Partners in diabetes epidemic: A global perspective.

There is a recent increase in the worldwide prevalence of both obesity and diabetes. In this review we assessed insulin signaling, genetics, environment, lipid metabolism dysfunction and mitochondria as the major determinants in diabetes and to identify the potential mechanism of gut microbiota in diabetes diseases. We searched relevant articles, which have key information from laboratory experiments, epidemiological evidence, clinical trials, experimental models, meta-analysis and review articles, in PubMed, MEDLINE, EMBASE, Google scholars and Cochrane Controlled Trial Database. We selected 144 full-length articles that met our inclusion and exclusion criteria for complete assessment. We have briefly discussed these associations, challenges, and the need for further research to manage and treat diabetes more efficiently. Diabetes involves the complex network of physiological dysfunction that can be attributed to insulin signaling, genetics, environment, obesity, mitochondria and stress. In recent years, there are intriguing findings regarding gut microbiome as the important regulator of diabetes. Valid approaches are necessary for speeding medical advances but we should find a solution sooner given the burden of the metabolic disorder - What we need is a collaborative venture that may involve laboratories both in academia and industries for the scientific progress and its application for the diabetes control.

KLF regulation of insulin pathway genes.

Alteration in lipid metabolism can result in fat accumulation in adipose tissues, which may lead to two most important human diseases, obesity and diabetes. A shift in lipid metabolism deregulates signaling pathways which regulates obesity and/or diabetes. In this study, we examined the components of insulin/ TGF-ß pathways and their genetic interaction with Krüppel-like transcription factors (KLFs). Their role in energy homeostasis were discussed. We separately created klf/daf genes double mutants by carrying out klfs RNAi on daf-2 (e1391), daf-4 (e1364), daf-7 (e1372); dpy-1 (e1), daf-14 (m77), daf-16 (mgDf50) mutants. And then conducted Oil O Red staining to assay the klf/daf RNAi worms for fat deposits and examine genetic interaction between klfs and daf genes. The results showed that worms bearing klf-1, 2, or 3 and daf-2, or daf-4 mutations deposit large, but similar fat levels as individual mutants. The results suggested that they target the same molecular pathway of fat storage. klf-1, 2 or 3 RNAi /daf-7 worms showed higher fat deposits in klf-1, 2, or 3 RNAi/daf-7 worms than klf-1, 2, or 3 RNAi or daf-7 mutants alone, which showed a functional interaction between klfs and daf-7 in perhaps TGF-ß-like pathway. Altogether our study suggests a direct role of klfs in insulin signaling pathway.

Salts and energy balance: A special role for dietary salts in metabolic syndrome.

BACKGROUND: Dietary salts sodium (Na+), potassium (K+), magnesium (Mg2+), and calcium (Ca2+) are important in metabolic diseases. Yet, we do not have sufficient understanding on the salts global molecular network in these diseases. In this systematic review we have pooled information to identify the general effect of salts on obesity, insulin resistance and hypertension. AIMS: To assess the roles of salts in metabolic disorders by focusing on their individual effect and the network effect among these salts. METHODS: We searched articles in PubMed, EMBASE and Google Scholar. We selected original laboratory research, systematic reviews, clinical trials, observational studies and epidemiological data that focused on dietary salts and followed the preferred reporting items for systematic review in designing the present systematic review. RESULTS: From the initial search of 2898 studies we selected a total of 199 articles that met our inclusion criteria and data extraction. Alterations in metabolic pathways associated with the sensitivity of sodium, potassium, magnesium and calcium may lead to obesity, hypertension, and insulin resistance. We found that the results of most laboratory research, animal studies and clinical trials are coherent but some research outcome are either inconsistent or inconclusive. CONCLUSION: Important of salts in metabolic disorder is evident. In order to assess the effects of dietary salts in metablic diseases, environmental factors, dietary habits, physical activity, and the microbiome, should be considered in any study. Although interest in this area of research continues to grow, the challenge is to integrate the action of these salts in metabolic syndrom.

Data on hypoxia-induced VEGF, leptin and NF-kB p65 expression.

The data set presented here is associated with the article "Intracellular calcium and NF-kB regulate hypoxia-induced leptin, VEGF, IL-6 and adiponectin secretion in human adipocytes" (Al-Anazi et al., 2018). Data illustrate hypoxia-induced VEGF and leptin expression in human adipocytes treated with the calcium chelator BAPTA-AM (1 µM). It also shows NF-κB p65 induced expression by hypoxia. Preadipocytes were differentiated for 14 days and then subjected to 0.5-1.5% oxygen in the presence and absence of BAPTA-AM or the NF-κB inhibitor SN50 for 48 h prior to RNA isolation and PCR analysis.

Intracellular calcium and NF-kB regulate hypoxia-induced leptin, VEGF, IL-6 and adiponectin secretion in human adipocytes.

AIMS: Hypoxia-induced adipokine release has been attributed mainly to HIF-1α. Here we investigate the role of intracellular calcium and NF-kB in the hypoxia-dependent release of leptin, VEGF, IL-6 and the hypoxia-induced inhibition of adiponectin release in human adipocytes. MAIN METHODS: We used intracellular calcium imaging to compare calcium status in preadipocytes and in adipocytes. We subjected both cell types to hypoxic conditions and measured the release of adipokines induced by hypoxia in the presence and absence of HIF-1α inhibitor YC-1, NF-κB inhibitor SN50 and intracellular calcium chelator BAPTA-AM. KEY FINDINGS: We demonstrate reduced intracellular calcium oscillations and increased oxidative stress as the cells transitioned from preadipocytes to adipocytes. We show that differentiation of preadipocytes to adipocytes is associated with distinct morphological changes in the mitochondria. We also show that hypoxia-induced secretion of leptin, VEGF, IL-6 and hypoxia-induced inhibition of adiponectin secretion are independent of HIF-1α expression. The hypoxia-induced leptin, VEGF and IL-6 release are [Ca++]i dependent whereas adiponectin is NF-kB dependent. SIGNIFICANCE: Our work suggests a major role for [Ca++]i in preadipocyte differentiation to adipocytes and that changes in mitochondrial morphology in the adipocytes might underlie the reduced calcium oscillations observed in the adipocytes. It also demonstrates that multiple signaling pathways are associated with the hypoxia-induced adipokine secretion.

Pistachio hull water-soluble polysaccharides as a novel prebiotic agent.

We isolated and characterized pistachio hull polysaccharides (PHP). The PHP was a heteropolysaccharide mainly contained 75.50% (w/w) total sugar and 9.51% (w/w) uronic acid. As determined by GPC analysis, the polysaccharide with a molecular weight of 3.71×106 D (83.2%) was the most dominant fraction. Moreover, HPLC analysis indicated that PHP was predominantly composed of xylose, glucose, arabinose, and fructose with a molar ratio of 1.00:2.50:19.67:28.81. FT-IR and NMR analysis also confirmed the results obtained by HPLC and characterized preliminary structure features of the PHP. Functional properties of the PHP including water holding capacity (WHC: 2.44±0.05g water/g DM), and oil holding capacity (OHC: 11.53±0.04g oil/g DM) were significant compared to inulin used as reference prebiotic (p<0.01). Furthermore, the PHP remained 94.37% undigested in the simulated digestion process and stimulated the growth of L. plantarum PTCC 1896 and L. rhamnosus GG and increased the acetate, propionate and butyrate production over inulin in vitro. Totally, the PHP showed a considerable prebiotic capability and high WHC, OHC suggesting that the PHP is a potent pharmaceutical with good technological properties which can be used in food and drug industries.

Defective lipid metabolism associated with mutation in klf-2 and klf-3: important roles of essential dietary salts in fat storage.

BACKGROUND: Dietary salts are important factors in metabolic disorders. They are vital components of enzymes, vitamins, hormones, and signal transduction that act synergistically to regulate lipid metabolism. Our previous studies have identified that Krüppel-like factor -3 (KLF-3) is an essential regulator of lipid metabolism. However, it is not known if KLF-2 also regulates lipid metabolism and whether KLF-2 and -3 mediate the effects of dietary salts on lipid metabolism. METHODS: In this study, we used klf mutants [homozygous klf-2 (ok1043) V and klf-3 (ok1975) II mutants] to investigate the role of dietary salts in lipid metabolism. All gene expression was quantified by qRT-PCR. Localization of KLF-2 was analyzed by the expression of klf-2::gfp (in pPD95.75 vector) using a fluorescent microscope. Fat storage was measured by Oil Red O staining. RESULTS: Klf-2 was identified to express in the intestine during all stages of Caenorhabditis elegans development with peak expression at L3 stage. Mutation of klf-2 increased fat accumulation. Under regular growth media free of Ca2+, the expression of both klf-2 and -3 was inhibited slightly; further their expression reduced significantly in WT worms fed on 10X Ca2+ diet. When klf-3 was mutated, the expression of klf-2 increased under 10X Ca2+ diet; but when klf-2 was mutated, the expression of klf-3 was not altered under 10X Ca2+ diet. Overall, Mg2+ and K+ were less effective on the gene expression of klfs. KLF target gene Ce-C/EBP-2 showed elevated expression in WT and klf-3 (ok1975) worms with changed Ca2+ concentrations but not in klf-2 (ok1043) worms. However, high Ca+2 diet exhibited inhibitory effect on Ce-SREBP expression in WT worms. CONCLUSION: Dietary Ca2+ is most effective on fat storage and klf-2 expression, wherein high Ca2+ diet decreased klf-2 expression and reduced fat buildup. Mechanistic study identified Ce-C/EBP (C48E7.3; lpd-2) and Ce-SREBP (Y47D3B.7; lpd-1) as the target genes of klf-2 and/or klf-3 to mediate lipid metabolism. This study identifies a new function of klf-2 in inhibiting fat buildup and reveals the interplay between dietary salts and klf-2 and klf-3 in lipid metabolism.

Human cancer: is it linked to dysfunctional lipid metabolism?

BACKGROUND: Lipid metabolism dysfunction leading to excess fat deposits (obesity) may cause tumor (cancer) development. Both obesity and cancer are the epicenter of important medical issues. Lipid metabolism and cell death/proliferation are controlled by biochemical and molecular pathways involving many proteins, and organelles; alteration in these pathways leads to fat accumulation or tumor growth. Mammalian Krüppel-like factors, KLFs play key roles in both lipid metabolism and tumor development. SCOPE OF REVIEW: Substantial epidemiological and clinical studies have established strong association of obesity with a number of human cancers. However, we need more experimental verification to determine the exact role of this metabolic alteration in the context of tumor development. A clear understanding of molecules, pathways and the mechanisms involved in lipid metabolism and cell death/proliferation will have important implications in pathogenesis, and prevention of these diseases. MAJOR CONCLUSION: The regulatory role of KLFs, in both cell death/proliferation and lipid metabolism suggests a common regulation of both processes. This provides an excellent model for delivering a precise understanding of the mechanisms linking altered expression of KLFs to obesity and tumor development. GENERAL SIGNIFICANCE: Currently, mouse and rats are the models of choice for investigating disease mechanisms and pharmacological therapies but a genetic model is needed for a thorough examination of KLF function in vivo during the development of an organism. The worm Caenorhabditis elegans is an ideal model to study the connectivity between lipid metabolism and cell death/proliferation.

Regulation of lipoprotein assembly, secretion and fatty acid ß-oxidation by Krüppel-like transcription factor, klf-3.

Lipid metabolism is coordinately regulated through signaling networks that integrate biochemical pathways of fat assimilation, mobilization and utilization. Excessive diversion of fat for storage is a key risk factor for many fat-related human diseases. Dietary lipids are absorbed from the intestines and transported to various organs and tissues to provide energy and maintain lipid homeostasis. In humans, disparity between triglycerides (TG) synthesis and removal, via mitochondrial ß-oxidation and VLDL (very low density lipoprotein) secretion, causes excessive TG accumulation in the liver. The mutation in Caenorhabditis elegans KLF-3 leads to high TG accumulation in the worm's intestine. Our previous data suggested that klf-3 regulates lipid metabolism by promoting fatty acid ß-oxidation. Depletion of cholesterol in the diet has no effect on fat deposition in klf-3 (ok1975) mutants. Addition of vitamin D in the diet, however, increases fat levels in klf-3 worms. This suggests that excess vitamin D may be lowering the rate of fatty acid ß-oxidation, with the eventual increase in fat accumulation. We also demonstrate that mutation in klf-3 reduces expression of C. elegans dsc-4 and/or vit genes, the orthologs of mammalian microsomal triglyceride transfer protein and apolipoprotein B, respectively. Both microsomal triglyceride transfer protein and apolipoprotein B are essential for mammalian lipoprotein assembly and transport, and mutation in both dsc-4 (qm182) and vit-5 (ok3239) results in high fat accumulation in worm intestine. Genetic interactions between klf-3 and dsc-4, as well as vit-5 genes, suggest that klf-3 may have an important role in regulating lipid assembly and secretion.

A C. elegans model to study human metabolic regulation.

Lipid metabolic disorder is a critical risk factor for metabolic syndrome, triggering debilitating diseases like obesity and diabetes. Both obesity and diabetes are the epicenter of important medical issues, representing a major international public health threat. Accumulation of fat in adipose tissue, muscles and liver and/or the defects in their ability to metabolize fatty acids, results in insulin resistance. This triggers an early pathogenesis of type 2 diabetes (T2D). In mammals, lipid metabolism involves several organs, including the brain, adipose tissue, muscles, liver, and gut. These organs are part of complex homeostatic system and communicate through hormones, neurons and metabolites. Our study dissects the importance of mammalian Krüppel-like factors in over all energy homeostasis. Factors controlling energy metabolism are conserved between mammals and Caenorhabditis elegans providing a new and powerful strategy to delineate the molecular pathways that lead to metabolic disorder. The C. elegans intestine is our model system where genetics, molecular biology, and cell biology are used to identify and understand genes required in fat metabolism. Thus far, we have found an important role of C. elegans KLF in FA biosynthesis, mitochondrial proliferation, lipid secretion, and ß-oxidation. The mechanism by which KLF controls these events in lipid metabolism is unknown. We have recently observed that C. elegans KLF-3 selectively acts on insulin components to regulate insulin pathway activity. There are many factors that control energy homeostasis and defects in this control system are implicated in the pathogenesis of human obesity and diabetes. In this review we are discussing a role of KLF in human metabolic regulation.

Regulation of fat storage and reproduction by Krüppel-like transcription factor KLF3 and fat-associated genes in Caenorhabditis elegans.

Coordinated regulation of fat storage and utilization is essential for energy homeostasis, and its disruption is associated with metabolic syndrome and atherosclerosis in humans. Across species, Krüppel-like transcription factors (KLFs) have been identified as key components of adipogenesis. In humans, KLF14 acts as a master transregulator of adipose gene expression in type 2 diabetes and cis-acting expression quantitative trait locus associated with high-density lipoprotein cholesterol. Herein we report that, in Caenorhabditis elegans, mutants in klf-3 accumulate large fat droplets rich in neutral lipids in the intestine; this lipid accumulation is associated with an increase in triglyceride levels. The klf-3 mutants show normal pharyngeal pumping; however, they are sterile or semisterile. We explored important genetic interactions of klf-3 with the genes encoding enzymes involved in fatty acid (FA) ß-oxidation in mitochondria or peroxisomes and FA synthesis in the cytosol, namely acyl-CoA synthetase (acs-1 and acs-2), acyl-CoA oxidase (F08A8.1 and F08A8.2), and stearoyl-CoA desaturase (fat-7). We show that mutations or RNA interference in these genes increases fat deposits in the intestine of acs-1, acs-2, F08A8.1, and F08A8 animals. We further show that acs-1 and F08A8.1 influence larval development and fertility, respectively. Thus, KLF3 may regulate FA utilization in the intestine and reproductive tissue. We demonstrate that depletion of F08A8.1 activity, but not of acs-1, acs-2, F08A8.2, or fat-7 activity, enhances the fat phenotype of the klf-3 mutant. Taken together, these results suggest that klf-3 regulates lipid metabolism, along with acs-1, acs-2, F08A8.1, and F08A8.2, by promoting FA ß-oxidation and, in parallel, may contribute to normal reproductive behavior and fecundity in C. elegans.

Partner in fat metabolism: role of KLFs in fat burning and reproductive behavior.

The abnormalities caused by excess fat accumulation can result in pathological conditions which are linked to several interrelated diseases, such as cardiovascular disease and obesity. This set of conditions, known as metabolic syndrome, is a global pandemic of enormous medical, economic, and social concern affecting a significant portion of the world's population. Although genetics, physiology and environmental components play a major role in the onset of disease caused by excessive fat accumulation, little is known about how or to what extent each of these factors contributes to it. The worm, Caenorhabditis elegans offers an opportunity to study disease related to metabolic disorder in a developmental system that provides anatomical and genomic simplicity relative to the vertebrate animals and is an excellent eukaryotic genetic model which enable us to answer the questions concerning fat accumulation which remain unresolved. The stored triglycerides (TG) provide the primary source of energy during periods of food deficiency. In nature, lipid stored as TGs are hydrolyzed into fatty acids which are broken down through ß-oxidation to yield acetyl-CoA. Our recent study suggests that a member of C. elegans Krüppel-like factor, klf-3 regulates lipid metabolism by promoting FA ß-oxidation and in parallel may contribute in normal reproduction and fecundity. Genetic and epigenetic factors that influence this pathway may have considerable impact on fat related diseases in human. Increasing number of studies suggest the role of mammalian KLFs in adipogenesis. This functional conservation should guide our further effort to explore C. elegans as a legitimate model system for studying the role of KLFs in many pathway components of lipid metabolism.

Krüppel-like family of transcription factors: an emerging new frontier in fat biology.

In mammals, adipose tissue stores energy in the form of fat. The ability to regulate fat storage is essential for the growth, development and reproduction of most animals, thus any abnormalities caused by excess fat accumulation can result in pathological conditions which are linked to several interrelated diseases, such as cardiovascular diseases, diabetes, and obesity. In recent years significant effort has been applied to understand basic mechanism of fat accumulation in mammalian system. Work in mouse has shown that the family of Krüppel-like factors (KLFs), a conserved and important class of transcription factors, regulates adipocyte differentiation in mammals. However, how fat storage is coordinated in response to positive and negative feedback signals is still poorly understood. To address mechanisms underlying fat storage we have studied two Caenorhabditis elegans KLFs and demonstrate that both worm klfs are key regulators of fat metabolism in C. elegans. These results provide the first in vivo evidence supporting essential regulatory roles for KLFs in fat metabolism in C. elegans and shed light on the human counterpart in disease-gene association. This finding allows us to pursue a more comprehensive approach to understand fat biology and provides an opportunity to learn about the cascade of events that regulate KLF activation, repression and interaction with other factors in exerting its biological function at an organismal level. In this review, we provide an overview of the most current information on the key regulatory components in fat biology, synthesize the diverse literature, pose new questions, and propose a new model organism for understanding fat biology using KLFs as the central theme.

Mutation in Caenorhabditis elegans Krüppel-like factor, KLF-3 results in fat accumulation and alters fatty acid composition.

In vertebrates, adipose tissue stores energy in the form of fat. Fat storage is tightly controlled by and dynamically balanced with energy expenditure under physiological settings; the perturbation of fat in either excess (obese) or deficit (lipodystrophy) has devastating pathologic consequences in the fueling of homeostasis and organismal fitness. The process by which fat storage is coordinated through positive and negative feedback signals is still poorly understood. To address potential mechanisms underlying fat storage we study a Caenorhabditis elegans Krüppel-like transcription factor, Ce-klf-3 and demonstrate that klf-3 is a hitherto unrecognized key regulator of fat metabolism in C. elegans. The Ce-klf-3 is highly expressed during larval development and predominantly present in intestine: the site of fat digestion, absorption, storage, and utilization. We found a strong positive correlation between klf-3 expression and fat deposition in a worm's intestine. Significantly, a klf-3 (ok1975) loss-of-function mutation, characterized by the deletion of a 1658-bp sequence spanning the 3' end of exon 2 through to the 5' end of exon 3 of klf-3, enhanced fat deposition in the intestine and caused severe defects in worm reproduction. Although klf-3 mutants seemed very similar to wild type worms in appearance and life span, 70% of mutants became semi-sterile, each producing 40-50 viable progenies, and the remaining 30% were rendered completely sterile toward adulthood. Notably, both mutant types displayed extensive deposition of fat in the intestine. Our study also demonstrates that klf-3 is critical for maintaining normal fatty acid composition by regulating genes involved in a fatty acid desaturation pathway. Strikingly, klf-3 mutant animals with impaired fatty acid beta-oxidation pathway genes resulted in fat accumulation in the mutant worm. We present the first clear in vivo evidence supporting essential regulatory roles of KLF-3 in fat storage in C. elegans and shed light on the human equivalent in disease-gene association.

Functional analysis of the cathepsin-like cysteine protease genes in adult Brugia malayi using RNA interference.

BACKGROUND: Cathepsin-like enzymes have been identified as potential targets for drug or vaccine development in many parasites, as their functions appear to be essential in a variety of important biological processes within the host, such as molting, cuticle remodeling, embryogenesis, feeding and immune evasion. Functional analysis of Caenorhabditis elegans cathepsin L (Ce-cpl-1) and cathepsin Z (Ce-cpz-1) has established that both genes are required for early embryogenesis, with Ce-cpl-1 having a role in regulating in part the processing of yolk proteins. Ce-cpz-1 also has an important role during molting. METHODS AND FINDINGS: RNA interference assays have allowed us to verify whether the functions of the orthologous filarial genes in Brugia malayi adult female worms are similar. Treatment of B. malayi adult female worms with Bm-cpl-1, Bm-cpl-5, which belong to group Ia of the filarial cpl gene family, or Bm-cpz-1 dsRNA resulted in decreased numbers of secreted microfilariae in vitro. In addition, analysis of the intrauterine progeny of the Bm-cpl-5 or Bm-cpl Pro dsRNA- and siRNA-treated worms revealed a clear disruption in the process of embryogenesis resulting in structural abnormalities in embryos and a varied differential development of embryonic stages. CONCLUSIONS: Our studies suggest that these filarial cathepsin-like cysteine proteases are likely to be functional orthologs of the C. elegans genes. This functional conservation may thus allow for a more thorough investigation of their distinct functions and their development as potential drug targets.

A Krüppel-like factor in Caenorhabditis elegans with essential roles in fat regulation, cell death, and phagocytosis.

We demonstrate that a Caenorhabditis elegans Krüppel-like transcription factor is involved in fat regulation, cell death, and phagocytosis in C. elegans. Suppression of C. elegans klf-1 function by RNA interference (RNAi) results in increased fat storage in the intestine of the RNAi worm that directly or indirectly causes germ cells to die. These dead cells are not engulfed or phagocytosed in the RNAi worm. High-level expression of Ce-klf-1 during larval development, as well as its specific localization in the worm's intestine, supports a direct role for Ce-klf-1 in fat regulation. The C. elegans klf-1 encodes a C(2)H(2) zinc finger protein that is known to act as transcriptional modulator of tissue-specific expression. Members of the Krüppel-like factor (KLF) family play a variety of important roles in vertebrate tissue differentiation. KLFs have recently been implicated in energy and glucose homeostasis through their expression in pancreas, adipose, liver, and muscle tissues. The extensive fat storage and increased cell death in the Ce-klf-1 RNAi worm is important in that it may explain the connection between Ce-klf-1 signaling, cell death, and fat storage. This is the first evidence involving Ce-KLF-1 protein in such functions. In future studies, a thorough analysis of cellular functions of other members of C. elegans Krüppel-like transcription factors together with their interactions and pathway activities with other molecular partners should yield significant insights into mammalian KLF proteins.

Characterization of STIP, a multi-domain nuclear protein, highly conserved in metazoans, and essential for embryogenesis in Caenorhabditis elegans.

We report here the identification and characterization of STIP, a multi-domain nuclear protein that contains a G-patch, a coiled-coil, and several short tryptophan-tryptophan repeats highly conserved in metazoan species. To analyze their functional role in vivo, we cloned nematode stip-1 genes and determined the spatiotemporal pattern of Caenorhabditis elegans STIP-1 protein. RNA analyses and Western blots revealed that stip-1 mRNA was produced via trans-splicing and translated as a 95-kDa protein. Using reporter constructs, we found STIP-1 to be expressed at all developmental stages and in many tissue/cell types including worm oocyte nuclei. We found that STIP-1 is targeted to the nucleus and forms large polymers with a rod-like shape when expressed in mammalian cells. Using deletion mutants, we mapped the regions of STIP-1 involved in nuclear import and polymer assembly. We further showed that knockdown of C. elegans stip-1 by RNA interference arrested development and resulted in morphologic abnormalities around the 16-cell stage followed by 100% lethality, suggesting its essential role in worm embryogenesis. Importantly, the embryonic lethal phenotype could be faithfully rescued with Drosophila and human genes via transgenic expression. Our data provide the first direct evidence that STIP have a conserved essential nuclear function across metazoans from worms to humans.

The Caenorhabditis elegans CPI-2a cystatin-like inhibitor has an essential regulatory role during oogenesis and fertilization.

In the present study, we characterized a sterile cpi-2a(ok1256) deletion mutant in Caenorhabditis elegans and showed that CPI-2a has an essential regulatory role during oogenesis and fertilization. We have also shown that the CPI2a inhibitor and both Ce-CPL-1 and Ce-CPZ-1 enzymes are present in the myoepithelial sheath surrounding germ cells, oocytes, and embryos as well as in the yolk granules within normal oocytes. Staining of mutant worms with anti-yolk protein antibodies has indicted that the proteins are not present in the mature oocytes. Moreover, green fluorescent protein expression was absence or reduced in cpi-2a/yp170:gfp mutant oocytes, although it was expressed in one of the successfully developed embryos. Based on these results, we hypothesize that the sterility in cpi-2a(ok1256) mutant worms is potentially caused by two possible mechanisms: 1) defects in the uptake and/or processing of yolk proteins by the growing oocytes and 2) indirect induction of defects in cell-cell signaling that is critical for promoting germ line development, oocyte maturation, ovulation, and fertilization. A defect in any of these processes would have detrimental effects on the development of normal embryos and consequently normal production of progenies as we observed in cpi-2a mutant worms. This is the first study that demonstrates the expression of cysteine proteases and their endogenous inhibitor in the gonadal sheath cells surrounding germ cells and oocytes, which indirectly have established their potential involvement in proteolytic processing of molecules within the gonadal sheath cells, such as components of the extracellular matrix or the cytoskeletal proteins, which are essential for proper cell-cell signaling activities of the gonadal sheath cells during normal maturation and ovulation processes.

CeRh1 (rhr-1) is a dominant Rhesus gene essential for embryonic development and hypodermal function in Caenorhabditis elegans.

Rhesus (Rh) proteins share a conserved 12-transmembrane topology and specify a family of putative CO(2) channels found in diverse species from microbes to human, but their functional essentiality and physiological importance in metazoans is unknown. To address this key issue and analyze Rh-engaged physiologic processes, we sought to explore model organisms with fewer Rh genes yet are tractable to genetic manipulations. In this article, we describe the identification in nematodes of two Rh homologues that are highly conserved and similar to human Rh glycoproteins, and we focus on their characterization in Caenorhabditis elegans. RNA analysis revealed that CeRh1 is abundantly expressed in all developmental stages, with highest levels in adults, whereas CeRh2 shows a differential and much lower expression pattern. In transient expression in human cells, both CeRh1 and CeRh2-GFP fusion proteins were routed to the plasma membrane. Transgenic analysis with GFP or LacZ-fusion reporters showed that CeRh1 is mainly expressed in hypodermal tissue, although it is also in other cell types. Mutagenesis analysis using deletion constructs mapped a minimal promoter region driving CeRh1 gene expression. Although CeRh2 was dispensable, RNA interference with CeRh1 caused a lethal phenotype mainly affecting late stages of C. elegans embryonic development, which could be rescued by the CbRh1 homologue from the worm Caenorhabditis briggsae. Taken together, our data provide direct evidence for the essentiality of the CeRh1 gene in C. elegans, establishing a useful animal model for investigating CO(2) channel function by cross-species complementation.

RNA interference targeting cathepsin L and Z-like cysteine proteases of Onchocerca volvulus confirmed their essential function during L3 molting.

We describe the successful use of RNA interference (RNAi) to investigate gene function in the human filarial parasite Onchocerca volvulus third-stage larvae (L3). We targeted two specific gene products, the O. volvulus cathepsin L (Ov-CPL) and cathepsin Z-like (Ov-CPZ) cysteine proteases, which were proposed to function during O. volvulus L3 molting. We show that fluorescent-labeled Cy3-dsRNA corresponding to cpl or cpz regions encoding the mature enzymes can enter the larvae. The molting rate of larvae treated overnight with 0.5 mg ml(-1) cpl was reduced by 92% and 86% in comparison to normal control worms. It appeared that although the larvae started the molting process the last stage of molting, ecdysis was inhibited. The effect was gene specific, as larvae that did not molt in the presence of cpl or cpz dsRNA expressed the other cysteine protease, CPZ and CPL, respectively. This was confirmed by immunoelectron microscopy using antibodies directed against each enzyme. Our present study validate conclusively that both enzymes are essential for the molting of O. volvulus L3 to fourth-stage larvae. We also confirmed that the activity of the enzymes is specific to the changes that occur during the molting process on days 1-3, when the separation between the cuticles is in progress. The development of RNAi in O. volvulus L3 could further help study many of the abundant L3 and molting L3 genes identified through the filarial genome project, many of which, although have no attributed function, were identified as vaccine candidates or potential drug targets.