Pilot validation of on-field STR typing and human identity testing by MinION nanopore sequencing.

Nanopore sequencing technology has broad application prospects in forensic medicine due to its small size, portability, fast speed, real-time result analysis capabilities, single-molecule sequencing abilities, and simple operation. Here, we demonstrate for the first time that nanopore sequencing platforms can be used to identify individuals in the field. Through scientific and reasonable design, a nanopore MinION MK1B device and other auxiliary devices are integrated into a portable detection box conducive to individual identification at the accident site. Individual identification of 12 samples could be completed within approximately 24 h by jointly detecting 23 short tandem repeat (STR) loci. Through double-blinded experiments, the genotypes of 49 samples were successfully determined, and the accuracy of the STR genotyping was verified by the gold standard. Specifically, the typing success rate for 1150 genotypes was 95.3%, and the accuracy rate was 86.87%. Although this study focused primarily on demonstrating the feasibility of full-process testing, it can be optimistically predicted that further improvements in bioinformatics workflows and nanopore sequencing technology will help enhance the feasibility of Oxford Nanopore Technologies equipment for real-time individual identification at accident sites.

Ginsenoside Rd Protects SH-SY5Y Cells against 1-Methyl-4-phenylpyridinium Induced Injury.

Ginsenoside Rd (GSRd), one of the main active monomer compounds from the medical plant Panax ginseng, has been shown to promote neuronal survival in models of ischemic cerebral damage. As an extending study, here we examined whether GSRd could exert a beneficial effect in an experimental Parkinson disease (PD) model in vitro, in which SH-SY5Y cells were injured by 1-methyl-4-phenylpyridinium (MPP+), an active metabolic product of the classical Parkinsonian toxin1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Our results, from the addition of different concentrations of GSRd (1, 10 and 50 µM), showed that GSRd at 1 and 10 µM could significantly attenuate MPP+-induced cell death. This protective effect may be ascribed to its ability to reduce intracellular reactive oxygen species levels, enhance antioxidant enzymatic activities, preserve the activity of respiratory complex I, stabilize the mitochondrial membrane potential and increase intracellular ATP levels. Additionally, the PI3K/Akt survival-signaling pathway was also involved in the protective effect of GSRd. Finally, using a mouse PD model in vivo, we also found that GSRd obviously reversed the loss of tyrosine hydroxylase-positive cells in substanitia nigra induced by MPTP. Thus, our findings demonstrated that GSRd showed a significant neuro-protective effect against experimental PD models, which may involve its antioxidant effects and mitochondrial function preservation.

Evaluation of chromID strepto B as a screening media for Streptococcus agalactiae.

BACKGROUND: Streptococcus agalactiae (Group B Streptococcus, GBS), a leading cause of sepsis and meningitis in infants, can be transmitted vertically from mother to infant during passage through the birth canal. Detection of GBS colonization in perinatal women is a major strategy for the prevention of postpartum neonatal disease. The U.S. Centers for Disease Control and Prevention recommends that all women undergo vaginal-rectal screening for GBS colonization at 35-37 weeks of gestation. ChromID Strepto B (STRB) is a chromogenic GBS screening media on which GBS colonies appear pink or red, while other bacteria are either inhibited or form colonies in other colors. In this study, we compared STRB with a conventional GBS detection method using 5% sheep blood agar (BA) followed by a selective enrichment broth. METHODS: Anovaginal swabs were collected from 1425 women during weeks 35 to 37 of their pregnancies. The swabs were used to inoculate both STRB and BA plates after enrichment with selective Todd Hewitt Broth (THB). A GBS latex agglutination test was used to confirm the identity of isolates from each plate. RESULTS: GBS was recovered from 319 (22.4%) samples with one or both media: 318 on STRB compared to 299 using BA. One false negative was observed on STRB, and 20 false negatives were observed on BA. In addition, non-hemolytic GBS was recovered from 19 (6.0%) samples using STRB. CONCLUSIONS: STRB offers effectiveness and convenience over BA for GBS screening in clinical laboratories. STRB produces fewer false negatives, has a higher detection rate and uses a simple color screen that is ideal for technician-level applications. We recommend STRB as the media of choice for GBS screening.

Conditioned medium of Wnt/ß-catenin signaling-activated olfactory ensheathing cells promotes synaptogenesis and neurite growth in vitro.

Olfactory ensheathing cells (OECs), the major glia cells in the olfactory system, have been extensively studied because of their ability to promote axonal growth and regeneration. Whether it could facilitate synaptogenesis is an important, but remains as yet an unanswered question. We have identified a subgroup of Wnt signaling-activated OECs, spatiotemporal distribution of which in the olfactory bulb suggests a role for these cells in both axonal growth and synaptogenesis. In the present study, we explored this possibility in vitro. OECs were primarily cultured, in which Wnt signaling was activated by overexpressing ß-catenin, and inhibited by dominant negative TCF4. Neurite growth and synaptogenesis were assessed by co-culturing neurons with conditioned medium from control OECs (cOECs CM), Wnt/ß-catenin signaling-activated OECs (wOECs CM), or Wnt signaling-inhibited OECs (wiOECs). The results showed that although cOECs CM enhances axonal growth, wOECs CM exhibited a stronger axonal growth-promoting effect, than cOECs CM. More importantly, wOECs CM stimulates synatpogenesis, demonstrated by the expression of Synaptophysin and whole-cell patch clamp recording. In contrast, both cOECs CM and wiOECs CM do not affect synaptogenesis. Our data, for the first time, demonstrated that, in comparison with regularly cultured OECs, wOECs CM are more effective in enhancing axonal growth, and can promote synaptogenesis, probably by secreting factors. These results suggest a potential application of wOECs for treating spinal cord injury.

NDRG2 regulates the formation of reactive astrocyte-derived progenitor cells via Notch signaling pathway after brain traumatic injury in rats.

In response to traumatic brain injury, a subpopulation of cortical astrocytes is activated, resulting in acquisition of stem cell properties, known as reactive astrocytes-derived progenitor cells (Rad-PCs). However, the underlying mechanisms remain largely unknown during this process. In this study, we examined the role of N-myc downstream-regulated gene 2 (NDRG2), a differentiation- and stress-associated molecule, in Rad-PCs after cortical stab injury in adult rats. Immunohistochemical analysis showed that in the cerebral cortex of normal adult rats, NDRG2 was exclusively expressed in astrocytes. After liu cortical injury, the expression of NDRG2 was significantly elevated around the wound and most cells expressing NDRG2 also expressed GFAP, a reactive astrocyte marker. Importantly, NDRG2-expressing cells were co-labeled with Nestin, a marker for neural stem cells, some of which also expressed cell proliferation marker Ki67. Overexpression of NDRG2 further increased the number of NDRG2/Nestin double-labeling cells around the lesion. In contrast, shRNA knockdown of NDRG2 decreased the number of NDRG2+/Nestin+ cells. Intracerebroventricular administration of stab-injured rats with a Notch antagonist, DAPT, led to a significant decrease in Nestin+/NDRG2+ cells around the injured boundary, but did not affect NDRG2+ cells. Moreover, overexpression or knockdown of NDRG2 led to up- and down-regulation of the expression of Notch intracellular domain NICD and Notch target gene Hes1, respectively. Taken together, these results suggest that NDRG2 may play a role in controlling the formation of Rad-PCs in the cerebral cortex of adult rats following traumatic injury, and that Notch signaling pathway plays a key role in this process.

Cerebrospinal Fluid from Healthy Pregnant Women Does Not Harbor a Detectable Microbial Community.

Cerebrospinal fluid (CSF) circulating in the human central nervous system has long been considered aseptic in healthy individuals, because normally, the blood-brain barrier can protect against microbial invasions. However, this dogma has been called into question by several reports that microbes were identified in human brains, raising the question of whether there is a microbial community in the CSF of healthy individuals without neurological diseases. Here, we collected CSF samples and other samples, including one-to-one matched oral and skin swab samples (positive controls), from 23 pregnant women aged between 23 and 40 years. Normal saline samples (negative controls), sterile swabs, and extraction buffer samples (contamination controls) were also collected. Twelve of the CSF specimens were also used to evaluate the physiological activities of detected microbes. Metagenomic and metatranscriptomic sequencing was performed in these 116 specimens. A total of 620 nonredundant microbes were detected, which were dominated by bacteria (74.6%) and viruses (24.2%), while in CSF samples, metagenomic sequencing found only 26 nonredundant microbes, including one eukaryote, four bacteria, and 21 viruses (mostly bacteriophages). The beta diversity of microbes compared between CSF metagenomic samples and other types of samples (except negative controls) was significantly different from that of the CSF self-comparison. In addition, there was no active or viable microbe in the matched metagenomic and metatranscriptomic sequencing of CSF specimens after subtracting those also found in normal saline, DNA extraction buffer, and skin swab specimens. In conclusion, our results showed no strong evidence of a colonized microbial community present in the CSF of healthy individuals. IMPORTANCE The microbiome is prevalent throughout human bodies, with profound health implications. However, it remains unclear whether it is present and active in human CSF, which has been long considered aseptic due to the blood-brain barrier. Here, we applied unbiased metagenomic and metatranscriptomic sequencing to detect the presence of a microbiome in CSF collected from 23 pregnant women with matched controls. Analysis of 116 specimens found no strong evidence to support the presence of a colonized microbiome in CSF. Our findings will strengthen our understanding of the internal environment of the CSF in healthy people, which has strong implications for human health, especially for neurological infections and disorders, and will help further disease diagnostics, prevention, and therapeutics in clinical settings.

Granulocyte colony-stimulating factor treatment prevents cognitive impairment following status epilepticus in rats.

Status epilepticus (SE) rendered selective neuronal loss and cognitive impairments. Previous studies proved that granulocyte colony-stimulating factor (G-CSF) acted as a neuroprotectant in some nervous diseases. However, no investigations were focused on whether G-CSF could protect the hippocampus from SE. In this study, we administered recombinant human G-CSF into Sprague-Dawley rats with lithium-pilocarpine-induced SE subcutaneously for three times. The Morris water maze was employed to determine spatial learning ability from the 15th to 20th days after the treatment. The quantitative terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) staining and levels of apoptosis-related molecules including cleaved caspase-3, Bcl-xL and Bax on hippocampal CA1 region were examined by immunohistochemical staining at the 3rd and the 5th day after the treatment. Moreover, the phosphorylation of AKT was evaluated with Western blot at the 6th, 24th and 48th hours after the treatment to explore apoptosis and detect the protective effects of G-CSF. We found G-CSF treatment prevented SE-induced cognitive impairments with the decreased escape latency time on the 17th (29.86+/-9.09 vs. 38.33+/-6.94, p<0.05) and 18th days (23.83+/-6.17 vs. 33.52+/-8.48, p<0.05). The reduced TUNEL staining demonstrated reduced neuronal apoptosis occurrences. The anti-apoptotic effects were associated with decreased cleaved caspase-3 and Bax expression and increased phosphorylation of AKT and Bcl-xL expression. Taken together, our results suggested that systemic G-CSF treatment conducted neuroprotective function following SE through an anti-apoptotic pathway and prevented cognitive impairments, which may provide novel insights into pathogenesis and treatment following SE injury.

Comparison of rapamycin and methylprednisolone for treating inflammatory muscle disease in a murine model of experimental autoimmune myositis.

Idiopathic inflammatory myopathies (IIMs) are a group of autoimmune inflammatory muscle diseases. Rapamycin has been shown to ameliorate inflammation and improve muscle function in a mouse model of experimental autoimmune myositis (EAM). In the present study, the therapeutic effect of rapamycin was compared with methylprednisolone (MP) on EAM. Mice were injected with myosin for 10 days to induce EAM and were subsequently treated with rapamycin (1.5 mg/kg), MP (40 mg/kg) or placebo (DMSO) for 14 days. The rapamycin-treated group exhibited significantly decreased severe inflammation and improved muscle strength compared with the MP-treated group. The plasma transforming growth factor-ß (TGF-ß) concentration in the rapamycin-treated group was significantly higher compared with the placebo group. However, both treatment groups exhibited significantly lower plasma interleukin-10 levels compared with the placebo group. Moreover, splenic regulatory T cell frequency in both the rapamycin- and MP-treated animals was significantly lower than that in the animals of the placebo group. Rapamycin showed better immune suppressive effects than MP in this model of EAM, and these effects were likely to be mediated by the TGF-ß signaling pathway.

Protopanaxadiol ginsenoside Rd protects against NMDA receptor-mediated excitotoxicity by attenuating calcineurin-regulated DAPK1 activity.

Neuroprotective strategies in the treatment of stroke have been attracting a great deal of attentions. Our previous clinical and basic studies have demonstrated that protopanaxadiol ginsenoside-Rd (Rd), a monomer compound extracted from Panax ginseng or Panax notoginseng, has neuroprotective effects against ischemic stroke, probably due to its ability to block Ca2+ overload, an usual consequence of the overactivation of NMDA receptor (NMDAR). As an extending study, we explored here whether Rd exerted its neuroprotection as a novel NMDAR blocker. Our whole-cell patch-clamp results showed that Rd reduced NMDAR currents of cultured rat cortical neurons (EC50 = 7.7 µM) dose-dependently by acting on extrasynaptic NMDAR NR2b subunit. However, unexpectedly, cell transfection and radioligand binding assays revealed that Rd did not bind to the NMDAR channel directly. Alternatively, it inhibited the phosphorylation of NR2b at Ser-1303, a target of death associated protein kinase 1 (DAPK1). Moreover, cell-based and cell-free enzymatic assays showed that Rd did not inhibit the activity of DAPK1 directly, but blocked the activity of calcineurin, a key phosphatase for activating DAPK1. Importantly, other protopanaxadiol ginsenosides were also found to have potential inhibitory effects on calcineurin activity. Furthermore, as expected, calcineurin inhibition by cyclosporin A could mimic Rd's effects and protect against NMDA-, oxygen glucose deprivation- or transient ischemic stroke-induced neuronal injury. Therefore, our present study provided the first evidence that Rd could exert an inhibitive effect on NMDAR-triggered currents and sequential excitotoxicity through mitigation of DAPK1-mediated NR2b phosphorylation by attenuating calcineurin activity.

Identification of candidate genes for human pituitary development by EST analysis.

BACKGROUND: The pituitary is a critical neuroendocrine gland that is comprised of five hormone-secreting cell types, which develops in tandem during the embryonic stage. Some essential genes have been identified in the early stage of adenohypophysial development, such as PITX1, FGF8, BMP4 and SF-1. However, it is likely that a large number of signaling molecules and transcription factors essential for determination and terminal differentiation of specific cell types remain unidentified. High-throughput methods such as microarray analysis may facilitate the measurement of gene transcriptional levels, while Expressed sequence tag (EST) sequencing, an efficient method for gene discovery and expression level analysis, may no-redundantly help to understand gene expression patterns during development. RESULTS: A total of 9,271 ESTs were generated from both fetal and adult pituitaries, and assigned into 961 gene/EST clusters in fetal and 2,747 in adult pituitary by homology analysis. The transcription maps derived from these data indicated that developmentally relevant genes, such as Sox4, ST13 and ZNF185, were dominant in the cDNA library of fetal pituitary, while hormones and hormone-associated genes, such as GH1, GH2, POMC, LHbeta, CHGA and CHGB, were dominant in adult pituitary. Furthermore, by using RT-PCR and in situ hybridization, Sox4 was found to be one of the main transcription factors expressed in fetal pituitary for the first time. It was expressed at least at E12.5, but decreased after E17.5. In addition, 40 novel ESTs were identified specifically in this tissue. CONCLUSION: The significant changes in gene expression in both tissues suggest a distinct and dynamic switch between embryonic and adult pituitaries. All these data along with Sox4 should be confirmed to further understand the community of multiple signaling pathways that act as a cooperative network that regulates maturation of the pituitary. It was also suggested that EST sequencing is an efficient means of gene discovery.

Simultaneous Detection of Five Pathogens from Cerebrospinal Fluid Specimens Using Luminex Technology.

Early diagnosis and treatment are crucial for the outcome of central nervous system (CNS) infections. In this study, we developed a multiplex PCR-Luminex assay for the simultaneous detection of five major pathogens, including Mycobacterium tuberculosis, Cryptococcus neoformans, Streptococcus pneumoniae, and herpes simplex virus types 1 and 2, which frequently cause CNS infections. Through the hybridization reaction between multiplex PCR-amplified targets and oligonucleotide "anti-TAG" sequences, we found that the PCR-Luminex assay could detect as low as 10¹-10² copies of synthetic pathogen DNAs. Furthermore, 163 cerebrospinal fluid (CSF) specimens from patients with suspected CNS infections were used to evaluate the efficiency of this multiplex PCR-Luminex method. Compared with Ziehl-Neelsen stain, this assay showed a high diagnostic accuracy for tuberculosis meningitis (sensitivity, 90.7% and specificity, 99.1%). For cryptococcal meningitis, the sensitivity and specificity were 92% and 97.1%, respectively, compared with the May Grunwald Giemsa (MGG) stain. For herpes simplex virus types 1 and 2 encephalitis, the sensitivities were 80.8% and 100%, and the specificities were 94.2% and 99%, respectively, compared with Enzyme Linked Immunosorbent Assay (ELISA) assays. Taken together, this multiplex PCR-Luminex assay showed potential efficiency for the simultaneous detection of five pathogens and may be a promising supplement to conventional methods for diagnosing CNS infections.

Development of an improved animal model of experimental autoimmune myositis.

Multiple animal models of experimental autoimmune myositis (EAM) have been developed. However, these models vary greatly in the severity of disease and reproducibility. The goal of this study was to test whether vaccination twice with increased dose of rat myosin and pertussis toxin (PT) could induce EAM with severer disease in mice. BALB/c mice were injected with 1 mg rat myosin in 50% complete Freund's adjuvant (CFA) weekly for four times and one time of PT (EAM) or twice with 1.5 mg myosin in CFA and PT (M-EAM). In comparison with that in the CFA and PT injected controls, vaccination with rat myosin and injection PT significantly reduced the muscle strength and EMG duration, elevated serum creatine kinase levels, promoted inflammatory infiltration in the muscle tissues, leading to pathological changes in the muscle tissues, demonstrating to induce EAM. Interestingly, we found that vaccination twice with the high dose of myosin and PT prevented EAM-related gain in body weights and caused significantly less muscle strength in mice. More importantly, all of the mice receiving high dose of myosin and PT survived while 3 out of 16 mice with four times of low dose of myosin died. Finally, vaccination with high dose of myosin promoted CD4(+) and CD8(+) T cell infiltration in the muscle tissues and up-regulated MHC-I expression in the muscle tissues of mice. Hence, the new model of EAM is a time-saving, efficient and easily replicable tool for studying autoimmune myositis.

Active phagocytosis of Mycobacterium tuberculosis (H37Ra) by T lymphocytes (Jurkat cells).

This study aimed to co-culture Jurkat T lymphocytes with inactivated Mycobacterium tuberculosis (Mtb H37Ra), explore whether T lymphocytes could phagocytose H37Ra cells, and determine the underlying mechanism. Jurkat T lymphocytes were co-cultured with H37Ra cells, and confocal laser scanning microscopy, electron microscopy, and flow cytometry techniques were used to identify phagocytosis and elucidate its mechanism. After Jurkat T lymphocytes phagocytosed H37Ra cells, the cell body became larger, with abundant cytoplasm, the portion of the nucleus closest to the bacterium deformed, long and short pseudopodia were extended, and the folds of the cell membrane formed depressions that created phagocytic vesicles surrounding the bacterium. The macropinocytosis inhibitor amiloride and the cytoskeletal inhibitor cytochalasin D were found to inhibit phagocytic efficacy; serum complements might enhance phagocytosis through opsonization. Jurkat T lymphocytes could actively phagocytose inactivated Mtb via the macropinocytotic mechanism. Actin remodeling played an important role in the macropinocytotic process. Serum complements may regulate phagocytosis.

Osteoclastogenic potential of bone marrow cells increases with age in elderly women with fracture.

The most reliable explanation for decreasing bone mass in elderly women is an imbalance of osteoclastic resorption and osteoblastic formation resulting from a relative increase in osteoclastic resorption. However, it is not clear whether an increase in osteoclastic bone resorption with age is due to increased osteoclast formation or to osteoclastic bone resorption activity. In this study, using a human bone marrow culture system, we attempt to clarify the increase in osteoclast formation with age. The mononuclear cell-rich fraction from bone marrow, obtained from the proximal region of the femur from female elderly patients with fracture, were cultured for 14 days in the presence of 1,25 dihydroxyvitamin D(3). Tartrate-resistant acid phosphatase-positive multinucleated cells were counted as osteoclasts. In our investigation, human osteoclast formation in the bone marrow culture increased with age in elderly women (age 64-96 years). The osteoclast formation was positively correlated with macrophage-colony stimulation factor and prostaglandin E(2) production in bone marrow culture. Also, osteoclast formation ex vivo was negatively correlated with bone mineral density of the lumbar spine (L2-L4). The above results indicate that the osteoclastogenic potential of bone marrow cells increases with aging in elderly women with fracture, and suggest that a decrease in bone mass of elderly women may be due to an increase in osteoclast population associated with aging.

Senescence marker protein-30 as a novel antiaging molecule.

Senescence marker protein-30 (SMP30), composed of 299 amino acids, has an approximate molecular mass of 32-34 kDa and has a pI 4.9 in charge. The amino acid alignment from various animal species revealed a highly conserved structure. SMP30 has an enzyme activity hydrolyzing sarin, soman, and tabun, known as lethal toxic nerve chemicals. We analyzed the organophosphatase activity of SMP30 using DFP as a substrate. This DFPase activity is revealed in a dose-dependent manner in the presence of magnesium ions. We investigated the intracellular localization of SMP30. It is localized in both the cytoplasm and nucleus. To confirm the presence of SMP30 in the nucleus, we prepared nuclear and cytoplasmic extracts from isolated cultured hepatocytes. Western blotting showed that SMP30 was detected in both extracts. Because the expression is reduced by carbon tetrachloride, one can speculate that the expression is modulated by oxidative stress increased with aging.

BCL11B is required for positive selection and survival of double-positive thymocytes.

Transcriptional control of gene expression in double-positive (DP) thymocytes remains poorly understood. We show that the transcription factor BCL11B plays a critical role in DP thymocytes by controlling positive selection of both CD4 and CD8 lineages. BCL11B-deficient DP thymocytes rearrange T cell receptor (TCR) alpha; however, they display impaired proximal TCR signaling and attenuated extracellular signal-regulated kinase phosphorylation and calcium flux, which are all required for initiation of positive selection. Further, provision of transgenic TCRs did not improve positive selection of BCL11B-deficient DP thymocytes. BCL11B-deficient DP thymocytes have altered expression of genes with a role in positive selection, TCR signaling, and other signaling pathways intersecting the TCR, which may account for the defect. BCL11B-deficient DP thymocytes also presented increased susceptibility to spontaneous apoptosis associated with high levels of cleaved caspase-3 and an altered balance of proapoptotic/prosurvival factors. This latter susceptibility was manifested even in the absence of TCR signaling and was only partially rescued by provision of the BCL2 transgene, indicating that control of DP thymocyte survival by BCL11B is nonredundant and, at least in part, independent of BCL2 prosurvival factors.

Quantitation of myelin oligodendrocyte glycoprotein and myelin basic protein in the thymus and central nervous system and its relationship to the clinicopathologic features of autoimmune encephalomyelitis.

There is controversy whether the amount of autoantigens expressed in the thymus regulates negative selection of autoreactive T cells and determine susceptibility or resistance to experimental autoimmune encephalomyelitis (EAE). In the present study, we have addressed this issue by quantifying neuroantigens in the thymus of two EAE-susceptible (LEW and LEW.1AV1) and one EAE-resistant (BN) rat strains. We further examined whether amounts of neuroantigens in various parts of the central nervous system (CNS) affect the clinical course and lesion distribution of acute and chronic EAE. Real-time PCR and histologic analyses showed that there was no significant difference in the amount and distribution of myelin oligodendrocyte glycoprotein and myelin basic protein in the thymus and CNS among the three strains and that both acute and chronic EAE lesions in the CNS were preferentially distributed in the area where neuroantigens were abundantly present. These findings suggest that susceptibility or resistance to EAE is not regulated by the amount of the neuroantigens expressed in the thymus. Furthermore, the lesion distribution, but not the clinical course, of EAE is related to the neuroantigen expression in the CNS.

Citrullination preferentially proceeds in glomerular Bowman's capsule and increases in obstructive nephropathy.

BACKGROUND: Peptidylarginine deiminases (PADs) are a group of posttranslational modification enzymes that citrullinate (deiminate) protein arginine residues, yielding citrulline residues. Citrullination of arginine residues abolishes their positive charge, markedly altering their structure. We undertook this study to investigate the actions of PADs in the kidney. METHODS: In male rats, we ligated the unilateral ureter, then analyzed the obstructed and contralateral kidneys 1 week later. Controls were rats simultaneously given sham operations. In another experiment, we ligated unilateral ureters of eight rats, four of which received a ureter-bladder anastomosis 1 week later. These rats were subjected to histologic examinations 5 weeks after unilateral ureteral obstruction (UUO). RESULTS: Reverse transcription-polymerase chain reaction (RT-PCR) revealed that, of PADs (type I, II, III, and IV), only PAD type II was expressed in kidneys. Western blot study showed that PAD type II expression and citrullinated protein content increased greatly in kidneys that underwent unilateral ureteral ligation compared to that in contralateral or sham-operated kidneys. Immunohistochemical analyses revealed that PAD type II was preferentially expressed by parietal epithelial cells and that only in Bowman's capsule were proteins citrullinated. Additionally, these PAD type II and citrullinated proteins in obstructed nephropathy were significantly attenuated by the release of the obstruction. Proteome analysis revealed that one of citrullinated proteins in the kidney should be actin. CONCLUSION: This result indicates that PAD type II and citrullinated proteins are suitable markers of Bowman's capsule. Not only are these markers preferentially expressed in Bowman's capsules but their expression is also increased in damaged kidneys by UUO, features that promise the further clarification of kidney diseases.

Association between bone loss and promoter polymorphism in the IL-6 gene in elderly Japanese women with hip fracture.

Recently, a G/C polymorphism was found at position -573 of the interleukin-6 ( IL-6) gene promoter. We investigated how this genetic polymorphism relates to IL-6 production and osteoporosis in elderly Japanese women. Genomic DNA was extracted from an aliquot of monocytes in the bone marrow; the monocytes were simultaneously used to form osteoclast-like multinucleated cells (MNCs) and to produce IL-6. Of the 47 subjects with fractures, 96% had a C allele at position -573 of the IL-6 gene. Only 2 subjects possessed homozygotes of G at that position. We investigated IL-6 levels, MNC formation in bone marrow culture, and femoral neck bone mineral density (BMD) in the subjects with the GC and CC genotypes. There were no significant differences between these genotypes as regards IL-6 levels, MNC formation, and femoral neck BMD. However, in the CC genotype, there was a negative relationship between femoral neck BMD and IL-6 levels, and between femoral neck BMD and MNC formation, whereas in the GC genotype and combined (GC + CC) genotypes, femoral neck BMD tended to be related to IL-6 levels and MNC formation. Moreover, the stromal cells in the CC genotype showed higher IL-1alpha-stimulated IL-6 production than did the stromal cells in the GC genotype. Our findings suggest that important information might be obtained not only by continued comparison of different genotypes but also by comparative study within each particular genotype.