmation. However, ALA importantly increased the photosynthetic pigment contents inboth genotypes irrespective of doses. Nevertheless, GST might have assisted the plants to escape from the herbicidaleffect by detoxification. However, in the combined stress condition, high ALA concentration may have somepositive role to play. Our findings also showed that BHM-9 performed better than BHM-7. Therefore, ALA atlower concentration was effective for single stress of saline and drought, while higher concentration can improveplant survival under combined stress.     

Investigation of the antioxidant defensive role of both AD-MSCs and BM-MSCs in modulating the alteration in the oxidative stress status in various STZ-diabetic rats’ tissues

Diabetes mellitus (DM) could negatively affect patients’ health via inducing a lot of serious functional hazards in many tissues’ cells at molecular levels. Recently, many scientists had proposed stem cell therapy being an appropriate alternative treatment protocol for numerous health threatening issues including diabetes. Therefore, the current study was designed to investigate the antioxidant potentiality of two MSCs types in alleviating tissues’ oxidative stress dramatic elevation resulting as a consequence of Type 1 DM induction. In our 4 weeks study, animals were divided into four groups: control group, STZ-diabetic group (D), D+AD-MSCs group and D+BM-MSCs group. Data reported that diabetic rats treated with either AD-MSCs or BM-MSCs exhibited a marvelous body tissues (Pancreas, Liver and Kidney) enhancing capabilities in attenuating the oxidative stress status; as evidenced by XO, ROS, and MDA levels down-regulation; with a general concomitant elevation in the antioxidants’ content; evidenced by many enzymatic and non-enzymatic antioxidants up-regulation; relative to the diabetic untreated group. Interestingly, comparing both treatments with each other and to control group, most of the measured parameters were reverted back to near normal levels after AD-MSCs injection; which clearly point out their stunning health benefits and superiority as anti-diabetic agent in overcoming different tissues’ complications; owing to their marked cytoprotective and regenerative potentialities.     

Oxidative stress indicators in human and bottlenose dolphin leukocytes in response to a pro-inflammatory challenge

Marine mammals undergo cycles of tissue ischemia and reperfusion during the dive response. Reperfusioninjury can result in oxidative tissue damage and the activation of a pro-inflammatory immune response. The risk ofoxidative damage is reduced by antioxidants. Our hypothesis is that the reported higher antioxidant defenses withinmarine mammal tissues provide additional protection in situations that produce oxidative stress, like inflammation, incomparison to terrestrial mammal tissues. Leukocytes were isolated from the whole blood of Pacific bottlenosedolphins (Tursiops truncatus gilli) and humans (Homo sapiens) and were exposed to lipopolysaccharides (LPS, 10 µg/mL)in vitro to simulate a pro-inflammatory challenge. Oxidative stress indicators, including superoxide radical (O₂^•−)production, activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase(GR), and glutathione S-transferase (GST), as well as oxidative protein damage, were quantified by spectrophotometry.Following 48 h under experimental conditions, bottlenose dolphin leukocytes produced 1.9 times more O₂^•− butdisplayed 2.0 times lower protein carbonyl concentrations compared to human leukocytes. Following 48 h underexperimental conditions, bottlenose dolphin leukocytes displayed 7.9, 2.0, 11.1, and 3.3 times more activities of CAT,GPx, GR, and GST, respectively, compared to human leukocytes. These results suggest that, under cell culture conditions,the antioxidant defenses in bottlenose dolphin leukocytes provide additional protection against pro-inflammatorychallenges in comparison to human leukocytes, likely as an adaptive advantage.     

Expression of brain natriuretic peptide in the rat heart studies during heart growth and in relation to sympathectomy

Brain natriuretic peptide (BNP) might be of importance during heart development and is described to be increasingly expressed in congestive heart failure and to affect the progress of this condition. However, details in the normal expression of BNP are still unclear in various parts of the adult and growing heart, including the conduction system. In this study, we investigated the expression of BNP in relation to that of atrial natriuretic peptide (ANP) in the growing as well as in the adult rat heart. The effects of chemical sympathectomy in adult rats were also examined. Contrary to previous BNP immunohistochemical studies, the BNP antiserum was preabsorbed with an excess of ANP before staining to abolish the crossreactivity with ANP. There was a pronounced BNP immunoreaction in the auricles, the trabeculated ventricular walls, and the peripheral parts of the conduction system at 0-1 days postnatally. The degree of immunoreaction gradually decreased with increasing age. A similar developmental pattern was seen concerning ANP expression, but the magnitude of the latter clearly exceeded that for BNP. Immunoreaction for BNP was never detected in the atrioventricular (AV) node and AV bundle at any stage. In contrast to the situation for ANP previously observed, no obvious changes in BNP immunoreaction patterns were observed in response to sympathectomy. This is the first study to thoroughly demonstrate the expression of BNP in the various regions of the rat heart during growth and in the normal and sympathectomized adult stage. The observations are related to possible functions of natriuretic peptides in the growing and adult heart.     

Association between preterm birth risk and polymorphism and expression of the DNA repair genes <i>OGG1</i> and <i>APE1</i> in Saudi women

Genomic instability and mutations caused by increases in oxidative stress during pregnancy can damage the fetoplacental unit and can upshot preterm birth. Oxidative damage to DNA may possibly be involved in etiology of preterm birth (PTB) which can be repaired by DNA repair gene. In the present study, we assessed the association of base excision repair gene family by analyzing the association of single nucleotide polymorphisms and genes expression in 8-oxoguanine glycosylase-1 (OGG1) and apurinic-apyrimidinic endonuclease 1 (APE1) genes with risk of preterm birth in Saudi women. We analyzed genotypes of four single nucleotide polymorphisms (SNPs) (rs1052133, rs293795, rs2072668 and rs2075747) in OGG1 gene and three SNPs (rs1130409, rs3136814, and rs3136817) in APE1 gene using TaqMan Genotyping assay kits in 50 pairs of preterm cases and individually matched controls. Also, gene expression level was explored by RT-PCR in 10 pairs of preterm placental tissues and individually matched normal placental tissues. Two OGG1 SNP, rs1052133 (OR=0.497; c2=1.11; p=0.292) and rs2072668 (OR=0.408; c2=1.90; p=0.167) and one APE1 SNP rs3136817 (OR=0.458; c2=0.40; p=0.527) showed nonsignificant protective effect against PTB development. The expression of both genes under study was found lower in the PTB patients. Genotype and allele frequencies of both gene SNPs did not show any association with the risk of preterm delivery in Saudi women (P˃0.05). However, synthesis and release of OGG1 and APE1 proteins decreased in preterm placental tissues compared to term delivery reflects the probability of being one of the mechanisms leading to preterm birth.     

Morphological alterations of the glomerular (visceral) epithelium in response to pathological and experimental situations

The glomerular (visceral) layer of Bowman's capsule is comprised of a unique population of cells which have been termed “podocytes.:” Arising from these cells are large major processes and numerous smaller foot processes which completely surround underlying glomerular capillary loops. Podocyte foot processes interdigitate with each other and are separated by spaces (filtration slits) which are designed to facilitate flow of a large amount of filtrate across the glomerular wall. Podocytes exhibit dramatic morphological changes in response to the nephrotic syndrome and some forms of acute renal failure and may play an important role in the pathophysiology of these conditions. In vitro and in vivo studies have shown that a reduction in the sialic acid component of a thick anionic surface coat plays a major role in the morphological changes that these cells exhibit in the nephrotic syndrome. Also, it has been shown that filamentous actin concentrated mainly within podocyte foot processes are the contractile elements responsible for altering the shapes of these processes. There is evidence to suggest that by altering the shapes of their foot processes, podocytes in the normal kidney are able to alter the number of fully patent filtration slits and thereby actively regulate the rate of solute efflux across the glomerular wall. In vitro and in vivo studies have indicated that cytoplasmic microtubules are probably not involved in alterations of the podocyte foot processes but do appear important in maintaining the morphological integrity of podocyte cell bodies and their major processes. In the present paper, the morphological changes which glomerular podocytes exhibit in response to the nephrotic syndrome, various forms of acute renal failure, and during in vitro incubation are discussed along with studies of the possible roles of cytoplasmic microtubules, microtubules, and the glomerular anionic surface coat in these changes.     

Proteomics in investigation of protein nitration in kidney disease: technical challenges and perspectives from the spontaneously hypertensive rat

Kidneys are the mammalian organs with widest range of oxidative status ranging from the well-perfused cortex to the relatively anoxic medulla. This organ is of key interest from the perspective of hypertension, an important contributor to human mortality, and there has been growing use of the spontaneously hypertensive rat (SHR) as a model to explore oxidative stress in hypertensive kidney. Nitrosative stress is often associated with oxidative stress and, like oxidative stress, can lead to covalent modification of protein side-chains. It is especially relevant to kidney because of high levels of both nitrite/nitrate and nitric oxide synthase in medulla. Because of their relatively low abundance and their well-known role in signal transduction, nitration of tyrosines to 3-nitrotyrosines (3NT) is of particular interest in this regard. This modification has the potential to contribute to changes in regulation, in protein activity and may provide a means of specific targeting of key proteins. Mass spectrometry (MS) offers a promising route to detecting this modification. This review surveys protein nitration in kidney disease and highlights opportunities for MS detection of nitrated residues in the SHR.     

Bushen Yizhi Formula regulates the IRE1α pathway to alleviate endoplasmic reticulum stress in an Alzheimer’s disease rat model

Background: While the Bushen Yizhi Formula can treat Alzheimer’s disease (AD), the yet to be ascertainedspecific mechanism of action was explored in this work. Methods: Different concentrations of the Bushen YizhiFormula and amyloid-beta peptide (Aβ) were used to treat rat pheochromocytoma cells (P12) and humanneuroblastoma cells (SH-SY5Y). Cell morphological changes were observed to determine the in vitro cell damage. CellCounting Kit (CCK)-8 assay and flow cytometry were employed to identify cell viability and apoptosis/cell cycle,respectively. Western blotting and immunohistochemistry were employed to measure the expressions of endoplasmicreticulum stress (ERS)-related proteins (GRP78 and CHOP), p-IRE1α, IRE1α, ASK1, p-JNK, JNK, Bax, Bcl-2, XBP-1,and Bim. Fura 2-acetoxymethyl ester (Fura-2/AM) was used to determine the intracellular calcium (Ca²⁺)concentration. Also, an AD model was constructed by injecting Aβ into the CA1 area of the hippocampus in SpragueDawley rats. AD model rats were gavaged with different concentrations of Bushen Yizhi Formula for 14 consecutivedays. The Morris water maze experiment was conducted to test the learning and memory of rats. Hematoxylin &Eosin (H&E) and Terminal-deoxynucleotidyl Transferase (TdT)-mediated dUTP Nick-End Labeling (TUNEL)staining were done to determine histopathological changes in the brain. Results: Bushen Yizhi Formula relieved theAβ-induced effects including cell injury, decreased viability, increased apoptosis, G0/G1 phase cell cycle arrest, upregulation of GRP78, CHOP, p-IRE1α, p-JNK, Bax, XBP-1 and Bim, as well as down-regulation of Bcl-2. Theseresults were also seen with IRE1α silencing. While Aβ suppressed the learning and memory abilities of rats, theBushen Yizhi Formula alleviated these effects of Aβ. Brain nerve cell injury induced by Aβ could also be treated withBushen Yizhi Formula. Conclusion: Bushen Yizhi Formula could influence ERS through the IRE1α signaling pathwayto achieve its therapeutic effects on AD.     

Localization of calretinin in the rat ovary and in relation to nerve cell bodies in dorsal root and paravertebral ganglia projecting to the ovary

Retrograde tracing with True Blue was combined with immunocytochemistry to determine the source of any calretinin-immunoreactive (CR-ir) nerves projecting to the rat ovary. In the ovary, a strong signal for calretinin immunoreactivity was localized in interstitial gland cells; however, no intraovarian CR-ir nerves could be demonstrated. When the superior ovarian nerve was isolated, cut, and True Blue applied to the proximal end, the fluorescent dye was retrogradely transported to a population of cells located in T-12, T-13, and L-1 dorsal root and paravertebral ganglia. There was virtually no dual labeling of cells in these ganglia with calretinin (< 0.009% dual labeling in dorsal root and <0.014% in paravertebral ganglia). However, greater than two-thirds of the True Blue-labeled cells were immediately adjacent to CR-ir cells in dorsal root ganglia. This arrangement is suggestive of a paracrine mechanism between CR-ir cells and cells projecting to the ovary. In paravertebral ganglia, 63% of cells projecting to the ovary were surrounded completely or partially by beaded CR-ir nerve fibers. The source of these fibers (sensory or preganglionic sympathetic) is unknown but hypothesized to be preganglionic. Collectively, these observations suggest a participatory role for calretinin in ovarian function, either directly via effects on the interstitial gland or indirectly by influencing neurons projecting to the ovary.     

GABAergic synapses in the brain identified with antisera to GABA and its synthesizing enzyme,glutamate decarboxylase

GABA is a known inhibitory neurotransmitter in the mammalian brain. The site of GABAergic synapses can be determined with immunocytochemical methods that localize either GABA or its synthesizing enzyme, glutamate decarboxylase (GAD). In general, GABAergic axon terminals contain pleomorphic synaptic vesicles and form symmetric synapses. However, a small number of GABAergic axon terminals in selected brain regions (spinal cord, cerebellum, superior colliculus, striatum, globus pallidus, inferior olive, and substantia nigra) form asymmetric synapses. GAD- and GABA-immunoreactive processes that contain synaptic vesicles participate in every known morphological type of chemical synapse. These include axosomatic, axodendritic, axospinous, initial segment, axoaxonic, dendrodendritic, serial, reciprocal, and ribbon synapses. Although GABAergic synapses form a heterogeneous group, they most commonly form axosomatic, axodendritic, and initial segment synapses in the brain and spinal cord. These findings provide helpful guidelines for the identification of GABAergic synapses in future studies.     

Morphological alterations in the synganglion and integument of Rhipicephalus sanguineus ticks exposed to aqueous extracts of neem leaves (Azadirachta indica A. JUSS)

Currently, the necessity of controlling infestation by ticks, especially by Rhipicephalus sanguineus, has led researchers and public health managers around the world to search for new and more efficient control methods. This way, we can highlight neem (Azadirachta indica A. Juss) leaf, bark, and seed extracts, which have been very effective on tick control, and moreover causing less damage to the environment and to the host. This study showed the potential of neem as a control method for R. sanguineus through morphological and morphometric evaluation of the integument and synganglion of females, in semiengorged stage. To attain this, routine techniques of optical microscopy, scanning electron microscopy and morphometry of the cuticle and subcuticle of the integument were applied. Expressive morphological alterations were observed in both organs, presenting a dose‐dependent effect. Integument epithelial cells and nerve cells of the synganglion showed signs of cell vacuolation, dilated intercellular boundaries, and cellular disorganization, alterations not previously reported in studies with neem. In addition, variations in subcuticle thickness were also observed. In general, the effects of neem are multiple, and affect the morphology and physiology of target animals in various ways. The results presented in this work are the first evidence of its effects in the coating and nervous system of ticks, thus allowing an indication of neem aqueous extracts as a potential control method of the brown dog tick and opening new perspectives on acaricide use. Microsc. Res. Tech. 77:989–998, 2014. © 2014 Wiley Periodicals, Inc.     

Elemental changes in the brain, muscle, and gut cells of the housefly, Musca domestica, exposed to heavy metals

The toxic effects of heavy metals on organisms are well established. However, their specific action at the cellular level in different tissues is mostly unknown. We have used the housefly, Musca domestica, as a model organism to study the toxicity of four heavy metals: copper (Cu), zinc (Zn), cadmium (Cd), and lead (Pb). These have been fed to larvae at low and high, semi-lethal concentrations, and their accumulation in the head, thorax, and abdomen was subsequently measured in adult flies. In addition, their impact on the cellular concentration of several elements important for cell metabolism-sodium (Na+), magnesium (Mg++), phosphorous (P), sulphur (S), chloride (Cl-) and potassium (K+)-were measured in neural cells, muscle fibers, and midgut epithelial cells. Our study showed that the heavy metals accumulate mainly in the abdomen, in which the concentrations of two of the xenobiotic metals, Cd and Pb, were 213 and 23 times more concentrated, respectively, than in controls. All the heavy metals affected the cellular concentration of light elements in all cell types, but the changes observed were dependent on tissue type and were specific for each heavy metal, and its concentration.     

An easy brain thick-section-clearing protocol to observe antigens in the brains of neurological disease mouse models by conventional epifluorescence and confocal laser scanning microscopy

We developed a brain thick-section-clearing (BTS-C) procedure to observe coronal sections of the entire mouse brain with conventional epifluorescence microscopy (EFM). After clearing with the BTS procedure, the white light transmittance of the 1-mm-thick mouse brain sections was more than 96%. The distribution patterns of amyloid plaques or α-synuclein in 1-mm-thick brain sections of five different mouse strains cleared with this procedure were easily observed by EFM. In addition, the detailed distribution of antigens at higher magnifications was revealed by observing the same slides with a confocal laser scanning microscope (CLSM). In conclusion, we have shown that the BTS protocol can replace laborious and time-consuming semi-thin-sectioning procedures, and the cleared 1-mm-thick sections can be examined by EFM and CLSM to elucidate the distribution patterns of antigens.     

General approach to residual stresses determination in thin-walled structures by combining the hole drilling method and reflection hologram interferometry

Main questions, which are connected with a determination of residual stresses by combining the hole-drilling method and whole-field displacement measurements, are considered in detail. The relations, which are needed for converting initial experimental data into stress values of interest, are presented for thin-walled plane structures. Required input data are obtained by simultaneous measurements of probe hole distortions in two principal strain directions on opposite sides of thin plane specimen. Emphasis is made on a careful quantitative formulation of the inverse problem, a solution of which is capable of deriving both membrane and bending residual stress components with the highest possible accuracy. It is shown that an availability of two-side initial data is both an essential and a necessary condition of such formulation. Detailed analysis of accuracy of the results obtained is performed proceeding from three different criteria. All presented considerations are supported with a set of high-quality actual interferograms and analogous reference fringe patterns related to weld-induced residual stress fields.     

Autophagosomal glycogen‐degrading activity and its relationship to the general autophagic activity in newborn rat hepatocytes: The effects of parenteral glucose administration

The effects of the administration of parenteral glucose on the postnatal glycogen autophagic activity and its relationship to the general autophagic activity, were studied in newborn rat liver using electron microscopy and biochemical methods. Glucose abolished the normal postnatal hypoglycemia and preserved the hepatocytic hyaloplasmic glycogen to the levels of birth. It also inhibited the normal postnatal increase in the number and volume of autophagic vacuoles. Glucose especially decreased the rate of postnatal development of the glycogen‐containing autophagic vacuoles. This decrease was greater than that of the autophagic vacuoles in general. In the control animals at the age of 6 h, the total volume of the glycogen‐containing autophagic vacuoles accounted for 87% of the autophagic vacuoles in general, whereas in the glucose‐treated animals of the same age, for only 62%. The results of this and previous studies support the view that the general autophagic activity that develops in the immediate postnatal period in rat hepatocytes is mainly expressed as glycogen autophagic activity selectively inhibited by glucose. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.