Cytoplasm Of Rats Research Articles

Brain Metabolic DNA in Rat Cytoplasm.

Brain metabolic DNA (BMD) is not involved in cell division or DNA repair but is modulated by memory acquisition, sleep processing, and circadian oscillations. Using routine methods of subcellular fractionation, newly synthesized BMD from male rats is shown to be localized in crude nuclear, mitochondrial, and microsomal fractions and in two fractions of purified nuclei. Sub-fractionation of the mitochondrial fraction indicates the prevalent localization of BMD in free mitochondria and to a lesser degree in synaptosomes and myelin. Cesium density profiles of homogenate, subcellular fractions, and purified nuclei obtained after incorporation periods from 30min to 4h indicate that BMD synthesis takes place by reverse transcription in cytoplasmic organelles. Following the acquisition of the double-stranded structure, BMD is transferred to nuclei. Kinetic analyses lasting several weeks highlight the massive BMD turnover in subcellular fractions and purified nuclei and its dependence on age. Data are in agreement with the role of BMD as a temporary information store of cell responses of potential use in comparable forthcoming experiences.

Screening for lactic acid bacteria in traditional fermented Tibetan yak milk and evaluating their probiotic and cholesterol-lowering potentials in rats fed a high-cholesterol diet

The objectives of the present study were to screen cholesterol-lowering lactic acid bacteria from traditional fermented Tibetan yak milk, and to evaluate their potential use as cholesterol-reducing probiotics in rats. A total of 115 isolates were evaluated for their cholesterol-lowering and probiotic profiles by in vitro assays, and a strain with good performance in in vitro test was selected to test its probiotic effects in rats fed a high cholesterol diet. Results showed that the strain of L. plantarum Lp3 had a high cholesterol-reducing rate (73.3%) and survival rate in simulated gastrointestinal fluids. Administration of Lp3 to rats fed a high-cholesterol diet promoted notable declines in serum and liver cholesterol and triglycerides levels, and reduced lipid deposition in the cytoplasm of rat’s liver tissue. These results suggest that the strain of L. plantarum Lp3 could be a potential probiotic to treat hyperlipidemia and to be used in functional food.

Effects of electroacupuncture combined with dietary control on liver endoplasmic reticulum stress in rats with non-alcoholic fatty liver disease

To observe the effects of electroacupuncture (EA) combined with dietary control on liver endoplasmic reticulum stress (ERS) in rats with non-alcoholic fatty liver disease (NAFLD), and to explore the action mechanism of EA on NAFLD. Sixty male SD rats were randomly divided into a common diet group (15 rats) and a model group (45 rats, fed with high-fat diet to induce NAFLD model). After model establishment, 10 rats in the common diet group were randomly selected as a control group, and 40 rats in model group were randomly divided into a high-fat diet (HFD) group, a low-fat diet (LFD) group, an EA-1 group and an EA-2 group, 10 rats in each one. From the 2nd day of model establishment, rats in the HFD group and EA-1 group were fed with high-fat diet, while rats in the LFD group and EA-2 group were fed with common diet. Rats in the EA-1 group and EA-2 group were treated with acupuncture at unilateral "Zusanli" (ST 36), "Sanyinjiao" (SP 6) and "Taichong" (LR 3), besides,"Zusanli" (ST 36) and "Sanyinjiao" (SP 6) were connected with EA. The treatment was given once a day, and two sides of acupoints were selected alternatively. Each treatment last 20 min for totally 4 weeks. The rats in the remaining groups were treated with identical fixation method. After treatment, the liver tissues of rats in each group were collected and made into ultra-thin slice to perform transmission electron microscope (TEM) on the endoplasmic reticulum morphology; western blot and real-time PCR method were applied to observe the protein and gene expression of the specific ERS protein, glucose-regulated protein 78 (GRP78). Under TEM, plenty of rough endoplasmic reticulum in alignment was observed in the cytoplasm of rats in the control group, accompanied with indistinct adhering ribosome; the rough endoplasmic reticulum in the cytoplasm in the HFD group was in expansion and obvious breakage, presenting small vacuoles in irregular arrangement; the expansion of rough endoplasmic reticulum in the LFD group and EA-1 group was relieved without appa-rent breakage and disarrangement; the rough endoplasmic reticulum in the EA-2 group was in alignment without obvious expansion. The protein expression of GRP78 was little in the control group, while the protein and mRNA expression of GRP78 were obviously increased in the remaining groups (all P<0.01). The protein and mRNA expression of GRP78 in the LFD group and EA-1 group were significantly lower than those in the HFD group (all P<0.01); those in the EA-2 group were lower than those in the LFD group and EA-1 group (all P<0.01). EA effectively improves the liver ERS in NAFLD rats, which can be one of the action mechanisms of EA on NAFLD. In addition, the efficacy is more significant with diet control.

Discovery and Structure-Guided Optimization of Diarylmethanesulfonamide Disrupters of Glucokinase-Glucokinase Regulatory Protein (GK-GKRP) Binding: Strategic Use of a N → S (nN → σ*S-X) Interaction for Conformational Constraint.

The HTS-based discovery and structure-guided optimization of a novel series of GKRP-selective GK-GKRP disrupters are revealed. Diarylmethanesulfonamide hit 6 (hGK-hGKRP IC50 = 1.2 μM) was optimized to lead compound 32 (AMG-0696; hGK-hGKRP IC50 = 0.0038 μM). A stabilizing interaction between a nitrogen atom lone pair and an aromatic sulfur system (nN → σ*S-X) in 32 was exploited to conformationally constrain a biaryl linkage and allow contact with key residues in GKRP. Lead compound 32 was shown to induce GK translocation from the nucleus to the cytoplasm in rats (IHC score = 0; 10 mg/kg po, 6 h) and blood glucose reduction in mice (POC = -45%; 100 mg/kg po, 3 h). X-ray analyses of 32 and several precursors bound to GKRP were also obtained. This novel disrupter of GK-GKRP binding enables further exploration of GKRP as a potential therapeutic target for type II diabetes and highlights the value of exploiting unconventional nonbonded interactions in drug design.

An improved postconditioning algorithm: Gradually increased reperfusion provides improved cardioprotection in rats

The aim of the present study was to investigate whether a gradually increasing reperfusion algorithm, in which the brief reperfusion was lengthened as the duration of each reperfusion/reocclusion cycle remained fixed, enhances cardioprotection. Rats were randomized into 5 groups: the sham, reperfusion injury (R/I), gradually decreased reperfusion (GDR; 30/10‑25/15‑15/25‑10/30 sec of reperfusion/reocclusion), equal reperfusion (ER; 4 20/20‑sec reperfusion/reocclusion cycles) and gradually increased reperfusion (GIR; 10/30‑15/25‑25/15‑30/10 sec of reperfusion/reocclusion). The rats were sacrificed to measure serum markers, apoptotic indices and infarct size. Western blot analyses were used to analyze the expression of molecules involved in important signaling pathways. All the three postconditioning patterns were found to provide cardioprotection (P<0.05 compared with the R/I group). GIR provided optimum cardioprotection, followed by ER and then GDR. Apoptotic index and serum marker levels were significantly reduced in the GIR compared with the ER group (P<0.05). The enhanced cardioprotection provided by GIR was accompanied by significantly increased levels of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and Bcl‑2, as well as lower levels of p38/c‑Jun N‑terminal kinase (JNK) phosphorylation, tumor necrosis factor α (TNFα), caspase‑8, Bax, caspase‑9 and cytochrome c (Cyt‑c) in the cytoplasm of rats (P<0.05, all compared with ER). The infarct size in the rats of the GIR group was also smaller compared with that in the rats of the ER group, but this difference was not significant (16.30±5.22 vs. 20.57±6.32%, P>0.05). All the variables measured in the present study were significantly improved in the GIR group compared with the GDR group (P<0.05). In conclusion, the association between brief reperfusion and reocclusion is an important factor in postconditioning algorithms. Additionally, GIR results in improved cardioprotection compared with that achieved by the remaining algorithms examined.

Vacuolation of Sensory Ganglion Neuron Cytoplasm in Rats with Long-term Exposure to Organophosphates

Cytoplasmic vacuolation of sensory neurons has been reported to occur within the dorsal root ganglia in studies investigating various neuropathic conditions including the effects of neurotoxic chemicals. In this study, we investigated this lesion in adult (98-119 days old) male Long-Evans rats, after multiple exposures to two organophosphates (tri-ortho-tolyl phosphate [TOTP] and chlorpyrifos) and the modifying effects of concurrent corticosterone. Tri-ortho-tolyl phosphate was administered by gavage (75, 150, or 300 mg/kg) every other day between days 14 and 28 and between days 49 and 63, chlorpyrifos (60 mg/kg) was administered subcutaneously on days 7 and 42, and corticosterone was provided in the drinking water throughout the study at a concentration of 400 microg/mL. Although relatively uncommon, there was an increase in frequency of cytoplasmic vacuoles seen in treatment groups having multiple exposures to TOTP. They were characterized as peripherally located, single-limiting membrane-bound structures in the neuronal perikarya. There was no associated cell death, even when vacuoles were large. This is the initial report of an association of this change following exposure to neurotoxic organophosphates.

Electron probe microanalysis of the elemental (K, P) content in the cytoplasm of cardiomyocyte of pregnant rat after an acute hypoxia incident

The cytoplasmic concentrations of elements (K, P) in the cytoplasm of rat (Wistar) cardiomyocytes have been determined by electron probe microanalysis. The intracellular content of potassium and phosphorus was determined in early organogenesis and in unfertilized animals. A decrease in the cytoplasmic concentration of phosphorus and an increase in potassium concentration were shown in pregnant animals. Acute hypobaric hypoxia was shown to change the cytoplasmic contents of both elements. The data obtained are discussed in terms of activation of the ion transport system aimed at compensation for cell acidosis and lactosis induced by development of hypoxic deenergization in pregnant animals.

Expression of germ cell nuclear factor in rat epididymis during postnatal development

To explore the expression and distribution of germ cell nuclear factor (GCNF), an orphan receptor of the nuclear receptor superfamily which is supposed to have multifunction including spermatogenesis, neurogenesis and embryo development, in adult rat epididymis and young rats during postnatal development. Whole epididymis was collected from SD rats' aged 7, 14, 28, 45, 60 and 90 days, and 18 months. The protein expression of GCNF during rat epididymal development and its cellular localizations were examined by Western blotting and immunohistochemistry respectively. GCNF was immunolocalized in the nuclei of epididymal epithelial cells with negatively stained cytoplasm in rats during postnatal development. Study of postnatal development revealed that the density of GCNF protein (about 55000) was age-dependent. GCNF protein was first detected in the rats aged day 14. The number and intensity of positive cells increased along with differentiation and proliferation of the epithelium, and peaked when the rats were aged 45 days and then decreased gradually. The results of Western-blotting confirmed those of immunohistochemistry. In adult rats, basal cell, apical cell, narrow cell, and clear cell and halo cell. GCNF in the principal cells exhibited a region-specific and cell-specific expression pattern that was observed from the age of 14 days. The GCNF expression was gradually decreased from the initial segment to proximal cauda epididymis. There was a predominant expression in the proximal caput region. The principal cells in the same segment also displayed a variable staining pattern, with the nucleus being intensely, moderately, or weekly reactive. In the distal cauda epididymis, only the nuclei of clear cells displayed an intensive staining, as compared with very weak staining in the principal cells. The expression of GCNF may be concerned with the differentiation and development of epididymal epithelium, and its expression pattern is epididymal segment-specific and cell-specific.

Molecular Crowding and Viscosity as Determinants of Translational Diffusion of Metabolites in Subcellular Organelles

The role of molecular crowding and viscosity on the apparent translational diffusion coefficient (ADC) of small metabolites was investigated in different subcellular organelles using the pulse-field gradient spin-echo1H NMR technique. ADCs of metabolites with increasing radius of gyration (0.7 Å <RG< 4.5 Å) were measured in the cytoplasm of rat or chicken erythrocytes, in the nucleus of chicken erythrocytes, and in isolated rat liver mitochondria. Metabolite ADCs in these systems were compared with the corresponding ADCs determined in model solutions of increasing bulk viscosity but different molecular crowding. For solutions having the same viscosity, metabolite ADCs decreased with increasing concentration of cosolutes. This effect is adequately described by the modified Stokes–Einstein relationship, ADC =k/RG(1 + 2.5Φ), wherekis a constant for a given temperature and Φ is an obstruction factor reporting the fractional volume of solution occupied by cosolutes, a measure of the molecular crowding in the solution. Cytoplasmic values of Φ for metabolites of different sizes did not depend exclusively on metaboliteRGbut on additional factors including the chemical nature of the metabolite, the presence of diffusional barriers, and metabolite-specific binding sites. In the case of water, nuclear Φ values approached those of the extracellular space while mitochondrial Φ values were significantly higher than those of the cytoplasm. Taken together, these results reveal important differences in molecular crowding within the different subcellular compartments, suggesting considerable diffusional heterogeneity for small metabolites within the different intracellular organelles.

Multiple protein domains determine the cell type-specific nuclear distribution of the catalytic subunit required for apolipoprotein B mRNA editing.

Apolipoprotein B (apoB) mRNA editing catalyzed by apoB mRNA editing catalytic subunit 1 (APOBEC-1) has been proposed to be a nuclear process. To test this hypothesis, the subcellular distribution of hemagglutinin- (HA) tagged APOBEC-1 expressed in transiently transfected hepatoma cells was determined by indirect immunofluorescence microscopy. HA-APOBEC-1 was detected in both the nucleus and cytoplasm of rat and human hepatoma cells. Mutagenesis of APOBEC-1 demonstrated that the N-terminal 56 amino acids (1-56) were necessary for the nuclear distribution of APOBEC-1, but this region did not contain a functional nuclear localization signal (NLS). However, we identified a 24-amino acid domain in the C terminus of APOBEC-1 with characteristics of a cytoplasmic retention signal (CRS) or a nuclear export signal (NES). These data suggest, therefore, that the nuclear import of APOBEC-1 may not be mediated by a positive NLS; rather, it may be achieved by overcoming the effect of a CRS/NES. We also demonstrated that the nuclear distribution of APOBEC-1 occurred only in cell lines that were capable of editing apoB RNA. We propose that the cellular distribution of APOBEC-1 is determined by multiple domains within this protein, and a nuclear localization of the enzyme may be regulated by cell type-specific factors that render these cells uniquely editing competent.

Development of rat × mouse hybrid embryos produced by microsurgery

Experimental production of hybrid embryos between Mus musculus L. and Rattus norvegicus L. was achieved by nuclear transplantation using both ovulated oocytes in metaphase II and pronuclear zygotes. Recipient egg-cells were of mouse origin in all cases. The developmental potential of hybrids was examined in vivo. Nucleo-cytoplasmic hybrids resulting from the introduction of rat metaphase II chromosomes into enucleated mouse oocytes, which were subsequently activated, were regularly blocked at the 1- or 2-cell stage. Nuclear (genetic) hybrids produced by transfer of a rat nucleus (in the form of metaphase II chromosomes or a pronucleus) into a nucleated mouse recipient (oocyte or zygote) were capable of development to the 5- to 8-cell stage. Transplantation of rat cytoplasm alone to intact metaphase II oocytes, followed by oocyte activation, generated cytoplasmic hybrids which developed to the morula stage. In control experiments (nuclear transfer between mouse oocytes or zygotes), a high proportion of embryos formed morulae and blastocysts. These results demonstrate that the rat nucleus is incapable of functioning in mouse cytoplasm, that introduction of the rat genome into intact mouse egg-cells impairs normal development, and that transfer of foreign (rat) cytoplasm into mouse egg-cells affects preimplantation development of manipulated embryos.

Developmental Abilities of Rat and Mouse Pronuclear Eggs Following Interspecific Nuclear or Cytoplasmic Transplantation.

Homologous and heterologous replacement of a small amount of cytoplasm or both pronuclei were performed in rat and mouse pronuclear eggs. Rat eggs after receiving rat or mouse cytoplast never developed beyond the 2-cell stage in vitro. On the other hand, the in-vitro development of mouse eggs that received rat cytoplasm was possible, but it was greatly inhibited compared with those that received mouse cytoplasm. None of enucleated rat eggs that received rat or mouse pronuclei also developed beyond the 2-cell stage. Although few enucleated mouse eggs after receiving rat pronuclei developed beyond the 2-cell stage, high proportion (64%) of those that received mouse pronuclei could develop to blastocysts in vitro. When 2-cell embryos obtained 18 h after fusing with donor karyoplast were transferred to pseudopregnant rats, 2 (9%) of 23 enucleated rat embryos and 1 (7%) of 15 enucleated mouse embryos each fused with heterologous karyoplast developed to early blastocyst 48-55 h after transfer. These results may indicate that the embryonic genome can be activated in the heterospecific cytoplasm.

Purification, characterization, cloning, and amino acid sequence of the bifunctional enzyme 5,10-methylenetetrahydrofolate dehydrogenase/5,10-methenyltetrahydrofolate cyclohydrolase from Escherichia coli.

We have purified the enzyme 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) from Escherichia coli to homogeneity by a newly devised procedure. The enzyme has been purified at least 2,000-fold in a 31% yield. The specific activity of the enzyme obtained is 7.4 times greater than any previous preparation from this source. The purified enzyme is specific for NADP. The protein also contains 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9) activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and behavior on a molecular sieving column suggest that the enzyme is a dimer of identical subunits. We have cloned the E. coli gene coding for the enzyme through the use of polymerase chain reaction based on primers designed from the NH2 terminal analysis of the isolated enzyme. We sequenced the gene. The derived amino acid sequence of the enzyme contains 287 amino acids of Mr 31,000. The sequence shows 50% identity to two bifunctional mitochondrial enzymes specific for NAD, and 40-45% identity to the presumed dehydrogenase/cyclohydrolase domains of the trifunctional C1-tetrahydrofolate synthase of yeast mitochondria and cytoplasm and human and rat cytoplasm. An identical sequence of 14 amino acids with no gaps is present in all 7 sequences.

Secretory proteins of the lung in rodents: immunocytochemistry.

The reactivity of rabbit antisera to rat lung secretory proteins with other rodent species was evaluated by immunocytochemistry. Rabbit anti-rat surfactant apoprotein antiserum reacts with the cytoplasm of rat, mouse, and hamster type II pneumocytes and is specific for these cells. Rabbit antiserum to rat Clara cell secretory proteins stains rat, mouse, and hamster Clara cells. Rabbit antisera specific to the two antigenic types of rat Clara cell antigens were also both reactive with rat, mouse, and hamster Clara cells. An antiserum to the non-serum proteins of hamster lung lavage was also prepared and shown to be specifically reactive with hamster Clara cells. The availability of specific reagents for secretory proteins of rodent lungs is expected to facilitate studies of the respective cell types in various pathologic states.

Ultrastructure of the rat mammary gland after lactogenesis induced by perphenazine.

Summary. The ultrastructural response of the rat mammary gland was studied in normal oestrous rats given 0·0 2·5, 5·0 or 10·0 mg perphenazine/kg body weight daily for 5 days starting 12 hr after ovulation. Growth of the parenchymal tissue was largely limited to alveolar development. Tissue from controls receiving only injections of the 0·03 n-HCl vehicle was similar to that from rats at 7 days' gestation, the earliest stage of pregnancy studied. Protein granules appeared in the Golgi complexes in rats given the 2·5 mg/kg dose and the occurrence increased with increasing dose levels, but these granules did not appear in the lumina until the dose reached 5·0 mg/kg. Large lipid droplets appeared in the cytoplasm in rats given the 5·0 mg/kg dose or more, but were not found in the lumina except at the highest dose. Despite the presence of abundant protein and lipid accumulations in the cytoplasm and lumina in mammary tissue of rats given the 10·0 mg/kg dose and the abundance of free ribosomes in the parenchymal cells, the rough endoplasmic reticulum was grossly underdeveloped as compared with mammary glands removed from pregnant rats 2 days before parturition. Evidence is presented from these and collateral studies to suggest that progesterone levels, elevated by the perphenazine treatment, are responsible for inhibition of rough endoplasmic reticulum development and, thus, the capacity for both protein and lipid biosynthesis.

Fluorouracil effect on cytochrome c reductase activity of rat liver cell endoplasmic reticulum following partial hepatectomy.

FUra (5-fluorouracil) prevents the appearance of 29 and 18S ribosomal RNA in the liver cell cytoplasm of rat, but permits the appearance of smooth-surfaced endoplasmic reticulum, as is clearly seen following partial hepatectomy. The present investigation revealed that the liver microsomal protein of rats treated with the antimetabolite FUra has a high concentration of NADPH oxidating enzyme in sham as well as in partially hepatectomized rats. There was even a tendency towards increased total amount of NADPH oxidating microsomal enzyme per liver in the FUra-treated rats as compared with control animals.

Evaluation of Radio-protective Efficacy of Cysteamine (with Rat-liver-cell Glycogen as Reference System)

SummaryA technique for localization of glycogen in the hepatic-cell cytoplasm of rats was utilized for determination of the radio-protective efficacy of cysteamine with 15 mg dose per animal.Radiation-induced glycogenesis was at its maximum peak 24 hours after irradiation with 300 r, 500 r and 800 r.After irradiation with 500 r, protective efficacy was maximal at 4 days and 13 days after irradiation. After 300 r and 800 r, efficacy was maximal at 4 days and 3 days after irradiation, respectively.