Although it has been known for many years that T. spiralis muscle larvae (ML) can not invade intestinal epithelial cells unless they are exposed to the intestinal milieu and activated into intestinal infective larvae (IIL), which genes in IIL are involved in the process of invasion is still unknown. In this study, suppression subtractive hybridization (SSH) was performed to identify differentially expressed genes between IIL and ML. SSH library was constructed using cDNA generated from IIL as the ‘tester’. About 110 positive clones were randomly selected from the library and sequenced, of which 33 T. spiralis genes were identified. Thirty encoded proteins were annotated according to Gene Ontology Annotation in terms of molecular function, biological process, and cellular localization. Out of 30 annotated proteins, 16 proteins (53.3%) had binding activity and 12 proteins (40.0%) had catalytic activity. The results of real-time PCR showed that the expression of nine genes (Ts7, Ndr family protein; Ts8, serine/threonine-protein kinase polo; Ts11, proteasome subunit beta type-7; Ts17, nudix hydrolase; Ts19, ovochymase-1; Ts22, fibronectin type III domain protein; Ts23, muscle cell intermediate filament protein OV71; Ts26, neutral and basic amino acid transport protein rBAT and Ts33, FACT complex subunit SPT16) from 33 T. spiralis genes in IIL were up-regulated compared with that of ML. The present study provide a group of the potential invasion-related candidate genes and will be helpful for further studies of mechanisms by which T. spiralis infective larvae recognize and invade the intestinal epithelial cells.
AIM: To investigate the cytotoxic mechanism of caribbean maitotoxin (MTX-C) in mammalian cells.
METHODS: We used whole-cell patch-clamp techniques and fluorescence calcium imaging to determine the cellular toxic mechanisms of MTX-C in insulin secreting HIT-T15 cells, which is a system where the effects of MTX have been observed. HIT-T15 cells stably express L-type calcium current, making it a suitable model for this study. Using the fluorescence calcium indicator Indo-1 AM, we found that there is a profound increase in HIT-T15 intracellular free calcium 3 min after application of 200 nmol/L MTX-C.
RESULTS: About 3 min after perfusion of MTX-C, a gradual increase in free calcium concentration was observed. This elevation was sustained throughout the entire recording period. Application of MTX-C did not elicit the L-type calcium current, but large cationic currents appeared after applying MTX-C to the extracellular solution. The current-voltage relationship of the cation current is approximately linear within the voltage range from -60 to 50 mV, but flattened at voltages at -80 and -100 mV. These results indicate that MTX-C induces a non-voltage activated, inward current under normal physiological conditions, which by itself or through a secondary mechanism results in a large amount of cationic influx. The biophysical mechanism of MTX-C is different to its isoform, pacific maitotoxin (MTX-P), when the extracellular calcium is removed.
CONCLUSION: We conclude that MTX-C causes the opening of non-selective, non-voltage-activated ion channels, which elevates level of intracellular calcium concentration and leads to cellular toxicities.
Maitotoxin; Calcium fluorescence; High voltage gated Ca2+ channels; Whole cell patch clamp; Insulin secreting cells
Treatment with the alpha-glucosidase inhibitor (AGI) acarbose is associated with a significant reduction the risk of cardiovascular events. However, the underlying mechanisms of this effect are unclear. AGIs were recently suggested to participate in stimulating glucagon-like peptide 1 (GLP-1) secretion. We therefore examined the effects of a 24-week treatment of acarbose on endogenous GLP-1, nitric oxide (NO) levels, nitric oxide synthase (NOS) activity, and carotid intima-media thickness (CIMT) in newly diagnosed patients with type 2 diabetes (T2D).
Blood was drawn from 24 subjects (14 male, 10 female, age: 50.7 ± 7.36 years, BMI: 26.64 ± 3.38 kg/m2, GHbA1c: 7.00 ± 0.74%) with drug-naïve T2D at 0 and 120 min following a standard mixed meal for the measurements of active GLP-1, NO and NOS. The CIMT was measured prior to and following 24 weeks of acarbose monotherapy (mean dose: 268 mg daily).
Following 24 weeks of acarbose treatment, both fasting and postprandial plasma GLP-1 levels were increased. In patients with increased postprandial GLP-1 levels, serum NO levels and NOS activities were also significantly increased and were positively related to GLP-1 levels. Although the CIMT was not significantly altered following treatment with acarbose, a decreased CIMT was negatively correlated with increased GLP-1 levels.
Twenty-four weeks of acarbose monotherapy in newly diagnosed patients with T2D is associated with significantly increased levels of both fasting and postprandial GLP-1 as well as significantly increased NO levels and NOS activity for those patients in whom postprandial GLP-1 levels were increased. Therefore, the benefits of acarbose on cardiovascular risk may be related to its stimulation of GLP-1 secretion.
Glucagon-like peptide 1; Carotid intima-media thickness; Nitric oxide type 2 diabetes; Acarbose
API-1 is a novel small molecule inhibitor of Akt, which acts by binding to Akt and preventing its membrane translocation, and has promising preclinical antitumor activity. In this study, we reveal a novel function of API-1 in regulation of c-FLIP levels and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, independent of Akt inhibition. API-1 effectively induced apoptosis in tested cancer cell lines including activation of caspase-8 and caspase-9. It reduced the levels of c-FLIP without increasing the expression of DR4 or DR5. Accordingly, it synergized with TRAIL to induce apoptosis. Enforced expression of ectopic c-FLIP did not attenuate API-1-induced apoptosis, but inhibited its ability to enhance TRAIL-induced apoptosis. These data indicate that downregulation of c-FLIP mediates enhancement of TRAIL-induced apoptosis by API-1, but is not sufficient for API-1-induced apoptosis. API-1-induced reduction of c-FLIP could be blocked by the proteasome inhibitor MG132. Moreover, API-1 increased c-FLIP ubiquitination and decreased c-FLIP stability. These data together suggest that API-1 downregulates c-FLIP by facilitating its ubiquitination and proteasome-mediated degradation. Since other Akt inhibitors including API-2 and MK2206 had minimal effects on reducing c-FLIP and enhancement of TRAIL-induced apoptosis, it is likely that API-1 reduces c-FLIP and enhances TRAIL-induced apoptosis independent of its Akt-inhibitory activity.
API-1; Akt; TRAIL; c-FLIP; apoptosis; cancer
Constitutively active KRAS mutations have been found to be involved in various processes of cancer development, and render tumor cells resistant to EGFR-targeted therapies. Mutation detection methods with higher sensitivity will increase the possibility of choosing the correct individual therapy. Here, we established a highly sensitive and efficient microfluidic capillary electrophoresis-based restriction fragment length polymorphism (µCE-based RFLP) platform for low-abundance KRAS genotyping with the combination of µCE and RFLP techniques. By using our self-built sensitive laser induced fluorescence (LIF) detector and a new DNA intercalating dye YOYO-1, the separation conditions of µCE for ΦX174 HaeIII DNA marker were first optimized. Then, a Mav I digested 107-bp KRAS gene fragment was directly introduced into the microfluidic device and analyzed by µCE, in which field amplified sample stacking (FASS) technique was employed to obtain the enrichment of the RFLP digestion products and extremely improved the sensitivity. The accurate analysis of KRAS statuses in HT29, LS174T, CCL187, SW480, Clone A, and CX-1 colorectal cancer (CRC) cell lines by µCE-based RFLP were achieved in 5 min with picoliter-scale sample consumption, and as low as 0.01% of mutant KRAS could be identified from a large excess of wild-type genomic DNA (gDNA). In 98 paraffin-embedded CRC tissues, KRAS codon 12 mutations were discovered in 28 (28.6%), significantly higher than that obtained by direct sequencing (13, 13.3%). Clone sequencing confirmed these results and showed this system could detect at least 0.4% of the mutant KRAS in CRC tissue slides. Compared with direct sequencing, the new finding of the µCE-based RFLP platform was that KRAS mutations in codon 12 were correlated with the patient’s age. In conclusion, we established a sensitive, fast, and cost-effective screening method for KRAS mutations, and successfully detected low-abundance KRAS mutations in clinical samples, which will allow provision of more precise individualized cancer therapy.
Objectives. The aim of this study was to evaluate the prevalence and the risk factors of prolonged QTc interval among Chinese patients with type 2 diabetes. Methods. The retrospective study included 3156 outpatients from the Diabetes Centre, the 306th Hospital of PLA, during the period from September 2003 to June 2010. QT interval was measured manually in the 12-lead conventional electrocardiogram. The QT interval corrected for heart rate (QTc) was calculated using Bazett's formula. Additional demographic and laboratory data were also collected. Potential risk factors of prolonged QTc interval were assessed using multivariable regression. Results. The prevalence of prolonged QTc interval among Chinese patients with type 2 diabetes was 30.1%. Height (OR 0.156, 95% CI 0.032~0.748), waist circumference (OR 1.025, 95% CI 1.010~1.040), diastolic blood pressure (OR 1.016, 95% CI 1.007~1.026), postprandial glucose (OR 1.040, 95% CI 1.022~1.059), fasting insulin (OR 1.014, 95% CI 1.003~1.025), and presence of microalbuminuria (OR 1.266, 95% CI 1.033~1.551) were significant risk factors. Conclusions. The prevalence of prolonged QTc interval among Chinese patients with type 2 diabetes is high. Risk factors for prolongation of QTc interval were low height, high waist circumference, increasing diastolic blood pressure levels, high postprandial glucose levels, high fasting insulin levels, and presence of microalbuminuria.
Diabetic HDL had diminished capacity to stimulate endothelial cell (EC) proliferation, migration, and adhesion to extracellular matrix. The mechanism of such dysfunction is poorly understood and we therefore sought to determine the mechanistic features of diabetic HDL dysfunction.
We found that the dysfunction of diabetic HDL on human umbilical vein endothelial cells (HUVECs) was associated with the down regulation of the HDL receptor protein, SR-BI. Akt-phosphorylation in HUVECs was induced in a biphasic manner by normal HDL. While diabetic HDL induced Akt phosphorylation normally after 20 minutes, the phosphorylation observed 24 hours after diabetic HDL treatment was reduced. To determine the role of SR-BI down regulation on diminished EC responses of diabetic HDL, Mouse aortic endothelial cells (MAECs) were isolated from wild type and SR-BI (−/−) mice, and treated with normal and diabetic HDL. The proliferative and migratory effects of normal HDL on wild type MAECs were greatly diminished in SR-BI (−/−) cells. In contrast, response to diabetic HDL was impaired in both types suggesting diminished effectiveness of diabetic HDL on EC proliferation and migration might be due to the down regulation of SR-BI. Additionally, SR-BI down regulation diminishes diabetic HDL’s capacity to activate Akt chronically.
Diabetic HDL was dysfunctional in promoting EC proliferation, migration, and adhesion to matrix which was associated with the down-regulation of SR-BI. Additionally, SR-BI down regulation diminishes diabetic HDL’s capacity to activate Akt chronically.
Previous studies suggest that oxidative stress plays an important role in the development of breast cancer. There is a significant inverse relationship between HDL and the risk and mortality of breast cancer. However, it is well known that under conditions of oxidative stress, such as breast cancer, HDL can be oxidatively modifiedand these modifications may have an effect on the functions of HDL. The purpose of this study is to determine the different effects of normal and oxidized (caused by hypochlorite-induced oxidative stress) HDL on breast cancer cell metastasis.
Human breast cancer cell lines were treated with normal and hypochlorite-oxidized HDL, and then cell metastasis potency in vivo and the abilities of migration, invasion, adhesion to HUVEC and ECM in vitro were examined. Integrin expression and PKC activity were evaluated, and PKC inhibitor and PKC siRNA was applied.
We found hypochlorite-oxidized HDL dramatically promotes breast cancer cell pulmonary metastasis (133.4% increase at P < 0.0 l for MDA-MB-231 by mammary fat pad injection; 164.3% increase at P < 0.01 for MCF7 by tail vein injection) and hepatic metastasis (420% increase at P < 0.0 l for MDA-MB-231 by mammary fat pad injection; 1840% fold increase at P < 0.001 for MCF7 by tail vein injection) in nude mice, and stimulates higher cell invasion (85.1% increase at P < 0.00 l for MDA-MB-231; 88.8% increase at P < 0.00 l for MCF7;), TC-HUVEC adhesion (43.4% increase at P < 0.00 l for MDA-MB-231; 35.2% increase at P < 0.00 l for MCF7), and TC-ECM attachment (41.0% increase at P < 0.00 l for MDA-MB-231; 26.7% increase at P < 0.05 for MCF7) in vitro compared with normal HDL. The data also shows that the PKC pathway is involved in the abnormal actions of hypochlorite-oxidized HDL.
Our study demonstrated that HDL under hypochlorite-induced oxidative stress stimulates breast cancer cell migration, invasion, adhesion to HUVEC and ECM, thereby promoting metastasis of breast cancer. These results suggest that HDL-based treatments should be considered for treatment of breast cancer patients.
Breast cancer; Oxidative stress; Metastasis; High-density lipoprotein
Alpha-albumin (AFM), a member of the albumin gene family that also includes albumin, alpha-fetoprotein, and vitamin D-binding protein, is expressed predominantly in the liver and activated at birth. Here, we identify two hepatocyte nuclear factor 1 (HNF1)-binding sites in the AFM promoter that are highly conserved in different mammals. These two sites bind HNF1α and HNF1β. The distal site (centered at −132, relative to AFM exon 1) is more important than proximal site (centered at −58), based on HNF1 binding and mutational analysis in transfected cells. Our data indicate that HNF1α is a more potent activator of AFM promoter than is HNF1β. However, HNF1β can act in a dominant manner to inhibit HNF1α-dependent transactivation of the AFM promoter when both proteins are expressed together. This suggests that the differential timing with which the albumin family genes are activated in the liver may be influenced by their responsiveness to HNF1α and HNF1β. Our comparison of HNF1-binding sites in the promoters in the albumin family of genes indicates that the primordial albumin-like gene contained two HNF1 sites; one of these sites was lost from the albumin promoter, but both sites still are present in other members of this gene family.
The N-myc downstream-regulated gene 2 (NDRG2) is involved in tumor cell differentiation and apoptosis, but its function in tumor angiogenesis remains to be established. Here, we employed adenovirus overexpressing NDRG2 (Ad-NDRG2) to efficiently up-regulate target gene expression in the NDRG2-low-expressing, breast cancer cell line MCF-7. Moreover, VEGF secretion was decreased in MCF-7 cells infected by Ad-NDRG2, and medium conditioned by these infected cells could significantly inhibit the proliferation, tube formation and invasion of human umbilical vein endothelial cells (HUVECs). Further study indicated that the angiogenesis promoting factors VEGF and HIF-1α were down-regulated, whereas the angiogenesis suppressing factors p53 and VHL were up-regulated in MCF-7 cells infected by Ad-NDRG2. Finally, in a nude mouse model, intratumoral injections of Ad-NDRG2 every 3 days for 20 days significantly inhibited the growth and angiogenesis of xenografted MCF-7 tumors. In summary, these data indicate that NDRG2 may be involved in angiogenesis by impacting the expression of angiogenesis related factors. Thus, specific overexpression of NDRG2 by adenovirus represents a promising approach for the treatment of tumor angiogenesis.
Auto-fluorescence bronchoscopy (AFB) has been used for the identification and localization of intra-epithelial pre-neoplastic and neoplastic lesions within the bronchus.
To determine the applicability of AFB for the detection and localization of precancerous and cancerous lesions, in addition to analyzing the morphologic presentation, their association to histological type and the variation between genders.
A five-year study involving 4983 patients, who underwent routine bronchoscopy [B] examination in a local tertiary teaching hospital, was done. The B examination was performed under intratracheal lidocaine, and samples were obtained using suitable approach. One thousand four hundred and eighty-five pathologically confirmed lung cancer patients were included in the study. The following parameters were studied: Morphological presentation, biopsy sites, histology. Differences between the groups were analyzed using Chi square test.
One thousand four hundred and eighty-five patients who had hyperplasia or neoplastic lesions were further confirmed as lung cancer pathologically. Lung cancer was more commonly found in the right lung (51.58% vs. 42.82%). The lesion occurred more frequently in the upper lobe than the lower lobe (44.17% vs. 22.42%). Male patients with squamous cell carcinoma showed upper lobe involvement more commonly, while the left main bronchus was more commonly involved in female patients. Adenocarcinoma mostly involved lesion of the upper lobe. Squamous cell carcinoma and small cell carcinoma were the major proliferative types (80.15% and 76.16% respectively).
AFB is efficient in the detection of pre-invasive and invasive lung lesions. The morphological presentation is associated to the histological type. There is variation in the presentation and histology of cancerous lung lesions between genders.
Auto-fluorescence bronchoscopy; gender; invasive lesion; lung cancer; screening
The first human case with trichinellosis was reported in 1964 in Tibet, China. However, up to the present, the etiological agent of trichinellosis has been unclear. The aim of this study was to identify a Tibet Trichinella isolate at a species level by PCR-based methods. Multiplex PCR revealed amplicon of the expected size (173 bp) for Trichinella spiralis in assays containing larval DNA from Tibet Trichinella isolate from a naturally infected pig. The Tibet Trichinella isolate was also identified by PCR amplification of the 5S ribosomal DNA intergenic spacer region (5S ISR) and mitochondrial large-subunit ribosomal RNA (mt-lsrDNA) gene sequences. The results showed that 2 DNA fragments (749 bp and 445 bp) of the Tibet Trichinella isolate were identical to that of the reference isolates of T. spiralis. The Tibet Trichinella isolate might be classifiable to T. spiralis. This is the first report on T. spiralis in southwestern China.
Trichinella spiralis; taxonomy; molecular identification; Tibet; China
It has been known for many years that Trichinella spiralis initiates infection by penetrating the columnar epithelium of the small intestine; however, the mechanisms used by the parasite in the establishment of its intramulticellular niche in the intestine are unknown. Although the previous observations indicated that invasion also occurs in vitro when the infective larvae are inoculated onto cultures of intestinal epithelial cells (e.g., human colonic carcinoma cell line Caco-2, HCT-8), a normal readily manipulated in vitro model has not been established because of difficulties in the culture of primary intestinal epithelial cells (IECs). In this study, we described a normal intestinal epithelial model in which T. spiralis infective larvae were shown to invade the monolayers of normal mouse IECs in vitro. The IECs derived from intestinal crypts of fetal mouse small intestine had the ability to proliferate continuously and express specific cytokeratins as well as intestinal functional cell markers. Furthermore, they were susceptible to invasion by T. spiralis. When inoculated onto the IEC monolayer, infective larvae penetrated cells and migrated through them, leaving trails of damaged cells heavily loaded with T. spiralis larval excretory-secretory (ES) antigens which were recognized by rabbit immune sera on immunofluorescence test. The normal intestinal epithelial model of invasion mimicking the natural environment in vivo will help us to further investigate the process as well as the mechanisms by which T. spiralis establishes its intestinal niche.
This study assessed the postoperative morbidity and mortality occurring in the first 30 days after radical gastrectomy by comparing gastric cancer patients who did or did not receive the FOLFOX7 regimen of neoadjuvant chemotherapy.
We completed a retrospective analysis of 377 patients after their radical gastrectomies were performed in our department between 2005 and 2009. Two groups of patients were studied: the SURG group received surgical treatment immediately after diagnosis; the NACT underwent surgery after 2-6 cycles of neoadjuvant chemotherapy.
There were 267 patients in the SURG group and 110 patients in the NACT group. The NACT group had more proximal tumours (P = 0.000), more total/proximal gastrectomies (P = 0.000) and longer operative time (P = 0.005) than the SURG group. Morbidity was 10.0% in the NACT patients and 17.2% in the SURG patients (P = 0.075). There were two cases of postoperative death, both in the SURG group (P = 1.000). No changes in complications or mortality rate were observed between the SURG and NACT groups.
The FOLFOX7 neoadjuvant chemotherapy is not associated with increased postoperative morbidity, indicating that the FOLFOX7 neoadjuvant chemotherapy is a safe choice for the treatment of local advanced gastric cancer.
Gastric cancer; neoadjuvant chemotherapy; complication; FOLFOX7; surgery
The asymmetric unit of the title compound, [CdCl2(C17H14N4)], contains two independent molecules in which the CdII ions are in distorted trigonal-bipyramidal CdN3Cl2 coordination environments. In the crystal structure, there is a π–π stacking interaction involving a pyridine ring and a symmetry-related benzene ring, with a centroid–centroid distance of 3.5088 (19) Å.
Vertilmicin is a novel aminoglycoside antibiotic with potent activity against gram-negative and -positive bacteria in vitro. In this study, we further evaluated the efficacy of vertilmicin in vivo in systemic and local infection animal models. We demonstrated that vertilmicin had relatively high and broad-spectrum activities against mouse systemic infections caused by Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus faecalis. The 50% effective doses of subcutaneously administered vertilmicin were 0.63 to 0.82 mg/kg, 0.18 to 0.29 mg/kg, 0.25 to 0.99 mg/kg, and 4.35 to 7.11 mg/kg against E. coli, K. pneumoniae, S. aureus, and E. faecalis infections, respectively. The therapeutic efficacy of vertilmicin was generally similar to that of netimicin, better than that of gentamicin in all the isolates tested, and better than that of verdamicin against E. coli 9612 and E. faecalis HH22 infections. The therapeutic efficacy of vertilmicin was further confirmed in local infection models of rabbit skin burn infection and mouse ascending urinary tract infection.
Vertilmicin is a new semisynthetic aminoglycoside with a structure similar to that of netilmicin except for a methyl group at the C-6′ position. In the present study, the in vitro antibacterial activity of vertilmicin was studied, and its susceptibility to modifications by the recombinant aminoglycoside bifunctional modifying enzyme AAC(6′)-APH(2″) was compared with those of verdamicin and netilmicin. A total of 1,185 clinical isolates collected from hospitals in Beijing between 2000 and 2001 were subjected to the in vitro antibacterial activity evaluations, including MIC, minimum bactericidal concentration (MBC), and time-kill curve tests. The MICs were evaluated in non-gentamicin-resistant (gentamicin-susceptible and gentamicin-intermediate) strains and gentamicin-resistant strains, respectively. For most of the non-gentamicin-resistant bacteria (except for the isolates of Pseudomonas spp.), the MIC90s of vertilmicin were in the range of 0.5 to 8 μg/ml, comparable to those of the reference aminoglycosides. For the gentamicin-resistant isolates, the three semisynthetic aminoglycosides (vertilmicin, netilmicin, and amikacin) demonstrated low MIC50s and/or MIC90s, as well as high percent susceptibility values. Among the study drugs, vertilmicin showed the lowest MIC90s, 16 μg/ml, for the gram-positive gentamicin-resistant isolates of Staphylococcus aureus and Staphylococcus epidermidis. Meanwhile, vertilmicin was a potent bactericidal agent, with MBC/MIC ratios in the range of 1 to 2 for Escherichia coli, Klebsiella pneumoniae, and S. aureus and 1 to 4 for S. epidermidis. The time-kill curve determination further demonstrated that this effect was rapid and concentration dependent. In evaluations of susceptibility to modifications by the recombinant AAC(6′)-APH(2″) with maximum rate of metabolism/Km measurements, vertilmicin exhibited susceptibilities to both acetylation and phosphorylation lower than those of netilmicin and verdamicin.
Chemokine receptors belong to a class of integral membrane G-protein coupled receptors (GPCRs) and are responsible for transmitting signals from the extracellular environment. However, the structural changes in the receptor, connecting ligand binding to G-protein activation, remain elusive for most GPCRs due to the difficulty to produce them for structural and functional studies. We here report high-level production in E.coli of 4 human GPCRs, namely chemokine receptors (hCRs) CCR5, CCR3, CXCR4 and CX3CR1 that are directly involved in HIV-1 infection, asthma and cancer metastasis. The synthetic genes of CCR5, CCR3, CXCR4 and CX3CR1 were synthesized using a two-step assembly/amplification PCR method and inserted into two different kinds of expression systems. After systematic screening of growth conditions and host strains, TB medium was selected for expression of pEXP-hCRs. The low copy number pBAD-DEST49 plasmid, with a moderately strong promoter tightly regulated by L-arabinose, proved helpful for reducing toxicity of expressed membrane proteins. The synthetic Trx-hCR fusion genes in the pBAD-DEST49 vector were expressed at high levels in the Top10 strain. After a systematic screen of 96 detergents, the zwitterionic detergents of the Fos-choline series (FC9-FC16) emerged as the most effective for isolation of the hCRs. The FC14 was selected both for solubilization from bacterial lysates and for stabilization of the Trx-hCRs during purification. Thus, the FC-14 solubilized Trx-hCRs could be purified using size exclusion chromatography as monomers and dimers with the correct apparent MW and their alpha-helical content determined by circular dichroism. The identity of two of the expressed hCRs (CCR3 and CCR5) was confirmed using immunoblots using specific monoclonal antibodies. After optimization of expression systems and detergent-mediated purification procedures, we achieved large-scale, high-level production of 4 human GPCR chemokine receptor in a two-step purification, yielding milligram quantities of CCR5, CCR3, CXCR4 and CX3CR1 for biochemical, biophysical and structural analysis.