Correlation between three assay systems for anti-Mullerian hormone (AMH) determination

PURPOSE: Analysis of anti-Müllerian hormone (AMH) is becoming of recognized importance in reproductive medicine, but assays are not standardized. We have evaluated the correlation between the new Gen II ELISA kit (Beckman-Coutler) and the older ELISA kits by Immunotech (IOT) and Diagnostic Systems Laboratories (DSL). METHODS: A total of 56 archived serum samples from patients with subfertility or reproductive endocrine disorders were retrieved and assayed in duplicate using the three AMH ELISA kits . The samples covered a wide range of AMH concentrations (1.9 to 142.5 pmol/L). RESULTS: We observed good correlations between the new (AMH Gen II) and old AMH assay kits by IOT and DSL (R(2) = 0.971 and 0.930 respectively). The regression equations were AMH (Gen II) = 1.353 × AMH (IOT) + 0.051 and AMH (Gen II) = 1.223 × AMH (DSL) – 1.270 respectively. CONCLUSIONS: AMH concentrations using the Gen II kit are slightly higher than those from the IOT and DSL kits. Standardization of assay results worldwide is urgently required but this analysis facilitates the interpretation of values obtained historically and in future studies using any of the 3 assays available. Meanwhile, adapting clinical cut-offs from previously published work by direct conversion is not recommended

Genetic causes of hypercalciuric nephrolithiasis

Renal stone disease (nephrolithiasis) affects 3–5% of the population and is often associated with hypercalciuria. Hypercalciuric nephrolithiasis is a familial disorder in over 35% of patients and may occur as a monogenic disorder that is more likely to manifest itself in childhood. Studies of these monogenic forms of hypercalciuric nephrolithiasis in humans, e.g. Bartter syndrome, Dent’s disease, autosomal dominant hypocalcemic hypercalciuria (ADHH), hypercalciuric nephrolithiasis with hypophosphatemia, and familial hypomagnesemia with hypercalciuria have helped to identify a number of transporters, channels and receptors that are involved in regulating the renal tubular reabsorption of calcium. Thus, Bartter syndrome, an autosomal disease, is caused by mutations of the bumetanide-sensitive Na–K–Cl (NKCC2) co-transporter, the renal outer-medullary potassium (ROMK) channel, the voltage-gated chloride channel, CLC-Kb, the CLC-Kb beta subunit, barttin, or the calcium-sensing receptor (CaSR). Dent’s disease, an X-linked disorder characterized by low molecular weight proteinuria, hypercalciuria and nephrolithiasis, is due to mutations of the chloride/proton antiporter 5, CLC-5; ADHH is associated with activating mutations of the CaSR, which is a G-protein-coupled receptor; hypophosphatemic hypercalciuric nephrolithiasis associated with rickets is due to mutations in the type 2c sodium–phosphate co-transporter (NPT2c); and familial hypomagnesemia with hypercalciuria is due to mutations of paracellin-1, which is a member of the claudin family of membrane proteins that form the intercellular tight junction barrier in a variety of epithelia. These studies have provided valuable insights into the renal tubular pathways that regulate calcium reabsorption and predispose to hypercalciuria and nephrolithiasis

Quantitative Proteomic and Interaction Network Analysis of Cisplatin Resistance in HeLa Cells

Cisplatin along with other platinum based drugs are some of the most widely used chemotherapeutic agents. However drug resistance is a major problem for the successful chemotherapeutic treatment of cancer. Current evidence suggests that drug resistance is a multifactorial problem due to changes in the expression levels and activity of a wide number of proteins. A majority of the studies to date have quantified mRNA levels between drug resistant and drug sensitive cell lines. Unfortunately mRNA levels do not always correlate with protein expression levels due to post-transcriptional changes in protein abundance. Therefore global quantitative proteomics screens are needed to identify the protein targets that are differentially expressed in drug resistant cell lines. Here we employ a quantitative proteomics technique using stable isotope labeling with amino acids in cell culture (SILAC) coupled with mass spectrometry to quantify changes in protein levels between cisplatin resistant (HeLa/CDDP) and sensitive HeLa cells in an unbiased fashion. A total of 856 proteins were identified and quantified, with 374 displaying significantly altered expression levels between the cell lines. Expression level data was then integrated with a network of protein-protein interactions, and biological pathways to obtain a systems level view of proteome changes which occur with cisplatin resistance. Several of these proteins have been previously implicated in resistance towards platinum-based and other drugs, while many represent new potential markers or therapeutic targets

A Pro-Cathepsin L Mutant Is a Luminal Substrate for Endoplasmic-Reticulum-Associated Degradation in C. elegans

Endoplasmic-reticulum associated degradation (ERAD) is a major cellular misfolded protein disposal pathway that is well conserved from yeast to mammals. In yeast, a mutant of carboxypeptidase Y (CPY*) was found to be a luminal ER substrate and has served as a useful marker to help identify modifiers of the ERAD pathway. Due to its ease of genetic manipulation and the ability to conduct a genome wide screen for modifiers of molecular pathways, C. elegans has become one of the preferred metazoans for studying cell biological processes, such as ERAD. However, a marker of ERAD activity comparable to CPY* has not been developed for this model system. We describe a mutant of pro-cathepsin L fused to YFP that no longer targets to the lysosome, but is efficiently eliminated by the ERAD pathway. Using this mutant pro-cathepsin L, we found that components of the mammalian ERAD system that participate in the degradation of ER luminal substrates were conserved in C. elegans. This transgenic line will facilitate high-throughput genetic or pharmacological screens for ERAD modifiers using widefield epifluorescence microscopy

Repeated BCG treatment of mouse bladder selectively stimulates small GTPases and HLA antigens and inhibits single-spanning uroplakins

<p>Abstract</p> <p>Background</p> <p>Despite being a mainstay for treating superficial bladder carcinoma and a promising agent for interstitial cystitis, the precise mechanism of Bacillus Calmette-Guerin (BCG) remains poorly understood. It is particularly unclear whether BCG is capable of altering gene expression beyond its well-recognized pro-inflammatory effects and how this relates to its therapeutic efficacy. The objective of this study was to determine differentially expressed genes in the mouse bladder following repeated intravesical BCG therapy.</p> <p>Methods</p> <p>Mice were transurethrally instilled with BCG or pyrogen-free on days 1, 7, 14, and 21. Seven days after the last instillation, urothelia along with the submucosa was removed and amplified ds-DNA was prepared from control- and BCG-treated bladder mucosa and used to generate suppression subtractive hybridization (SSH). Plasmids from control- and BCG-specific differentially expressed clones and confirmed by Virtual Northern were then purified and the inserts were sequenced and annotated. Finally, chromatin immune precipitation combined with real-time polymerase chain reaction assay (ChIP/Q-PCR) was used to validate SSH-selected transcripts.</p> <p>Results</p> <p>Repeated intravesical BCG treatment induced an up regulation of genes associated with antigen presentation (B2M, HLA-A, HLA-DQA1, HLA-DQB2, HLA-E, HLA-G, IGHG, and IGH) and representatives of two IFNγ-induced small GTPase families: the GBPs (GBP1, GBP2, and GBP5) and the p47GTPases (IIGTP1, IIGTP2, and TGTP). Genes expressed in saline-treated bladders but down-regulated by BCG included: the single-spanning uroplakins (UPK3a and UPK2), SPRR2G, GSTM5, and RSP 19.</p> <p>Conclusion</p> <p>Here we introduced a hypothesis-generator approach to determine key genes involved in the urothelium/sumbmucosa responses to BCG therapy. Urinary bladder responds to repeated BCG treatment by up-regulating not only antigen presentation-related genes, but also GBP and p47 small GTPases, both potentially serving to mount a resistance to the replication of the <it>Mycobacterium</it>. It will be of tremendous future interest to determine whether these immune response cascades play a role in the anti-cancer effects exerted by BCG.</p

The Caenorhabditis elegans HEN1 Ortholog, HENN-1, Methylates and Stabilizes Select Subclasses of Germline Small RNAs

Small RNAs regulate diverse biological processes by directing effector proteins called Argonautes to silence complementary mRNAs. Maturation of some classes of small RNAs involves terminal 2′-O-methylation to prevent degradation. This modification is catalyzed by members of the conserved HEN1 RNA methyltransferase family. In animals, Piwi-interacting RNAs (piRNAs) and some endogenous and exogenous small interfering RNAs (siRNAs) are methylated, whereas microRNAs are not. However, the mechanisms that determine animal HEN1 substrate specificity have yet to be fully resolved. In Caenorhabditis elegans, a HEN1 ortholog has not been studied, but there is evidence for methylation of piRNAs and some endogenous siRNAs. Here, we report that the worm HEN1 ortholog, HENN-1 (HEN of Nematode), is required for methylation of C. elegans small RNAs. Our results indicate that piRNAs are universally methylated by HENN-1. In contrast, 26G RNAs, a class of primary endogenous siRNAs, are methylated in female germline and embryo, but not in male germline. Intriguingly, the methylation pattern of 26G RNAs correlates with the expression of distinct male and female germline Argonautes. Moreover, loss of the female germline Argonaute results in loss of 26G RNA methylation altogether. These findings support a model wherein methylation status of a metazoan small RNA is dictated by the Argonaute to which it binds. Loss of henn-1 results in phenotypes that reflect destabilization of substrate small RNAs: dysregulation of target mRNAs, impaired fertility, and enhanced somatic RNAi. Additionally, the henn-1 mutant shows a weakened response to RNAi knockdown of germline genes, suggesting that HENN-1 may also function in canonical RNAi. Together, our results indicate a broad role for HENN-1 in both endogenous and exogenous gene silencing pathways and provide further insight into the mechanisms of HEN1 substrate discrimination and the diversity within the Argonaute family

Microfold (M) cells: important immunosurveillance posts in the intestinal epithelium

The transcytosis of antigens across the gut epithelium by microfold cells (M cells) is important for the induction of efficient immune responses to some mucosal antigens in Peyer’s patches. Recently, substantial progress has been made in our understanding of the factors that influence the development and function of M cells. This review highlights these important advances, with particular emphasis on: the host genes which control the functional maturation of M cells; how this knowledge has led to the rapid advance in our understanding of M-cell biology in the steady-state and during aging; molecules expressed on M cells which appear to be used as “immunosurveillance” receptors to sample pathogenic microorganisms in the gut; how certain pathogens appear to exploit M cells to infect the host; and finally how this knowledge has been used to specifically target antigens to M cells to attempt to improve the efficacy of mucosal vaccines