Increased endothelial and vascular smooth muscle cell adhesion on nanostructured titanium and CoCrMo

In the body, vascular cells continuously interact with tissues that possess nanostructured surface features due to the presence of proteins (such as collagen and elastin) embedded in the vascular wall. Despite this fact, vascular stents intended to restore blood flow do not have nanoscale surface features but rather are smooth at the nanoscale. As the first step towards creating the next generation of vascular stent materials, the objective of this in vitro study was to investigate vascular cell (specifically, endothelial, and vascular smooth muscle cell) adhesion on nanostructured compared with conventional commercially pure (cp) Ti and CoCrMo. Nanostructured cp Ti and CoCrMo compacts were created by separately utilizing either constituent cp Ti or CoCrMo nanoparticles as opposed to conventional micron-sized particles. Results of this study showed for the first time increased endothelial and vascular smooth muscle cell adhesion on nanostructured compared with conventional cp Ti and CoCrMo after 4 hours' adhesion. Moreover, compared with their respective conventional counterparts, the ratio of endothelial to vascular smooth muscle cells increased on nanostructured cp Ti and CoCrMo. In addition, endothelial and vascular smooth muscle cells had a better spread morphology on the nanostructured metals compared with conventional metals. Overall, vascular cell adhesion was better on CoCrMo than on cp Ti. Results of surface characterization studies demonstrated similar chemistry but significantly greater root-mean-square (rms) surface roughness as measured by atomic force microscopy (AFM) for nanostructured compared with respective conventional metals. For these reasons, results from the present in vitro study provided evidence that vascular stents composed of nanometer compared with micron-sized metal particles (specifically, either cp Ti or CoCrMo) may invoke cellular responses promising for improved vascular stent applications.     

Muscle interstitial glucose and lactate levels during dynamic exercise in humans determined by microdialysis.

The purpose of the present study was to use the microdialysis technique to determine skeletal muscle interstitial glucose and lactate concentrations during dynamic incremental exercise in humans. Microdialysis probes were inserted into the vastus lateralis muscle, and subjects performed knee extensor exercise at workloads of 10, 20, 30, 40, and 50 W. The in vivo probe recoveries determined at rest by the internal reference method for glucose and lactate were 28.7 +/- 2.5 and 32.0 +/- 2.7%, respectively. As exercise intensity increased, probe recovery also increased, and at the highest workload probe recovery for glucose (61.0 +/- 3.9%) and lactate (66. 3 +/- 3.6%) had more than doubled. At rest the interstitial glucose concentration (3.5 +/- 0.2 mM) was lower than both the arterial (5.6 +/- 0.2 mM) and venous (5.3 +/- 0.3 mM) plasma water glucose levels. The interstitial glucose levels remained lower (P < 0.05) than the arterial and venous plasma water glucose concentrations during exercise at all intensities and at 10, 20, 30, and 50 W, respectively. At rest the interstitial lactate concentration (2.5 +/- 0.2 mM) was higher (P < 0.05) than both the arterial (0.9 +/- 0. 2 mM) and venous (1.1 +/- 0.2 mM) plasma water lactate levels. This relationship was maintained (P < 0.05) during exercise at workloads of 10, 20, and 30 W. These data suggest that interstitial glucose delivery at rest is flow limited and that during exercise changes in the interstitial concentrations of glucose and lactate mirror the changes observed in the venous plasma water compartments. Furthermore, skeletal muscle contraction results in an increase in the diffusion coefficient of glucose and lactate within the interstitial space as reflected by an elevation in probe recovery during exercise.     

Nucleotide sequence of the rice cytochrome f gene and the presence of sequence variation near this gene

The rice (Oryza sativa L. var. Labelle) chloroplast (cp) gene encoding cytochrome f has been isolated and sequenced. The coding region of this rice gene displays 95.1%, 85.3% and 85.2% nucleotide sequence homology with that of wheat, pea and spinach, respectively. To examine the cpDNA sequence variation in rice, cpDNA from Labelle and its parents, Belle Patna and Dawn was compared. Using the cytochrome f gene as the probe for hybridization, we found several differences in the size and number of restriction fragments in the cp genome of three rice varieties. An additional restriction fragment found in the Belle Patna cp suggests that this cp genome is either heterogeneous or contains two copies of cytochrome f gene per cpDNA.     

Lateral diffusion of lipids and glycophorin in solid phosphatidylcholine bilayers. The role of structural defects

The lateral mobility of the lipid analog N-4-nitrobenzo-2-oxa-1,3 diazole phosphatidylethanolamine and of the integral protein glycophorin in giant dimyristoylphosphatidylcholine vesicles was studied by the photobleaching technique. Above the temperature of the chain-melting transition (Tm = 23 degrees C), the diffusion coefficient, Dp, of the protein [Dp = (4 +/- 2) X 10(-8) cm2/s at 30 degrees C] was within the experimental errors equal to the corresponding values DL of the lipid analog. In the P beta 1 phase the diffusion of lipid and glycophorin was studied as a function of the probe and the protein concentration. (a) At low lipid-probe content (cL less than 5 mmol/mol of total lipid), approximately 20% of the probe diffuses fast (D approximately equal to 10(-8) - 10(-9) cm2/s), while the mobility of the rest is strongly reduced (D less than 10(-10) cm2/s). At a higher concentration (cp approximately 20 mmol), all probe is immobilized (D less than 10(-10) cm2/s). (b) Incorporation of glycophorin up to cp = 0.4 mmol/mol of total lipid leads to a gradual increase of the fraction of mobile lipid probe due to the lateral-phase separation into a pure P beta 1 phase and a fraction of lipid that is fluidized by strong hydrophilic lipid-protein interaction. (c) The diffusion of the glycophorin molecules is characterized by a slow and a fast fraction. The latter increases with increasing protein content, which is again due to the lateral-phase separation caused by the hydrophilic lipid-protein interaction. The results are interpreted in terms of a fast transport along linear defects in the P beta 1 phase, which form quasi-fluid paths for a nearly one dimensional and thus very effective transport. Evidence for this interpretation of the diffusion measurements is provided by freeze-fracture electron microscopy.     

石化废水中酚对凤眼莲生长的影响

半动态酚毒性模拟试验表明：酚浓度高于600mg／l,凤眼莲几天内中毒死亡;酚浓度350mg／l以上亦造成不可恢复的损伤;酚浓度40mg／l以上,凤眼莲生长受到不同程度的抑制。同时叶绿素含量及a／b比率,光合作用速率及生长率等亦随酚浓度增高而降低。较强光照增加了酚的毒性影响。酚浓度25mg／l以下对凤眼莲无明显抑制影响。10mg／l以下浓度的酚还可以促进凤眼莲的生长。酚使凤眼莲无性繁殖能力下降但可促进其提早开花。     

Pair feeding-mediated changes in metabolism: stress response and pathophysiology in insulin-resistant, atherosclerosis-prone JCR:LA-cp rats

Rats of the JCR:LA-cp strain, which are homozygous for the cp gene (cp/cp), are obese, insulin-resistant, and hyperinsulinemic. They exhibit associated micro- and macrovascular disease and end-stage ischemic myocardial lesions and are highly stress sensitive. We subjected male cp/cp rats to pair feeding (providing the rats each day with the amount of food eaten by matched freely fed animals), a procedure that alters the diurnal feeding pattern, leading to a state of intermittent caloric restriction. Effects on insulin, glucose, and lipid metabolism, response to restraint stress, aortic contractile/relaxant response, and myocardial lesion frequency were investigated. Pair-fed young (12-wk-old) cp/cp rats had lower insulin and glucose levels (basal and following restraint), consistent with increased insulin sensitivity, but a greater increase in plasma nonesterified fatty acids in response to restraint. These effects were unrelated to lipolytic rates in adipose tissue but may be related to reduced fatty acid oxidation in skeletal muscle. Older (24-wk-old) pair-fed cp/cp rats had significantly reduced plasma triglyceride levels, improved micro- and macrovascular function, and reduced severity of ischemic myocardial lesions. These changes indicate a significant amelioration of end-stage disease processes in this animal model and the complexity of metabolic/physiological responses in studies involving alterations in food intake. The effects illustrate the sensitivity of the JCR:LA-cp rat, an animal model for the metabolic syndrome and associated cardiovascular disease, to the environmental and experimental milieu. Similar stress-related mechanisms may play a role in metabolically induced cardiovascular disease in susceptible human beings.     

Are turns required for the folding of ribonuclease T1?

Ribonuclease T1 (RNase T1) is a small, globular protein of 104 amino acids for which extensive thermodynamic and structural information is known. To assess the specific influence of variations in amino acid sequence on the mechanism for protein folding, circularly permuted variants of RNase T1 were constructed and characterized in terms of catalytic activity and thermodynamic stability. The disulfide bond connecting Cys-2 and Cys-10 was removed by mutation of these residues to alanine (C2, 10A) to avoid potential steric problems imposed by the circular permutations. The original amino-terminus and carboxyl-terminus of the mutant (C2, 10A) were subsequently joined with a tripeptide linker to accommodate a reverse turn and new termini were introduced throughout the primary sequence in regions of solvent-exposed loops at Ser-35 (cp35S1), Asp-49 (cp49D1), Gly-70 (cp70G1), and Ser-96 (cp96S1). These circularly permuted RNase T1 mutants retained 35-100% of the original catalytic activity for the hydrolysis of guanylyl(3'-->5')cytidine, suggesting that the overall tertiary fold of these mutants is very similar to that of wild-type protein. Chemical denaturation curves indicated thermodynamic stabilities at pH 5.0 of 5.7, 2.9, 2.6, and 4.6 kcal/mol for cp35S1, cp49D1, cp70G1, and cp96S1, respectively, compared to a value of 10.1 kcal/mol for wild-type RNase T1 and 6.4 kcal/mol for (C2, 10A) T1. A fifth set of circularly permuted variants was attempted with new termini positioned in a tight beta-turn between Glu-82 and Gln-85. New termini were inserted at Asn-83 (cp83N1), Asn-84 (cp84N1), and Gln-85 (cp85Q1). No detectable amount of protein was ever produced for any of the mutations in this region, suggesting that this turn may be critical for the proper folding and/or thermodynamic stability of RNase T1.     

Cardiovascular disease in the JCR:LA-cp rat

The JCR:LA-cp rat is one of a number of strains that carry the mutant autosomal recessive cp gene. Animals, of all strains, that are homozygous, for the gene (cp/cp) become obese, insulin resistant, and hypertriglyceridemic. Heterozygotes or homozygous normal rats (+/+)are lean and metabolically normal. The JCR:LA-cp rat is unique in the development of a frank vasculopathy with atherosclerotic lesions and associated ischemic myocardial lesions. The cardiovascular disease is strongly correlated with the hyperinsulinemia, which develops as the animals mature from 4 to 8 weeks of age. The hyperinsulinemia can be decreased by marked food restriction, ethanol consumption, or reduction of the postprandial glucose and insulin responses through the use of a-glucosidase inhibitors. Any treatment that reduces plasma insulin levels is associated with a reduction in cardiovascular disease. In contrast, a reduction in plasma triglyceride concentrations, alone, has no effect on end-stage lesions. JCR:LA-cp rats, particularly those that are cp/cp, are, however, sensitive to cholesterol in the diet, unlike other strains that are highly resistant. Further, the rats have abnormal vascular smooth muscle cells that, especially in the cp/cp animals, are hyperplastic and activated and migrate into the intimal space. Our findings suggest that susceptibility to cardiovascular disease requires hypermsulinemic stress coupled with excessive dietary intake and the presence of one or more other necessary, but not sufficient, genetic factors. One of these may be a genetic abnormality of vascular smooth muscle cells. A similar situation may occur in humans.     

Requirements for Borrelia burgdorferi plasmid maintenance

Borrelia burgdorferi has multiple linear and circular plasmids that are faithfully replicated and partitioned as the bacterium grows and divides. The low copy number of these replicons implies that active partitioning contributes to plasmid stability. Analyzing the requirements for plasmid replication and partition in B. burgdorferi is complicated by the complexity of the genome and the possibility that products may act in trans. Consequently, we have studied the replication-partition region (bbb10-13) of the B. burgdorferi 26kb circular plasmid (cp26) in Escherichia coli, by fusion with a partition-defective miniF plasmid. Our analysis demonstrated that bbb10, bbb11, and bbb13 are required for stable miniF maintenance, whereas bbb12 is dispensable. To validate these results, we attempted to inactivate two of these genes in B. burgdorferi. bbb12 mutants were obtained at a typical frequency, suggesting that the bbb12 product is dispensable for cp26 maintenance as well. We could not directly measure cp26 stability in the bbb12 mutant, because cp26 carries essential genes, and bacteria that have lost cp26 are inviable. Conversely, we were unable to inactivate bbb10 on cp26 of B. burgdorferi. Our results suggest that bbb12 is dispensable for cp26 maintenance, whereas bbb10, bbb11, and bbb13 play crucial roles in that process.     

Identification and characterization of columbid annexin Icp37. Insights into the evolution of annexin I phosphorylation sites.

Annexin I (AnxI) contains phosphorylation sites in its "hinge region" that have been implicated in the regulation of cell growth and/or differentiation. A pigeon (Columba livia) isoform of this protein, annexin Icp35 (cp35), has a very similar amino acid sequence overall but an unrelated sequence that lacks phosphorylation sites in the hinge region. We now report the identification and characterization of annexin Icp37 (cp37) from pigeon. Genomic cloning and Southern blot analysis demonstrated that cp37 and cp35 were encoded by separated genes. Prolactin induced the expression of cp35 mRNA but not cp37. The amino acid sequence of cp37 was deduced from a cDNA clone and found to share 93 and 75% sequence identity with cp35 and human AnxI, respectively. The amino acid sequence of cp37 bore similarities to both AnxI and cp35 in the critical hinge region. Like AnxI, cp37 contained consensus phosphorylation sites in its amino acid sequence and was phosphorylated on tyrosine by the EGF receptor/kinase and on serine by protein kinase C in vitro. Despite the functional similarities between cp37 and AnxI, the nucleotide sequence that encoded the hinge region of cp37 was very similar to the analogous region of cp35, but different from that of AnxI. We propose that certain features shared by cp37 and AnxI are the products of convergent evolution. The fact that evolution independently selected for two annexin I-like genes (cp37 and anxI) encoding analogous phosphorylation sites is strong evidence that phosphorylation is important for the regulation of the biological activity of these proteins.     

High glucose concentrations induce oxidative damage to mitochondrial DNA in explanted vascular smooth muscle cells

Oxidative stress is considered to be one of the mechanisms leading to atherosclerosis. It occurs in response to injury or to altered metabolic state. Alterations in cell growth (proliferation or apoptosis) can also contribute to the pathogenesis of atherosclerosis and is influenced by oxidative stress. Smooth muscle cells (SMC) from aortic explants of JCR:LA-cp homozygous cp/cp corpulent rats who are genetically predisposed to develop atherosclerosis exhibit increased SMC proliferation, which can be attenuated by exercise and food restriction. This study was conducted to characterize the effects fo oxidative stress and high glucose media on cell growth and its relationship to mitochondrial DNA integrity and gene expression in explanted aortic SMC from corpulent and lean JCR:LA-cp rats. The results show that SMC from the cp/cp rat appear to be resistant to oxidant-induced cell death and that they accumulate mitochondrial DNA mutations, probably as a result of a reduction in apoptosis. These data suggest that susceptibility to age- and glucose-related atherosclerosis may be related to alterations in redox signaling.     

Intra- and Interbacterial Genetic Exchange of Lyme Disease Spirochete <Emphasis Type="Italic">erp</Emphasis> Genes Generates Sequence Identity Amidst Diversity

All isolates of the spirochete Borrelia burgdorferi contain multiple, different plasmids of the cp32 family, each of which contains a locus encoding Erp surface proteins. Many of these proteins are known to bind host complement regulatory factor H, enabling the bacteria to avoid killing by the alternative complement pathway during vertebrate infection. In the present study, we characterized the erp loci and cp32 plasmids of strains N40, Sh-2-82, and 297 and compared them to the previously determined cp32 sequences of type strain B31. Bacteria of strain N40 contain 6 different cp32s, those of Sh-2-82 contain 10, and 297 bacteria contain 9 cp32s. Significant conservation between all strains was noted for the cp32 loci responsible for plasmid maintenance, indicating close relationships that appear to correspond with incompatibility groups. In contrast, considerable diversity was found between erp gene sequences, both within individual bacteria and between different strains. However, examples of identities among erp loci were found, with strains Sh-2-82, 297, and B31 each containing three identical loci that likely arose through intrabacterial genetic rearrangements. These studies also found the first evidence of large-scale genetic exchanges between Lyme disease spirochetes in nature, including the apparent transfer of an entire cp32 plasmid between two different bacteria.     

Serum lipids and lipoproteins in the atherosclerosis prone LA/N corpulent rat

The LA/N rat is one of two congenic strains bred from the original obese, hyperphagic and hypertensive rats of Koletsky. With the exception of hypertension the LA/N strain, when homozygous for the corpulent gene, is phenotypically similar to the parent Koletsky strain and prone to the development of vascular and myocardial lesions. Here we report a detailed analysis of the serum lipids, lipoproteins and apolipoproteins B, E and A-I levels in young adult homozygous corpulent (cp/cp) rats of both sexes and in lean males of the same age which were demonstrable non-carriers (+/+) of the cp gene. Both male and female cp/cp rats were hypertriglyceridemic (282-512 mg/100 ml) and moderately hypercholesterolemic (74-84 mg/100 ml). Elevations in these lipids reflected the presence of large (622 A), triacylglycerol-rich and apoprotein-poor VLDL containing both apolipoproteins Bh and B1 and increased phospholipid-rich HDL. Similar, but less pronounced, elevations in serum apolipoproteins B and E in the cp/cp rats when compared to the +/+ animals were also noted. Apolipoproteins A-I levels were 2.7-3-fold higher in cp/cp rats. The levels of VLDL were significantly higher in female cp/cp rats; however, the levels of IDL (intermediate-density lipoproteins), LDL and HDL were significantly lower than in the more atherosclerosis prone male cp/cp rats. Similarly, apolipoprotein A-I was higher and apolipoprotein B lower in the male cp/cp than in the female cp/cp rats. The LDL (d = 1.030-1.063 g/ml) in cp/cp rats, like that in normal animals, was heterogeneous and contained apolipoproteins Bh, E, A-I and C. This fraction was significantly elevated in male cp/cp rats when compared to females but still represented less than 13% of the total serum cholesterol and less than 6% of the total serum lipids in 3-month-old cp/cp animals. The ratio of cholesterol to phospholipids was significantly lower for all lipoproteins in cp/cp rats when compared to +/+ males and these ratios for female cp/cp rats were in all cases lower than those of male cp/cp animals.     

Increased hepatic VLDL secretion, lipogenesis, and SREBP-1 expression in the corpulent JCR:LA-cp rat.

The corpulent JCR:LA-cp rat (cp/cp) is a useful model for study of the metabolic consequences of obesity and hyperinsulinemia. To assess the effect of hyperinsulinemia on VLDL secretion in this model, we measured rates of secretion of VLDL in perfused livers derived from cp/cp rats and their lean littermates. Livers of cp/cp rats secreted significantly greater amounts of VLDL triglyceride and apolipoprotein, compared with lean littermates. The content of apoB, apoE, and apoCs in both perfusate and plasma VLDL was greater in the cp/cp rat, as was the apolipoprotein (apo)C, apoA-I, and apoA-IV content of plasma HDL. Triglyceride content was also greater in cp/cp livers, as was hepatic lipogenesis and expression of lipogenic enzymes and sterol regulatory element binding protein-1 (SREBP-1). Hepatic mRNAs for apoE, and apoA-I were higher in livers of cp/cp rats. In contrast, the steady state levels of apoC-II, apoC-III, and apoB mRNAs were unchanged. Thus, livers of obese hyperinsulinemic cp/cp JCR:LA-cp rats secrete a greater number of VLDL particles that are enriched in triglyceride, apoE, and apoC. Greater secretion of VLDL in the cp/cp rat in part results from higher endogenous fatty acid synthesis, which in turn may occur in response to increased expression of the lipogenic enzyme regulator SREBP-1c.     

Cation-dependent light-induced structural changes in visual receptor membranes

A stearamide spin probe was used to study the light-induced structural changes in Rod Outer Segment Membranes in the presence of sodium and calcium ions. The correlation time (τ_C) for the reorientation of the probe was calculated in the dark and light. In the presence of sodium ions, τ_C = 3.3 × 10^?9 sec in the dark, and 2.7 × 10^?9sec in the light while the opposite was noticed in the presence of calcium ions, τ_C = 2.9 × 10^?9 sec in the dark and 3.6 × 10^?9 sec in the light. The correlation times for reorientation of the probe were also calculated in aqueous glycerol solutions of varying viscosities at 20°C. Comparison of the values of τ_C (dark and light) suggests a change in local mobility in the ROS corresponding to a macroscopic viscosity difference of approximately 150 cp. The significance of calcium ion interaction with negatively charged groups and the formation of a Schiff base is emphasized.     

Characterization of the chloroplast genome sequence of oil palm (Elaeis guineensis Jacq.)

Oil palm (Elaeis guineensis Jacq.) is an economically important crop, which is grown for oil production. To better understand the molecular basis of oil palm chloroplasts, we characterized the complete chloroplast (cp) genome sequence obtained from 454 pyrosequencing. The oil palm cp genome is 156,973 bp in length consisting of a large single-copy region of?85,192 bp flanked on each side by inverted repeats of 27,071 bp with a small single-copy region of 17,639 bp joining the?repeats. The genome contains 112 unique genes: 79 protein-coding genes, 4 ribosomal RNA genes and 29 tRNA genes. By aligning the cp?genome sequence with oil palm cDNA sequences, we observed 18 non-silent and 10 silent RNA editing events among 19 cp protein-coding genes. Creation of an initiation codon by RNA editing in rpl2 has been reported in several monocots and was also found in the oil palm cp genome. Fifty common chloroplast protein-coding genes from 33 plant taxa were used to construct ML and MP?phylogenetic trees. Their topologies are similar and strongly support for the position of E. guineensis as the sister of closely related species Phoenix dactylifera in Arecaceae (palm families) of monocot subtrees.     

Effects of pelvic floor muscle contraction on anal canal pressure

The role of pelvic floor muscle contraction in the genesis of anal canal pressure is not clear. Recent studies have suggested that vaginal distension increases pelvic floor muscle contraction. We studied the effects of vaginal distension on anal canal pressure in 15 nullipara asymptomatic women. Anal pressure, rest, and squeeze were measured using station pull-through manometry techniques with no vaginal probe, a 10-mm vaginal probe, and a 25-mm vaginal probe in place. Rest and squeeze vaginal pressures were significantly higher when measured with the 25-mm probe compared with the 10-mm probe, suggesting that vaginal distension enhances pelvic floor contraction. In the presence of the 25-mm vaginal probe, rest and squeeze anal pressures in the proximal part of the anal canal were significantly higher compared with no vaginal probe or the 10-mm vaginal probe. On the other hand, distal anal pressures were not affected by any of the vaginal probes. Ultrasound imaging of the pelvic floor revealed that vaginal distension increased the anterior-posterior length of the puborectalis muscle. Atropine at 15 micro g/kg had no influence on the rest and squeeze anal pressures with or without vaginal distension. Our data suggest that pelvic floor contractions increase pressures in the proximal part of the anal canal, which is anatomically surrounded by the puborectalis muscle. We propose that pelvic floor contraction plays an important role in the fecal continence mechanism by increasing anal canal pressure.