Isolation of cDNA clones coding for mitochondrial 16S ribosomal RNA from the crustacean Artemia

cDNA clones coding for Artemia mitochondrial 16S ribosomal RNA (rRNA) have been isolated. The clones cover from nucleotide 650 of the RNA molecule to its 3' end. The comparison of Artemia sequence with both vertebrate and invertebrate mitochondrial 16S rRNA sequences has shown the existence of regions of high similarity between them. A model for the secondary structure of the 3' half of Artemia mitochondrial 16S rRNA is proposed. The size of the rRNA molecule has been estimated at 1.35 kb. Despite the similarity of the Artemia gene to insect rRNA in size, sequence and secondary structure, the G + C content of the Artemia gene (42%) is closer to that of mammals than to the insect genes. The number of mitochondria in Artemia has been estimated at 1500 per diploid genome in the cyst and 4000 in the nauplius. In contrast, the amount of mt 16S rRNA is constant at all stages of Artemia development.     

Molecular cloning and analysis of cDNA sequences for two ribosomal proteins from Artemia. The coordinate expression of genes for ribosomal proteins and elongation factor 1 during embryogenesis of Artemia

The large subunit of eukaryotic ribosomes contains acidic phosphoproteins which are related to L7/L12 from Escherichia coli. In the brine shrimp Artemia these proteins are designated eL12 and eL12'. We have isolated cDNA clones for these proteins from a cDNA bank that was constructed by the use of size-fractionated poly(A)-rich RNA (8-10S fraction) from Artemia and a synthetic oligonucleotide as primer. Clones containing DNA sequences coding for eL12 and eL12 were characterized by hybrid-selected translation and DNA sequencing. The proteins eL12 and eL12' share an identical peptide of 22 amino acids at their carboxy termini whereas the remaining part of the protein shows little sequence homology. The nucleotide sequences show a different codon use for the amino acids in the common carboxy terminus, thereby excluding a common exon coding for this part of both proteins. Despite the differences in amino acid sequence in the major part of eL12 and eL12' the proteins have a considerable degree of homology on the basis of the distribution of hydrophobic and hydrophilic amino acids over the polypeptide chains, in agreement with a related folding and function of both proteins. Relative levels of mRNA coding for eL12, eL12' and elongation factor 1 alpha were determined during the development of Artemia from a dormant cyst to a nauplius. The data show a coordinate expression of the genes for EF-1 alpha and both ribosomal proteins, excluding a differential expression of the genes for these related ribosomal proteins during embryogenesis. Analysis of the gene copy number for eL12 and eL12' indicates the presence of a few genes for each protein.     

The effects of chronic smoking on lung tissue and the role of alpha lipoic acid

Abstract

We investigated the effects of alpha lipoic acid (ALA) on blood and lung tissue exposed chronically to cigarette smoke (CS). Female Sprague-Dawley rats were divided into three groups. Group 1 was the control group (CON): fresh air was supplied twice daily and 0.1 ml physiological saline was given orally for 8 weeks. Group 2 was exposed to CS: 12 cigarettes were smoked daily at two sessions for 1 h and 0.1 ml saline was given orally for 8 weeks. Group 3 (CS + ALA) was exposed to 12 cigarettes daily in two sessions for 1 h and 100 mg/kg/day ALA was given orally for 8 weeks. DNA damage was assessed using comet analysis; oxidative damage was assessed using ischemia-modified albumin (IMA) from blood; and total oxidant status (TOS), total antioxidant status (TAS) and oxidative stress index (OSI) were measured in blood and lung tissue. Histopathological and immunohistochemical evaluation of hypoxia-inducible factor (HIF)-1α, and ?2α, caspase-3, vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF₂) were conducted using lung tissue. The oxidative markers, TOS, OSI and IMA, and the comet analysis score were increased and the TAS level was decreased in the blood of the CS group compared to the CON group. IMA levels in blood, and TOS and OSI levels in the lung were decreased significantly in the CS + ALA group compared to the CS group. We observed increased septal wall thickness, marked and diffuse inflammatory reaction, emphysema, and necrotic cell debris in bronchial and bronchiolar lumens in the CS group. HIF-1α, HIF-2α, caspase-3 and FGF₂ expressions were increased, while VEGF expression decreased in the lung tissues of the CS group compared to the CON group. ALA slightly ameliorated the damage caused by chronic exposure to CS in the lungs, but further investigation is needed to determine its possible protective effects at different dosages.     

Blood glucose control for individuals with type-2 diabetes: acute effects of resistance exercise of lower cardiovascular-metabolic stress

ABSTRACT: Moreira, SR, Sim?es, GC, Moraes, JFVN, Motta, DF, Campbell, CSG, and Sim?es, HG. Blood glucose control for individuals with type-2 diabetes: Acute effects of resistance exercise of lower cardiovascular-metabolic stress. J Strength Cond Res 26(10): 2806-2811, 2012-This study compared the effects of resistance exercise (RE) intensities on blood glucose (GLUC) of individuals without (ND) and with type-2 diabetes (T2D). Nine individuals with T2D and 10 ND performed: (a) RE circuit at 23% of 1 maximal repetition (1RM) (RE_L); (b) RE circuit at 43% 1RM (RE_M); and (c) control (CON) session. Blood lactate (LAC) and GLUC were measured before, during, and postinterventions. Double product (DP) and rate of perceived exertion (RPE) were recorded. The area under the curve (AUC) revealed the effects of RE circuits in reducing GLUC in individuals with T2D (RE_L: 12,556 ± 3,269 vs. RE_M: 13,433 ± 3,054 vs. CON: 14,576 ± 3,922 mg.dl.145 minutes; p < 0.05) with a lower AUC of GLUC in RE_L in comparison to RE_M. Similarly, for ND the RE_L reduced the AUC of GLUC when compared with RE_M and CON (RE_L: 10,943 ± 956 vs. RE_M: 12,156 ± 1,062 vs. CON: 11,498 ± 882 mg.dl.145 minutes; p < 0.05). The AUC of GLUC was higher for T2D compared with ND on CON condition (p = 0.02). However, after RE circuits the difference between groups for AUC of GLUC was abolished. The RE_M for T2D was more stressful when compared with RE_L for LAC (CON: 1.3 ± 0.5 vs. RE_L: 5.5 ± 1.5 vs. RE_M: 6.8 ± 1.3 mmol·L; p < 0.05), DP (CON: 8,415 ± 1,223 vs. RE_L: 15,980 ± 2,007 vs. RE_M: 18,047 ± 3,693 mmHg.bpm; p < 0.05), and RPE (RE_L: 11 ± 2 vs. RE_M: 13 ± 2 Borg Scale; p < 0.05). We concluded that RE_L and RE_M were effective in reducing GLUC for individuals with T2D, with lower cardiovascular-metabolic and perceptual stress being observed for RE_L. These data suggest that acute RE sessions at light or moderate intensities are effective for controlling GLUC in individuals with T2D.     

Increased hepatic expression of TLR2 and TLR4 in the hepatic inflammation-fibrosis-carcinoma sequence

We evaluated expression of TLR2, TLR4 and proinflammatory genes [NF-κB, TNF-α, cyclooxygenase-2 (COX-2)] in liver samples of patients in different stages of liver disease. Fifteen patients with unexplained transaminases elevation (reference group), 22 with viral chronic hepatitis (hepatitis group), 14 with virus-induced severe fibrosis/cirrhosis (cirrhosis group) and 10 with hepatocarcinoma (hepatocarcinoma group) were consecutively included in the study. Quantification of TLR2, TLR4, NF-κB, TNF-α and COX-2 mRNA was done by real-time RT-PCR and TLR2 and TLR4 protein expression was evaluated by immunohistochemistry. Compared with reference, TLR2 and TLR4 mRNA was increased in hepatitis (TLR2: 2.66?±?0.69; TLR4: 3.11?±?0.79; P?相似文献   

卤虫投喂下长吻(鱼危)仔稚鱼消化酶发育和口宽变化的研究

采用酶学和形态学测定方法, 研究在投喂卤虫条件下长吻(鱼危)仔鱼4种主要消化酶: 胃蛋白酶、胰蛋白酶、脂肪酶和淀粉酶的活性变化以及长吻(鱼危)仔鱼口宽、全长变化。实验共进行13d, 实验结果表明: (1)长吻(鱼危)仔鱼全长、口宽的发育与其日龄表现出明显的线性正相关(R_TL²=0.974, R_MW²=0.964)。口宽与全长比值(MW/TL)在仔鱼开口后急剧下降, 并自7日龄开始维持在0.07—0.08, 口宽和全长处于同步发育期并表现出明显的相关性(R²=0.948), 说明7日龄(/h, days post hatching)后口宽和全长处于同步发育期, 标志仔鱼转食的开始。(2)长吻(鱼危)仔鱼初次开口时即可检测出四种消化酶的活性。5—7/h时胰蛋白酶显著高于初孵仔鱼, 与此时仔鱼开始开口摄食的行为相一致。胃蛋白酶、脂肪酶活性在仔鱼孵化后第7天即开口的第3天, 淀粉酶活性在孵化后第6天, 显著高于初次孵化出来的仔鱼。8—13/h时, 胃蛋白酶、胰蛋白酶、脂肪酶和淀粉酶活性均在较高水平平稳的波动, 标志着消化道发育逐渐健全。     

Morphological development of larval cobia Rachycentron canadum and the influence of dietary taurine supplementation

The morphological development of larval cobia Rachycentron canadum from 3 days post hatch (dph) until weaning (27 dph) was examined using S.E.M. Two groups of fish were studied: a control group (CF), reared under standard feeding protocol, and a group in which prey items were enriched with supplemental taurine (4 g l(-1) day(-1) ; TF). TF fish grew faster (P < 0·001), attained greater size (mean ±s.e. 55·1 ± 1·5 v. 33·9 ± 1·0 mm total length) and had better survival (mean ±s.e. 29·3 ± 0·4 v. 7·1 ± 1·2 %) than CF fish. Canonical variance analysis confirmed findings with respect to differences in growth between the treatment groups with separation being explained by two cranial measurements. S.E.M. revealed that 3 dph larvae of R. canadum (in both groups) possess preopercular spines, superficial neuromasts on the head and body, taste buds in the mouth, an olfactory epithelium which takes the form of simple concave depressions, and primordial gill arches. Gill filaments start to form as early as 6 dph and lamellae buds are visible at 8 dph in both groups. In CF fish, the cephalic lateral line system continues its development at 12-14 dph with invagination of both supra- and infraorbital canals. At the same time, a thorn-like or acanthoid crest forms above the eye. At 14 dph, invaginations of the mandibular and preopercular canals are visible and around 22 dph enclosure of all cranial canals nears completion. In CF larvae, however, completely enclosed cranial canals were not observed within the course of the trial, i.e. 27 dph. In TF larvae, grooves of the cephalic lateral line system form 4 days earlier than observed in CF larvae of R. canadum (i.e. at 8 dph), with enclosure commencing at 16 dph, and completed by 27 dph. Along the flanks of 6 dph larvae of either treatment, four to five equally spaced neuromasts delineate the future position of the trunk lateral line. As myomeres are added to the growing larvae, new neuromasts appear such that at 16 dph a neuromast is associated with each myomere. By 27 dph, the trunk lateral line starts to invaginate in CF larvae, while it initiates closure in TF larvae. These findings elucidate important features of the larval development of R. canadum and show that dietary taurine supplementation benefits larval development, growth and survival in this species. Moreover, they suggest a conditional requirement for taurine in larval R. canadum.     

Expression of beta 1, beta 3, beta 4, and beta 5 integrins by human epidermal keratinocytes and non-differentiating keratinocytes

We have compared the adhesive properties and integrin expression profiles of cultured human epidermal keratinocytes and a strain of nondifferentiating keratinocytes (ndk). Both cell types adhered to fibronectin, laminin, and collagen types I and IV, but ndk adhered more rapidly and at lower coating concentrations of the proteins. Antibody blocking experiments showed that adhesion of both cell types to fibronectin was mediated by the alpha 5 beta 1 integrin and to laminin by alpha 3 beta 1 in synergy with alpha 2 beta 1. Keratinocytes adhered to collagen with alpha 2 beta 1, but an antibody to alpha 2 did not inhibit adhesion of ndk to collagen. Both cell types adhered to vitronectin by alpha v-containing integrins. Immunoprecipitation of surface-iodinated and metabolically labeled cells showed that in addition to alpha 2 beta 1, alpha 3 beta 1, and alpha 5 beta 1, both keratinocytes and ndk expressed alpha 6 beta 4 and alpha v beta 5. ndk expressed all these integrins at higher levels than normal keratinocytes. ndk, but not normal keratinocytes, expressed alpha v beta 1 and alpha v beta 3; they also expressed alpha 1 beta 1, an integrin that was not consistently detected on normal keratinocytes. Immunofluorescence experiments showed that in stratified cultures of normal keratinocytes integrin expression was confined to cells in the basal layer; terminally differentiating cells were unstained. In contrast, all cells in the ndk population were integrin positive. Our observations showed that the adhesive properties of ndk differ from normal keratinocytes and reflect differences in the type of integrins expressed, the level of expression and the distribution of integrins on the cell surface. ndk thus have a number of characteristics that distinguish them from normal basal keratinocytes.     

Allometric growth and functional development of the gut in developing cod Gadus morhua L. larvae

The development of the gut epithelium in cod Gadus morhua was studied during the larval period in intensive rearing systems. Cod larvae were fed enriched rotifers from mouth opening. On 17 days post‐hatch (dph) one group of larvae were fed Artemia sp. nauplii while another group were fed both rotifers and a formulated diet (co‐fed). At the end of the experiment (30 dph) larvae receiving live feed were almost three times larger than the co‐fed larvae, although no clear signs of pathological effects due to feeding regime were found in any larvae sampled for morphological studies. The midgut volume in larvae fed live feed increased by a factor of 38 during the experiment, and in particular volume increased rapidly between 24 and 30 dph. The enterocyte size increased between 12 and 24 dph from 652 ± 64 to 1479 ± 144 μm³ (mean ±s .e .). When enterocytes reached their maximum size, several morphological changes in the gut epithelium were initiated, such as increased number of mitochondria per enterocyte, increased size of the nuclei and a considerable increase in microvilli surface area. The mitochondrial membrane structures changed during the experiment, suggesting a maturation process of the mitochondria. The midgut development was strongly related to larval size rather than age. On 30 dph co‐fed larvae were equal in size to Artemia sp. fed larvae on 24 dph. This was reflected by equal values of estimated midgut volume, midgut length and total number of enterocytes and the number of mitochondria per enterocyte. The microvilli surface area, however, was significantly larger in co‐fed larvae on 24 dph compared to live‐feed larvae on 30 dph. This increase in absorptive surface was probably a response to suboptimal feeding conditions. The strong correlation between gut development and larval size and the lack of clear pathological effects, suggested that the gut tissue is flexible and can withstand periods of suboptimal nutrition at this stage.     

Molecular structure of the core-modified siRNA duplexes containing diastereomeric pair of [C6'(R)-OH]- versus [C6'(S)-OH]-carba-LNAs suggests a model for RNAi action

Molecular structures of native and a pair of modified small interfering RNA-RNA duplexes containing carbocyclic [6?'-(R)-OH/7?'-(S)-methyl]- and [6?'-(S)-OH/7?'-(S)-methyl]-carba-LNA-thymine nucleotides, which are two diastereomeric analogs of the native T nucleotide, incorporated at position 13 in the antisense (AS) strand of siRNA, have been simulated using molecular mechanics/dynamics techniques. The main aim of the project has been to find a plausible structural explanation of why modification of siRNA at T(13) position by the [6?'(R)-O-(p-Toluoyl)-7?'(S)-methyl]-carba-LNA-Thymine [IC(50) of 3.32 ± 0.17 nM] is ca 24 times more active as an RNA silencing agent against the target HIV-1 TAR RNA than the [6?'(S)-O-(p-Toluoyl)-7?'(S)-methyl]-counterpart [IC(50) of 79.8 ± 17 nM] [1]. The simulations reveal that introduction of both C6?'(R)-OH and C6?'(S)-OH stereoisomers does not lead even to local perturbation of the siRNA-RNA duplex structures compared to the native, and the only significant difference between 6?'(S)- and 6?'(R)-diastereomers found is the exposure of the 6?'-OH group of the 6?'(R)-diastereoisomer toward the edge of the duplex while the 6?'-hydroxyl group of the 6?'(S)-diastereoisomer is somewhat buried in the minor groove of the duplex. This rules out a hypothesis about any possible local distortion by the nature of chemical modification of the siRNA-target the RNA duplex, which might have influenced the formation of the effective RNA silencing complex (RISC) and puts some weight on the hypothesis about the 6?'-hydroxy group being directly involved with most probably Ago protein, since it is known from exhaustive X-ray studies [2, 3] that the core residues are indeed involved with hydrogen bonding with the internucleotidyl phosphates. Further systematic investigation is in progress to map the position-dependent functional and nonfunctional interactions of the modified [6?'(R or S)-O-(p-Toluoyl)-7?'(S)-methyl]-carba-LNA-T with the Ago2 protein of the RISC.     

The acute response of practical occlusion in the knee extensors

Training at low intensities with moderate vascular occlusion results in increased muscle hypertrophy, strength, and endurance. Elastic knee wraps, applied to the proximal portion of the target muscle, might elicit a stimulus similar to the KAATSU Master Apparatus. The purpose of this study was to test the hypothesis that intermittently occluding the leg extensors with elastic knee wraps would increase whole-blood lactate (WBL) over control (CON). Twelve healthy men and women participated in this study (age 21.2 ± 0.35 years, height 168.9 ± 2.60 cm, and body mass 71.2 ± 4.16 kg). One repetition maximum (1RM) testing for the leg extensors was performed on a leg extension machine for the first trial, followed by occlusion (OCC) and CON trials. Four sets of leg extension exercise (30-15-15-15) were completed with 150-second rest between sets at 30% 1RM. Whole-blood lactate, heart rate (HR), and ratings of perceived exertion (RPEs) were measured after every set of exercise and 3 minutes postexercise. Data were analyzed using repeated-measures analysis of variance with statistical significance set at p ≤ 0.05. Whole-blood lactate increased in response to exercise (p = 0.01) but was not different between groups (OCC 6.28 ± 0.66 vs. CON 5.35 ± 0.36 mmol·L, p = 0.051). Heart rate (OCC 128.86 ± 4.37 vs. CON 119.72 ± 4.10 b·min?1) was higher with OCC from sets 2-4 (p ≤ 0.03), with no difference 3 minutes postexercise (p = 0.29). Rating of perceived exertion was higher with OCC after every set (OCC 15.10 ± 0.31 vs. CON 12.16 ± 0.50, p = 0.01). In conclusion, no differences exist for WBL between groups, although there was a trend for higher levels with OCC. The current protocol for practical occlusion did not significantly increase metabolic stress more than normal low-intensity exercise. This study does not support the use of knee wraps as a mode of blood-flow restriction.