MiR-21-5p in macrophage-derived extracellular vesicles affects podocyte pyroptosis in diabetic nephropathy by regulating A20.

OBJECTIVES: Podocyte pyroptosis, characterized by inflammasome activation, plays an important role in inflammation-mediated diabetic nephropathy (DN). Our study aimed to investigate whether miR-21-5p in macrophage-derived extracellular vesicles (EVs) could affect podocyte injury in DN. METHODS: EVs were extracted after the treatment of RAW 264.7 (mouse macrophage line) with high glucose (HG). The podocyte pyroptosis was determined using the flow cytometry and the western blot. After the knockdown of miR-21-5p in HG-induced RAW264.7 cells, we injected the extracted EVs into DN model mice. RESULTS: The level of miR-21-5p was higher in HG-stimulated macrophage-derived EVs than in normal glucose-cultured macrophage-derived EVs. The co-culture of EVs and podocytes promoted reactive oxygen species (ROS) production and activation of inflammatory in MPC5 cells (mouse podocyte line). However, restraint of miR-21-5p in EVs reduced ROS production and inhibit inflammasome activation in MPC5 cells, thereby reducing podocytes injury. Meanwhile, we found that miR-21-5p inhibited the A20 expression through binding with its 3'-untranslated regions in MPC5 cells. Further studies showed that A20 was also involved in the regulation of miR-21-5p of RAW 264.7-derived EVs on MPC5 injury. At the same time, it was also proved in the DN model mice that miR-21-5p in macrophage-derived EVs could regulate podocyte injury. CONCLUSION: MiR-21-5p in macrophage-derived EVs can regulate pyroptosis-mediated podocyte injury by A20 in DN.

Reduced TRPC6 mRNA levels in the blood cells of patients with Alzheimer's disease and mild cognitive impairment.

Transient receptor potential canonical 6 (TRPC6) inhibits ß-amyloid (Aß) production. Hyperforin, the TRPC6 agonist, reduces Aß levels and improves cognitive performance in Alzheimer's disease (AD) models. However, it's unknown whether TRPC6 expression is changed in AD patients. In this case-control study, we measured TRPC6 expression levels in the peripheral blood cells of four independent AD sets from five hospitals and one mild cognitive impairment (MCI) set from a local community (229 AD, 70 MCI, 40 Parkinson disease and 359 controls from China, total n=698) using quantitative real-time PCR assay. We found a specific reduction of TRPC6 mRNA levels in four AD sets and one MCI set. The median TRPC6 mRNA levels were lower in the following: (1) combined AD patients than in age-matched controls (0.78 vs 1.73, P<0.001); (2) mild-to-moderate AD patients than in age-matched controls (0.81 vs 1.73, P<0.001); and (3) MCI patients than in age-matched controls (0.76 vs 1.72, P<0.001). In the receiver-operating characteristic curve analysis, the area under curve was 0.85 for combined AD, 0.84 for mild-to-moderate AD and 0.79 for MCI. In a subgroup of AD patients with brain Aß examination, TRPC6 was associated with standardized uptake value ratio of Pittsburgh Compound B (Spearman's r=-0.49, P=0.04) and cerebrospinal fluid Aß42 (Spearman's r=0.43, P=0.04). The TRPC6 reduction in AD patients was further confirmed in blood RNA samples from The Australian Imaging, Biomarkers and Lifestyle Flagship Study of Aging, in post-mortem brain tissues from The Netherlands Brain Bank and in induced pluripotent stem cells-derived neurons from Chinese donors. We conclude that TRPC6 mRNA levels in the blood cells are specifically reduced in AD and MCI patients, and TRPC6 might be a biomarker for the early diagnosis of AD.

Thermal stability control system of photo-elastic interferometer in the PEM-FTs.

A drifting model for the resonant frequency and retardation amplitude of a photo-elastic modulator (PEM) in the photo-elastic modulated Fourier transform spectrometer (PEM-FTs) is presented. A multi-parameter broadband-matching driving control method is proposed to improve the thermal stability of the PEM interferometer. The automatically frequency-modulated technology of the driving signal based on digital phase-locked technology is used to track the PEM's changing resonant frequency. Simultaneously the maximum optical-path-difference of a laser's interferogram is measured to adjust the amplitude of the PEM's driving signal so that the spectral resolution is stable. In the experiment, the multi-parameter broadband-matching control method is applied to the driving control system of the PEM-FTs. Control of resonant frequency and retardation amplitude stabilizes the maximum optical-path-difference to approximately 236 µm and results in a spectral resolution of 42 cm-1. This corresponds to a relative error smaller than 2.16% (4.28 standard deviation). The experiment shows that the method can effectively stabilize the spectral resolution of the PEM-FTs.

Relationship between the gene polymorphism of osteoprotegerin and bone mineral density in hemodialysis patients.

OBJECTIVE: To investigate the relationship between the gene polymorphism of osteoprotegerin (OPG) and bone mineral density (BMD) in hemodialysis patients. PATIENTS AND METHODS: A total of 147 patients with end-stage renal disease (ESRD) who were admitted to the Weifang People's Hospital for maintenance hemodialysis between January 2014 and December 2015 were enrolled. Peripheral blood was collected from the subjects for assay of the polymorphism of A163G and G1181C loci of OPG. The measurements of the levels of RANK, RANKL, TNF-α, IL-6, PINP, CTX-I, CTX-II and TRACP5 in the isolated serum were taken. RESULTS: For the polymorphism of A163G locus on the OPG gene, the BMDs of left femoral neck and lumbar poster anterior L1-L4 of the AA genotype were significantly higher than those of the AG and GG genotypes. There was no significant difference in comparison of BMDs at the forearm (distal 1/3) between the AA genotype and AG and GG genotypes. No significant differences were found in the comparison of BMDs at all sites between AG and GG genotypes. The serum level of RANKL of the AA genotype was significantly higher than levels of AG and GG genotypes, but the levels of RANK, TNF-α, IL-6, PINP, CTX-I, CTX-II and TRACP5 were prominently lower than those levels of AG and GG genotypes. For the polymorphism of G1181C locus on the OPG gene, the BMDs of left femoral neck and lumbar poster anterior L1-L4 of the CC genotype were significantly higher than the BMDs of GG and GC genotypes, There was no significant difference in the comparison of BMDs at the forearm (distal 1/3) between the CC genotype and GG and GC genotypes. No significant differences were found in the comparison of BMDs at all sites between GG and GC genotypes. The serum level of RANKL of the CC genotype was significantly higher than the level of GG and GC genotypes. However, the levels of RANK, TNF-α, IL-6, PINP, CTX-I, CTX-II and TRACP5 were prominently lower than those levels of GG and GC genotypes. CONCLUSIONS: The polymorphisms of A163G and G1181C loci on the OPG gene were correlated with the BMD of hemodialysis patients. The genotype AA of A163G and genotype CC of G1181C were identified as the protective factors for BMD.

Ion pair binding of Ca2+ and Cl- ions in micellar-packaged gramicidin A.

The two independent NMR experiments were performed to investigate the interaction between CaCl2 and the gramicidin A (GA) ion transport channel, using 13C-enriched GA and GA molecules incorporated into dodecylphosphocholine (DPC) micelles. The chemical shifts of C-13 labeled carbonyl carbons vs. CaCl2 concentration demonstrate that Ca2+ and Cl- ions interact as an ion pair within the GA structure with the Cl- ion located near the position of the carbonyl group of the Trp11 residue some 5.5 A from the mouth of the GA helix, and the Ca2+ ion bound at the position of the carbonyl group of the Trp15 residue some 2.5 A from the entrance to the helical pore. The measurements of the 35Cl line-widths and transverse relaxation times illustrate that the interaction occurs between Cl- ions and GA in DPC when in CaCl2 solution, that no interaction is detected between Cl- ions and GA in DPC when in NaCl solution, and that the interaction between Cl- ions and GA in DPC when in MgCl2 solution is much weaker than in CaCl2 solution. In short, a Cl- ion can enter the GA when it is paired with a divalent Ca2+ ion; and Ca2+ and Cl- ions as a pair exchange rapidly with sites of the GA dimer.

On the mechanism of channel-length dependence of gramicidin single-channel conductance.

Single-channel conductance data on four different gramicidin channel lengths demonstrate that conductance magnitude is neither inversely dependent on the square of the channel length nor on the image force arising from differences in the extent of lipid dimpling (Jordan and Vayl (1985) Biochim. Biophys. Acta 818, 416-420). Rather the conductance differences are consistent with the decreased off-rate constant for the singly occupied state as the ionic radius decreases from that of cesium ion to sodium ion coupled with the decreased probability of the doubly occupied channel due to increased ion-ion repulsion as the channel is shortened (Urry et al. (1984) Biochim. Biophys. Acta 774, 115-119).

The determination of binding constants of micellar-packaged gramicidin A by 13C-and 23Na-NMR.

Based on the malonyl gramicidin A structure of a single-stranded head-to-head hydrogen bonded right-handed, beta 6.3-helix in dodecyl phosphocholine (DPC) lipid micelles (Jing et al. (1994) Biophys. J. 66, A353), the determination of cation binding sites for gramicidin A (GA) in DPC micelles becomes a significant step in the study of ion transport through the model channel. First, the investigation of cation binding sites in DPC micellar packaged gramicidin A was achieved by 13C-NMR experiments at 30 degrees C using four C-13 labeled GA samples. Then, the analyses based on two different equations, one for single and one for double occupancy, were employed to evaluate the correct occupancy model for GA in DPC micelles. The results clearly indicate double occupancy to be correct for Na+ ion as well as for K+, Rb+, Cs+, and Tl+ ions. Finally, the binding constants for Na+ ion were also estimated by the measurement of the longitudinal relaxation time (T1) using 23Na-NMR of the same sample at the same ffmperature as used for the 13C-NMR study. The binding constants obtained from 23Na-NMR are essentially equivalent to those determined from the 13C-chemical shifts.

Mouse nestin cDNA cloning and protein expression in the cytoskeleton of transfected cells.

Complementary DNA (cDNA) corresponding to mouse nestin intermediate filament protein, a specific marker for neural stem cells, was isolated and characterized. The complete sequence comprised 5983 base pairs encoding 1821 amino acids, and the deduced polypeptide was similar to rat (84%), hamster (73%), and human (62%) nestin. Southern blots showed that mouse nestin was a single-copy gene, and Northern blots detected a 6.0 kilobase mRNA transcript. When the cDNA was overexpressed as an enhanced green fluorescent fusion protein in COS7 cells, nestin immunoreactivity appeared in the filamentous cytoskeletal network. Accordingly, biologically active mouse nestin cDNA may offer an important new tool for stem cell research.

The negative cell cycle regulator, Tob (transducer of ErbB-2), is a multifunctional protein involved in hippocampus-dependent learning and memory.

Tob (transducer of ErbB2) is a negative cell cycle regulator with anti-proliferative activity in the periphery. Using a behavioral screening paradigm to look for novel gene functions in the brain, we identified Tob as a brain-expressed protein involved in learning and memory. Behavioral training of fear-conditioning triggered a transient elevation of Tob protein, which preceded the formation of long-term memory. Functional perturbation of Tob by intra-CA1 infusion of antisense oligonucleotides in rats impaired spatial learning and memory in the Morris water maze and long-term memory for contextual fear conditioning, two behavioral paradigms that require the hippocampus. Furthermore, long-term potentiation was suppressed by Tob antisense infusion into the CA1 region. Together, these results indicate that the negative cell cycle regulator Tob is a multifunctional protein involved in hippocampus-dependent learning and memory.

Nestin expression during mouse eye and lens development.

Nestin is an intermediate filament protein, which is expressed predominantly in the developing central nervous system and skeletal muscles. In situ hybridization revealed that mouse nestin mRNA is in the optic stalk at 9.0 days post coitus (dpc) and in the lens vesicle at 10.0 dpc. From 14.5 dpc onward, nestin transcripts appear in lens fibers and neuroretina. Immunohistochemistry showed that nestin protein appears in the optic stalk at 9.5 dpc and in the posterior lens epithelium at 10.5 dpc. By 12.5 dpc, it is found in the lens, neuroretina, and optic stalk as well as in developing extrinsic ocular muscle, and it localizes in lens epithelium, optic disc, and optic nerve from 14.5 dpc to postnatal day 1. In adult eye, nestin protein appears in the optic nerve.

Murine tenascin: cDNA cloning, structure and temporal expression of isoforms.

Mouse tenascin (TN)-encoding cDNA clones were isolated from a cDNA library of the 2H6GR mammary tumor cell line. Nucleotide (nt) and deduced amino acid (aa) sequences revealed the characteristic primary structure, which begins with a signal peptide and TN unique sequences, follows with 14 1/2 epidermal growth factor (EGF)-like repeats and 13 fibronectin type-III repeats (FN repeat), and concludes with fibrinogen-homologous sequences. Similar to chicken and human TN, the mouse TN cDNA contains five consecutive insertional FN repeats, as well as eight constitutive FN repeats. Three different cDNA clones that may have been generated by alternative splicing of these insertional FN repeats were identified and characterized. Based upon the deduced as sequence, a polyclonal antibody was produced against a synthetic TN peptide. It specifically recognized two TN isoforms of 230 kDNA and 190 kDa in protein extracts of mouse tissues. The tissue distributions of mouse TN mRNAs, revealed by Northern blot analysis, suggest that there is tissue-specific expression of TN isoforms. Two distinct mRNA transcripts (7 kb and 5.5 kg) were detected in brain, skeletal muscle, digestive tract and bladder, but only one was observed in lung, kidney (7 kg) and thymus (5.5 kg). TN mRNA expression was down-regulated 1 month after birth in most tissues. However, the 5.5-kb transcript persisted in cerebellum, thymus, and colon. The spatial and temporal patterns of TN expression seem to be controlled at the level of transcription, because analysis of various tissues by Western blots showed the same pattern as that seen in Northern blots.

Evidence for a mouse brain-specific variant of alpha-tubulin.

While studying the neural precursor cell intermediate filament protein known as nestin in the developing mouse brain, we observed a strong cross-reaction of our nestin antibody with a 50 kDa protein that appeared on embryonic day 10 and continued to accumulate until postnatal day 1. Here we report evidence that this protein is a brain-specific variant form of alpha-tubulin and discuss its implications.

Maternal zinc deficiency impairs brain nestin expression in prenatal and postnatal mice.

Effects of maternal dietary zinc deficiency on prenatal and postnatal brain development were investigated in ICR strain mice. From d 1 of pregnancy (E0) until postnatal d 20 (P20), maternal mice were fed experimental diets that contained 1 mg Zn/kg/day (severe zinc deficient, SZD), 5 mg Zn/kg/day (marginal zinc deficient, MZD), 30 mg Zn/kg/day (zinc adequately supplied, ZA) or 100 mg Zn/kg/day (zinc supplemented, ZS and pair-fed, PF). Brains of offspring from these dietary groups were examined at various developmental stages for expression of nestin, an intermediate filament protein found in neural stem cells and young neurons. Immunocytochemistry showed nestin expression in neural tube 10.5 d post citrus (dpc) as well as in the cerebral cortex and neural tube from 10.5 dpc to postnatal d 10 (P10). Nestin immunoreactivities in both brain and neural tube of those zinc-supplemented control groups (ZA, ZS, PF) were stronger than those in zinc-deficient groups (SZD and MZD). Western blot analysis confirmed that nestin levels in pooled brain extracts from each of the zinc-supplemented groups (ZA, ZS, PF) were much higher than those from the zinc-deficient groups (SZD and MZD) from 10.5 dpc to P10. Immunostaining and Western blots showed no detectable nestin in any of the experimental and control group brains after P20. These observations of an association between maternal zinc deficiency and decreased nestin protein levels in brains of offspring suggest that zinc deficiency suppresses development of neural stem cells, an effect which may lead to neuroanatomical and behavioral abnormalities in adults.

Up-regulation of cystatin C expression in the murine hippocampus following perforant path transections.

Cystatins are endogenous cysteine protease inhibitors that modulate the turnover of intracellular and extracellular proteins. These inhibitors are strongly implicated in a variety of pathological processes such as tumor metastasis and many degenerating CNS disorders. Here we report the expression of cystatin C, a major cysteine protease inhibitor of mammalian animals, in the murine hippocampus at 3, 7, 15 and 30 days following perforant path transections. Northern blot analysis showed that cystatin C transcripts were up-regulated in a transient manner with a significant increase at 7 and 15 days post-lesion (219% and 185% of control, respectively) in the rat hippocampus after entorhinal deafferentation. In situ hybridization and immunohistochemistry confirmed the time-dependent up-regulation of both cystatin C mRNA and protein expressions in a mouse model which initiated at 3 days post-lesion, reached maximal levels 7-15 days post-lesion, and remained slightly elevated by day 30 post-lesion. The modulation of cystatin C expression was observed to occur specifically in the entorhinally denervated zones: the stratum lacunosum-moleculare of the hippocampus and the outer molecular layer of the dentate gyrus. Double labeling by either a combination of in situ hybridization for cystatin C with immunohistochemistry for glial fibrillary acidic protein or double immunofluorescence staining for both proteins in mouse hippocampus at 7 and 15 days post-lesion revealed that most cystatin C-expressing cells are astrocytes. From these results we suggest that the spatiotemporal up-regulation of cystatin C in the hippocampus is induced by entorhinal deafferentation and that cystatin C may be involved in the astroglia-mediated neural plasticity events in the hippocampus following perforant path transections.

Rational drug design of DNA oligonucleotides as HIV inhibitors.

DNA oligonucleotides as anti-HIV therapeutic agents have been developed for more than a decade. Numbers of oligonucleotides have been designed as potential anti-HIV inhibitors. Here we summarized the designed anti-viral oligonucleotides in last decade and divided the designed DNA HIV inhibitors into three categories: (i) antisense inhibitors, (ii) triplex inhibitors and (iii) G-quartet inhibitors, based upon their inhibitory mechanism and structures. Also we proposed a strategy of rational drug design of anti-HIV oligonucleotides, which includes several critical steps, such as (1) structure-based rational drug design, (2) chemical synthesis/combinational chemistry, (3) the determination of structural properties, (4) assays of the inhibition of HIV-1 IN and virus replication, and (5) 3D QSAR operation. This methodology has been used by the design of G-quartet inhibitors.

Developing G-quartet oligonucleotides as novel anti-HIV agents: focus on anti-HIV drug design.

Recently, a new class of oligonucleotides, forming G-quartet structures, has been developed as novel anti-HIV agents. Several critical structure-activity relationships between HIV-1 integrase and G-quartet oligonucleotides have been demonstrated. In addition the mechanism of the inhibition of HIV-1 integrase by G-quartet oligonucleotides, such as T30695 and its derivatives, has been explored. This review summarises the preliminary studies of developing G-quartet oligonucleotides as novel anti-HIV agents in several aspects including structure-activity relationship, stability-activity correlation, mechanism of HIV-1 integrase inhibition, substitution of phosphorothioates and targeting HIV-1 integrase in infected cells, which, hopefully, could help for developing a novel, efficient anti-HIV agent.

Structure-activity of inhibition of HIV-1 integrase and virus replication by G-quartet oligonucleotides.

As novel anti-HIV agents, the G-tetrad-forming oligonucleotides have been explored for their structure-activity relations with regard to inhibition of integrase (IN) (N. Jing, Expert Opin. Investig. Drugs (2000) 9, 1777-1785). We have now developed two families of G-quartet oligonucleotides: T40217-T40222, with potential formation of a tail-to-tail G-quartet dimer, and T40224-T40227, with phosphorothioate (PT) linkages in the guanine loops. The results obtained from biophysical measurements and the assays of the inhibition of HIV-1 IN and virus replication demonstrated that an increase in the length of the G-quartet structure from a monomer (15A) to a tail-to-tail dimer (47A) does not distinctly disrupt the inhibition of HIV-1 IN activity or the inhibition of HIV-1 replication in cell cultures. G-quartet oligonucleotides were observed to induce molecular aggregation of HIV-1 IN and interrupt the binding of viral DNA to HIV-1 IN. Also, PT substitutions did not confer any advantages compared with the regular phosphodiesters for the inhibition of HIV-1 replication by intramolecular G-quartets. The G-quartet motif is the primary requirement for the remarkable nuclease resistance and pronounced biological efficacy of these oligonucleotides.

Identification of a serine protease with nerve growth promoting activity from snake venom.

A new protein with nerve growth promoting activity was purified from the crude venom of the Agkistrodon halys Pallas, a Chinese snake. Its amino-terminal sequence unexpectedly showed high homology with serine proteases, suggesting that it is a new member of the serine protease family. It also cross-reacted with antibodies against thrombin-like enzyme and possessed weak arginine esterase activity, amounting to about 3% of the activity of trypsin. However, its nerve growth promoting activity was comparable to that of nerve growth factor (NGF). It was named NGF-like protease (NLP). Northern blot analysis further demonstrated different patterns of induction of c-myc, vgf and trkA mRNA transcription in PC12 pheochromocytoma cells treated with NGF and NLP, respectively. These data suggested that NLP represents a novel potent neurotrophic factor.

Mouse nestin protein localizes in growth cones of P19 neurons and cerebellar granule cells.

The neuronal growth cone, a highly motile structure at the distal tip of growing axons, contains filamentous actin and microtubules as its main cytoskeletal components. Using immunocytochemistry, we observed that nestin, which is the predominant intermediate filament protein in neuroepithelial cells and young neurons of the developing brain, appears to be strongly expressed in neurites and growth cones of neurons differentiating from P19 embryonic carcinoma cells in vitro. Double-staining of nestin and microtubule-associated protein-2 as well as nestin and growth-associated protein-43 revealed that nestin protein localizes in neurites and the central regions of growth cones of primary cultures of cerebellar granule cells from postnatal day 6 mice. These results suggest a role for nestin in growth cone guidance during axon elongation.

Identification of differentially expressed genes in the denervated rat hippocampus by cDNA arrays.

To elucidate the molecular mechanism underlying the physiological responses to injury in the central nervous system, gene expression profiles in rodent hippocampus following perforant path transection were investigated using cDNA array hybridization. Of the 8000 arrayed clones, 47 exhibited differential expression by >3-fold difference in the denervated hippocampus from control, with 15 up-regulated and 22 down-regulated. They can be functionally assigned into several classes, among which the most prominent are those coding proteins involved in macromolecules synthesis and processing. Northern blot analysis verified the validation of the aforementioned array data. These results throw some new light on the physiological responses of the hippocampus to entorhinal deafferentation at molecular level.