Two dinucleotide short tandem-repeat polymorphisms (STRPs) and a polymorphic Alu element spanning a 22-kb region of the PLAT locus on chromosome 8p12-q11.2 were typed in 1,287-1,420 individuals originating from 30 geographically diverse human populations, as well as in 29 great apes. These data were analyzed as haplotypes consisting of each of the dinucleotide repeats and the flanking Alu insertion/deletion polymorphism. The global pattern of STRP/Alu haplotype variation and linkage disequilibrium (LD) is informative for the reconstruction of human evolutionary history. Sub-Saharan African populations have high levels of haplotype diversity within and between populations, relative to non-Africans, and have highly divergent patterns of LD. Non-African populations have both a subset of the haplotype diversity present in Africa and a distinct pattern of LD. The pattern of haplotype variation and LD observed at the PLAT locus suggests a recent common ancestry of non-African populations, from a small population originating in eastern Africa. These data indicate that, throughout much of modern human history, sub-Saharan Africa has maintained both a large effective population size and a high level of population substructure. Additionally, Papua New Guinean and Micronesian populations have rare haplotypes observed otherwise only in African populations, suggesting ancient gene flow from Africa into Papua New Guinea, as well as gene flow between Melanesian and Micronesian populations.

Double-strand RNA (dsRNA)-mediated posttranscriptional gene silencing, also known as RNA interference (RNAi), is a powerful tool to inhibit gene expression in several experimental model systems, including Arabidopsis, Caenorhabditis, and Drosophila. We previously described that the microinjection of Mos dsRNA into fully grown mouse oocytes results in the specific degradation of Mos mRNA in a time- and concentration-dependent manner. We report here a transgenic RNAi approach that is suitable to study gene function during mouse oocyte development and differentiation. The oocyte-specific Zp3 promoter was used to drive the expression of a long hairpin dsRNA ( approximately 500 bp) targeting Mos mRNA. Transgenic founder animals appeared healthy, but while males were fertile, females were not, in accordance with the known Mos null phenotype. The amount of Mos mRNA in the transgenic F(1) females was reduced by >90%, whereas there was no decrease in the nontargeted tissue plasminogen activator (Plat) mRNA. Moreover, the maturation-associated increase in mitogen-activated protein (MAP) kinase activity was not observed, and the metaphase II eggs underwent spontaneous parthenogenetic activation, thus recapitulating the Mos null phenotype. This approach provides a powerful method to study the functions of any oocyte-synthesized gene during oocyte development and early embryogenesis.

Mutations to the prototypical members of the two general classes of polycystins, polycystin-1 encoded by PKD1 and polycystin-2 encoded by PKD2, underlie autosomal-dominant polycystic kidney disease. Here we report the identification of a pair of genes homologous to PKD1 from both the human and mouse genomes. PKD1L2 and PKD1L3 are located on human chromosome 16q22-q23 and mouse chromosome 8 and are alternatively spliced. The human and mouse forms of PKD1L2 are highly conserved, with each one consisting of 43 exons and approximately 2,460 codons. PKD1L3 shows regional sequence divergence, with the mouse form having two additional exons and a much larger exon 5. The predicted protein products of PKD1L2 and PKD1L3 contain the combination of GPS and PLAT/LH2 domains that uniquely define them as polycystin-1 family members. They are predicted to have 11 membrane-spanning regions with a large extracellular domain consistent with the proposed receptor function of this protein family. PKD1L2 and PKD1L3 contain strong ion channel signature motifs that suggest their possible function as components of cation channel pores. Polycystin-1-related proteins may not only regulate channels, but may actually be part of the pore-forming unit.

Malaria parasites suffer severe losses in the mosquito as they cross the midgut, haemolymph and salivary gland tissues, in part caused by immune responses of the insect. The parasite compensates for these losses by multiplying during the oocyst stage to form the infectious sporozoites. Upon human infection, malaria parasites are again attenuated by sustained immune attack. Here, we report a single copy gene that is highly conserved amongst Plasmodium species that encodes a secreted protein named PxSR. The predicted protein is composed of a unique combination of metazoan protein domains that have been previously associated with immune recognition/activation and lipid/protein adhesion interactions at the cell surface, namely: (i) scavenger receptor cysteine rich (SRCR); (ii) pentraxin (PTX); (iii) polycystine-1, lipoxygenase, alpha toxin (LH2/PLAT); (iv) Limulus clotting factor C, Coch-5b2 and Lgl1 (LCCL). In our assessment the PxSR molecule is completely novel in biology and is only found in Apicomplexa parasites. We show that PxSR is expressed in sporozoites of both human and rodent malaria species. Disruption of the PbSR gene in the rodent malaria parasite P. berghei results in parasites that form normal numbers of oocysts, but fail to produce any sporozoites. We suggest that, in addition to a role in sporogonic development, PxSR may have a multiplicity of functions.

BACKGROUND

Chronic inflammation is well known to be a risk factor for colon cancer. Previously we established a novel mouse model of inflammation-related colon carcinogenesis, which is useful to examine the involvement of inflammation in colon carcinogenesis. To shed light on the alterations in global gene expression in the background of inflammation-related colon cancer and gain further insights into the molecular mechanisms underlying inflammation-related colon carcinogenesis, we conducted a comprehensive DNA microarray analysis using our model.

METHODS

Male ICR mice were given a single ip injection of azoxymethane (AOM, 10 mg/kg body weight), followed by the addition of 2% (w/v) dextran sodium sulfate (DSS) to their drinking water for 7 days, starting 1 week after the AOM injection. We performed DNA microarray analysis (Affymetrix GeneChip) on non-tumorous mucosa obtained from mice that received AOM/DSS, AOM alone, and DSS alone, and untreated mice at wks 5 and 10.

RESULTS

Markedly up-regulated genes in the colonic mucosa given AOM/DSS at wk 5 or 10 included Wnt inhibitory factor 1 (Wif1, 48.5-fold increase at wk 5 and 5.7-fold increase at wk 10) and plasminogen activator, tissue (Plat, 48.5-fold increase at wk 5), myelocytomatosis oncogene (Myc, 3.0-fold increase at wk 5), and phospholipase A2, group IIA (platelets, synovial fluid) (Plscr2, 8.0-fold increase at wk 10). The notable down-regulated genes in the colonic mucosa of mice treated with AOM/DSS were the peroxisome proliferator activated receptor binding protein (Pparbp, 0.06-fold decrease at wk 10) and the transforming growth factor, beta 3 (Tgfb3, 0.14-fold decrease at wk 10). The inflammation-related gene, peroxisome proliferator activated receptor gamma (Ppargamma 0.38-fold decrease at wk 5), was also down-regulated in the colonic mucosa of mice that received AOM/DSS.

CONCLUSIONS

This is the first report describing global gene expression analysis of an AOM/DSS-induced mouse colon carcinogenesis model, and our findings provide new insights into the mechanisms of inflammation-related colon carcinogenesis and the establishment of novel therapies and preventative strategies against carcinogenesis.

Purified human leukocyte interferon produced by recombinant techniques (IFN-alpha A) was tested in vitro with chemotherapeutic drugs, vinblastine (VLB), vincristine (VCR), vindesine (VDS), vinzolidine (VZL), cis-platinum (PLAT), doxorubicin (DOXO), etoposide (VP-16), and melphalan (MEL). The activity of these agents alone or in combination was tested against various human tumor cell lines, using a modified soft agar clonogenic assay. Three human tumor cell lines (myeloma, RPMI 8226; breast, MCF-7; and colon, WiDR) showed sensitivity to these agents at clinically achievable drug concentrations. Statistically significant synergistic activity against in vitro colony formation was observed with the combination of VLB and IFN-alpha A. An additive or sub-additive effect was usually observed with the other agents tested. Continuous exposure of the 8226 myeloma cell line to both IFN-alpha A and PLAT showed evidence of a more significant potentiation. It is hypothesized that the synergistic effect observed between VLB and IFN-alpha A is due to some of their common mechanisms of action.

'Pure' familial spastic paraplegias (FSP) are neurodegenerative disorders that are clinically characterized by progressive spasticity of the lower limbs and are inherited as autosomal dominant (DFSP) or autosomal recessive (RFSP) traits. The primary defect in FSP is unknown. Genetic linkage analysis was applied to five RFSP families from Tunisia. In four of these five families tight linkage of the RFSP locus was established to the chromosome 8 markers, D8S260, D8S166, D8S285, PLAT, and D8S279. The RFSP locus in the fifth family was not linked to these markers which provided evidence of genetic locus heterogeneity in RFSP. Identification of the RFSP gene on chromosome 8 will help in understanding the genetic factors in motor neuron degeneration.

Laryngeal squamous cell carcinoma is very common in head and neck cancer, with high mortality rates and poor prognosis. In this study, we compared expression profiles of clinical samples from 13 larynx tumors and 10 non-neoplastic larynx tissues using a custom-built cDNA microarray containing 331 probes for 284 genes previously identified by informatics analysis of EST databases as markers of head and neck tumors. Thirty-five genes showed statistically significant differences (SNR>> or = | 1.0 |, p< or =0.001) in the expression between tumor and non-tumor larynx tissue samples. Functional annotation indicated that these genes are involved in cellular processes relevant to the cancer phenotype, such as apoptosis, cell cycle, DNA repair, proteolysis, protease inhibition, signal transduction and transcriptional regulation. Six of the identified transcripts map to intronic regions of protein-coding genes and may comprise non-annotated exons or as yet uncharacterized long ncRNAs with a regulatory role in the gene expression program of larynx tissue. The differential expression of 10 of these genes (ADCY6, AES, AL2SCR3, CRR9, CSTB, DUSP1, MAP3K5, PLAT, UBL1 and ZNF706) was independently confirmed by quantitative real-time RT-PCR. Among these, the CSTB gene product has cysteine protease inhibitor activity that has been associated with an antimetastatic function. Interestingly, CSTB showed a low expression in the tumor samples analyzed (p<0.0001). The set of genes identified here contribute to a better understanding of the molecular basis of larynx cancer, and provide candidate markers for improving diagnosis, prognosis and treatment of this carcinoma.

OBJECTIVE

We undertook a linkage disequilibrium (LD)-based genetic approach to investigate the hypothesis that common sequence variants in 5 thrombosis genes influence plasma hemostatic protein levels or risk of cardiovascular disease (CVD).

RESULTS

In a reference panel, we characterized LD structure at the fibrinogen gene cluster (fibrinogen-beta[FGB], FGA, and FGG), factor VII (F7), and tissue plasminogen activator (PLAT) loci. Forty-one tag single nucleotide polymorphisms (SNPs) were genotyped in 1811 unrelated Framingham Heart Study participants. There were significant associations of 9 FGB SNPs with fibrinogen level (minimum P=0.002) and of 7 F7 SNPs and factor VII level (minimum P<0.0001). SNPs at the PLAT locus were not associated with PLAT level. In stepwise analysis, a single FGB variant explained 1% of the residual variance in fibrinogen level, and 2 F7 SNPs together explained 10% of the residual variance in factor VII level. Two PLAT haplotypes were associated with CVD (multivariable-adjusted global P=0.0004).

CONCLUSIONS

A comprehensive survey of common sequence variation demonstrates that cis-regulatory SNPs explain a modest proportion of the residual variance in circulating fibrinogen and factor VII level and PLAT haplotypes increase the risk of CVD. Additional studies are warranted to confirm the association of PLAT sequence variation and risk of CVD.

The common genes responsible for the characteristics of primary cultured invasive phenotype hepatocellular carcinoma (HCC) cells were investigated. Primary cultured HCC cells from three patients were separated by Matrigel invasion into parent and invasive cells. Whole human genome oligo microarray was applied to detect the differentially expressed genes in invasive cells. A purchased HCC cell line (HA 22T/VGH) was studied for comparison. Forty genes were consistently up-regulated and 14 genes were consistently down-regulated among primary cultured invasive cells. Among these genes, only three up-regulated genes (CNN1, PLAT, SPARC) and one down-regulated tumor suppressor gene (MDFI) had same expressions in invasive cells originated from purchased cell line. For primary cultured invasive cells, differential expressions of several groups of common genes are known to have capacities to promote proliferation (CAV1, IL6, PLAT, RRAD, SRPX), remodeling of extracellular matrix (COL1A1, COL1A2, NID2, TNC, RELN, SPARC), migration (ACTG2, CAV1, CCL2, CCL26, CDC42EP3, CNN1, PHLDB2, PLAT, RRAD, SRPX), implantation (IL6), immune escape (CD70) and angiogenesis (CCL2, IL6, IL18, PLAT, SLIT3). Two genes related to signal transduction (AXL, RASL10B) and one related to metabolism (PTGS2) also showed consistent expressions. Differential expressions of these genes are capable for tumor invasiveness. In conclusion, the characteristics of invasive phenotype HCC cells are originated from differential expressions of several groups of genes rather than few target genes. This information may give us a new insight to design new stratagems in HCC treatment. Analysis of the results from a purchased cell line may have bias due to long-term repeated in vitro cultures.

The thrombogenic mechanism of arterial grafts has been studied by determining the relative utilization of platelets, fibrinogen and plasminogen by human arterial prostheses, and by direct examination of arterial grafts in a baboon model. Forty-one survival and turnover measurements of (51)Crplatelets, (131)I-fibrinogen and (125)I-plasminogen in ten patients with aortofemoral knitted Dacron prostheses demonstrated platelet consumption after graft placement (platelet survival 4.2 days +/- 0.5 and turnover 68,000 plat/ul/day +/-10,000 compared with 8.2 days +/- 0.3 and 35,000 plat/ul/day +/- 5,000 respectively for control subjects with stable vascular disease, p < 0.01). In vitro platelet function test results were normal. Platelet consumption was interrupted by dipyridamole or a combination of dipyridamole and acetylsalicylic acid, and platelet survival normalized spontaneously during nine months postoperatively. No significantly increased consumption of fibrinogen or plasminogen was found in these patients with arterial grafts. Placement of impervious knitted Dacron velour aortic grafts in baboons reproduced platelet consumption that progressively normalized over six weeks postoperatively. Platelet survival measurements correlated directly with endothelial cell coverage of the graft luminal surface in these animals implying that endothelialization of the graft surface was also occurring postoperatively in patients.

OBJECTIVE

To compare gene expression profiles of pancreatic adenocarcinoma tissue specimens, human pancreatic and colon adenocarcinoma and leukemia cell lines and normal pancreas samples in order to distinguish differentially expressed genes and to validate the differential expression of a subset of genes by quantitative real-time RT-PCR (RT-QPCR) in endoscopic ultrasound-guided fine needle aspiration (EUS-guided FNA) specimens.

METHODS

Commercially dedicated cancer cDNA macroarrays (Atlas Human Cancer 1.2) containing 1176 genes were used. Different statistical approaches (hierarchical clustering, principal component analysis (PCA) and SAM) were used to analyze the expression data. RT-QPCR and immunohistochemical studies were used for validation of results.

RESULTS

RT-QPCR validated the increased expression of LCN2 (lipocalin 2) and for the first time PLAT (tissue-type plasminogen activator or tPA) in malignant pancreas as compared with normal pancreas. Immunohistochemical analysis confirmed the increased expression of LCN2 protein localized in epithelial cells of ducts invaded by carcinoma. The analysis of PLAT and LCN2 transcripts in 12 samples obtained through EUS-guided FNA from patients with pancreatic adenocarcinoma showed significantly increased expression levels in comparison with those found in normal tissues, indicating that a sufficient amount of high quality RNA can be obtained with this technique.

CONCLUSIONS

Expression profiling is a useful method to identify biomarkers and potential target genes. Molecular analysis of EUS-guided FNA samples in pancreatic cancer appears as a valuable strategy for the diagnosis of pancreatic adenocarcinomas.

Many intriguing facets of lipoxygenase (LOX) catalysis are open to a detailed structural analysis. Polyunsaturated fatty acids with two to six double bonds are oxygenated precisely on a particular carbon, typically forming a single chiral fatty acid hydroperoxide product. Molecular oxygen is not bound or liganded during catalysis, yet it is directed precisely to one position and one stereo configuration on the reacting fatty acid. The transformations proceed upon exposure of substrate to enzyme in the presence of O2 (RH + O2 → ROOH), so it has proved challenging to capture the precise mode of substrate binding in the LOX active site. Beginning with crystal structures with bound inhibitors or surrogate substrates, and most recently arachidonic acid bound under anaerobic conditions, a picture is consolidating of catalysis in a U-shaped fatty acid binding channel in which individual LOX enzymes use distinct amino acids to control the head-to-tail orientation of the fatty acid and register of the selected pentadiene opposite the non-heme iron, suitably positioned for the initial stereoselective hydrogen abstraction and subsequent reaction with O2 . Drawing on the crystal structures available currently, this review features the roles of the N-terminal β-barrel (C2-like, or PLAT domain) in substrate acquisition and sensitivity to cellular calcium, and the α-helical catalytic domain in fatty acid binding and reactions with O2 that produce hydroperoxide products with regio and stereospecificity. LOX structures combine to explain how similar enzymes with conserved catalytic machinery differ in product, but not substrate, specificities.

Glioma-initiating cells (GIC) have stem-like cell properties thought to be sufficient for recurrence, progression, and drug resistance in glioblastomas. In the present study, we defined miRNA (miR)-340 as a differentially expressed miRNA in human GICs that inhibit GIC-mediated tumorigenesis. Furthermore, we defined tissue plasminogen activator (PLAT) as a critical direct target of miR340 for inhibition. Among miRNAs screened, we found that miR340 expression was decreased in all human GICs and in human glioblastoma tissues, compared with human neural stem cells and normal brain tissues. miR340 overexpression in GICs suppressed their proliferative, invasive, and migratory properties in vitro, triggering cell senescence in vitro and inhibiting GIC-induced tumorigenesis in mouse brains. shRNA-mediated silencing of PLAT in GICs phenocopied the effects of miR340 overexpression in vitro and in vivo, suggesting a potential role for tissue factor in stem-like cell function. Taken together, our results identified miR340 as a tumor suppressor that functions in GIC to enforce PLAT blockade and ablate their stem-like functions.

BACKGROUND

Predicting mortality has become a necessary step for selecting patients for clinical trials and defining outcomes. We examined whether stratification by tertiles of respiratory and ventilatory variables at the onset of acute respiratory distress syndrome (ARDS) identifies patients with different risks of death in the intensive care unit.

METHODS

We performed a secondary analysis of data from 220 patients included in 2 multicenter prospective independent trials of ARDS patients mechanically ventilated with a lung-protective strategy. Using demographic, pulmonary, and ventilation data collected at ARDS onset, we derived and validated a simple prediction model based on a population-based stratification of variable values into low, middle, and high tertiles. The derivation cohort included 170 patients (all from one trial) and the validation cohort included 50 patients (all from a second trial).

RESULTS

Tertile distribution for age, plateau airway pressure (P(plat)), and P(aO(2))/F(IO(2)) at ARDS onset identified subgroups with different mortalities, particularly for the highest-risk tertiles: age >> 62 years), P(plat) >> 29 cm H(2)O), and P(aO(2))/F(IO(2)) (< 112 mm Hg). Risk was defined by the number of coexisting high-risk tertiles: patients with no high-risk tertiles had a mortality of 12%, whereas patients with 3 high-risk tertiles had 90% mortality (P < .001).

CONCLUSIONS

A prediction model based on tertiles of patient age, P(plat), and P(aO(2))/F(IO(2)) at the time the patient meets ARDS criteria identifies patients with the lowest and highest risk of intensive care unit death.

Caenorhabditis elegans is a powerful model to study the molecular basis of autosomal dominant polycystic kidney disease (ADPKD). ADPKD is caused by mutations in the polycystic kidney disease (PKD)1 or PKD2 gene, encoding polycystin (PC)-1 or PC-2, respectively. The C. elegans polycystins LOV-1 and PKD-2 are required for male mating behaviors and are localized to sensory cilia. The function of the evolutionarily conserved polycystin/lipoxygenase/alpha-toxin (PLAT) domain found in all PC-1 family members remains an enigma. Here, we report that ATP-2, the beta subunit of the ATP synthase, physically associates with the LOV-1 PLAT domain and that this interaction is evolutionarily conserved. In addition to the expected mitochondria localization, ATP-2 and other ATP synthase components colocalize with LOV-1 and PKD-2 in cilia. Disrupting the function of the ATP synthase or overexpression of atp-2 results in a male mating behavior defect. We further show that atp-2, lov-1, and pkd-2 act in the same molecular pathway. We propose that the ciliary localized ATP synthase may play a previously unsuspected role in polycystin signaling.

We assessed feasibility and safety of extracorporeal carbon dioxide removal (ECCO₂R) to facilitate ultra-protective ventilation (V_T 4 mL/kg and P_PLAT ≤ 25 cmH₂O) in patients with moderate acute respiratory distress syndrome (ARDS).

Prospective multicenter international phase 2 study. Primary endpoint was the proportion of patients achieving ultra-protective ventilation with PaCO₂ not increasing more than 20% from baseline, and arterial pH > 7.30. Severe adverse events (SAE) and ECCO₂R-related adverse events (ECCO₂R-AE) were reported to an independent data and safety monitoring board. We used lower CO₂ extraction and higher CO₂ extraction devices (membrane lung cross-sectional area 0.59 vs. 1.30 m²; flow 300-500 mL/min vs. 800-1000 mL/min, respectively).

Ninety-five patients were enrolled. The proportion of patients who achieved ultra-protective settings by 8 h and 24 h was 78% (74 out of 95 patients; 95% confidence interval 68-89%) and 82% (78 out of 95 patients; 95% confidence interval 76-88%), respectively. ECCO₂R was maintained for 5 [3-8] days. Six SAEs were reported; two of them were attributed to ECCO₂R (brain hemorrhage and pneumothorax). ECCO₂R-AEs were reported in 39% of the patients. A total of 69 patients (73%) were alive at day 28. Fifty-nine patients (62%) were alive at hospital discharge.

Use of ECCO₂R to facilitate ultra-protective ventilation was feasible. A randomized clinical trial is required to assess the overall benefits and harms. CLINICALTRIALS.GOV: NCT02282657.

Mutations in genes encoding polycystin-1 (PC1) and polycystin-2 cause autosomal dominant polycystic kidney disease. The polycystin protein family is composed of Ca2+-permeable pore-forming subunits and receptor-like integral membrane proteins. Here we describe a novel member of the polycystin-1-like subfamily, polycystin-1L2 (PC1L2), encoded by PKD1L2, which has various alternative splicing forms with two translation initiation sites. PC1L2 short form starts in exon 12 of the long form. The longest open reading frame of PKD1L2 short form, determined from human testis cDNA, encodes a 1775-amino-acid protein and 32 exons, whereas the long form is predicted to encode a 2460-residue protein. Both forms have a small receptor for egg jelly domain, a G-protein-coupled receptor proteolytic site, an LH2/PLAT, and 11 putative transmembrane domains, as well as a number of rhodopsin-like G-protein-coupled receptor signatures. RT-PCR analysis shows that the short form, but not the long form, of human PKD1L2 is expressed in the developing and adult heart and kidney. Furthermore, by GST pull-down assay we observed that PC1L2 and polycystin-1L1 are able to bind to specific G-protein subunits. We also show that PC1 C-terminal cytosolic domain binds to Galpha12, Galphas, and Galphai1, while it weakly interacts with Galphai2. Our results indicate that both PC1-like molecules may act as G-protein-coupled receptors.

Combining different approaches (resequencing of portions of 54 obesity candidate genes, literature mining for pig markers associated with fat deposition or related traits in 77 genes, and in silico mining of porcine expressed sequence tags and other sequences available in databases), we identified and analyzed 736 SNP within candidate genes to identify markers associated with back fat thickness (BFT) in Italian Large White sows. Animals were chosen using a selective genotyping approach according to their EBV for BFT (276 with most negative and 279 with most positive EBV) within a population of ≈ 12,000 pigs. Association analysis between the SNP and BFT has been carried out using the MAX test proposed for case-control studies. The designed assays were successful for 656 SNP: 370 were excluded (low call rate or minor allele frequency <5%), whereas the remaining 286 in 212 genes were taken for subsequent analyses, among which 64 showed a P(nominal) value <0.1. To deal with the multiple testing problem in a candidate gene approach, we applied the proportion of false positives (PFP) method. Thirty-eight SNP were significant (P(PFP) < 0.20). The most significant SNP was the IGF2 intron3-g.3072G>A polymorphism (P(nominal) < 1.0E-50). The second most significant SNP was the MC4R c.1426A>G polymorphism (P(nominal) = 8.0E-05). The third top SNP (P(nominal) = 6.2E-04) was the intronic TBC1D1 g.219G>A polymorphic site, in agreement with our previous results obtained in an independent study. The list of significant markers also included SNP in additional genes (ABHD16A, ABHD5, ACP2, ALMS1, APOA2, ATP1A2, CALR, COL14A1, CTSF, DARS, DECR1, ENPP1, ESR1, GH1, GHRL, GNMT, IKBKB, JAK3, MTTP, NFKBIA, NT5E, PLAT, PPARG, PPP2R5D, PRLR, RRAGD, RFC2, SDHD, SERPINF1, UBE2H, VCAM1, and WAT). Functional relationships between genes were obtained using the Ingenuity Pathway Analysis (IPA) Knowledge Base. The top scoring pathway included 19 genes with a P(nominal) < 0.1, 2 of which (IKBKB and NFKBIA) are involved in the hypothalamic IKKβ/NFκB program that could represent a key axis to affect fat deposition traits in pigs. These results represent a starting point to plan marker-assisted selection in Italian Large White nuclei for BFT. Because of similarities between humans and pigs, this study might also provide useful clues to investigate genetic factors affecting human obesity.

We have established a 67-cM molecular genetic linkage map of mouse chromosome 8 by interspecific backcross analysis. Genes that were mapped in this study include Act-6, Aprt, Aprt-ps1, Emv-2, Es-N, Hp, Insr, Mt-1, Plat, Psx-8, Ucp, and Zfp-4. New regions of homology were established between mouse chromosome 8 and human chromosomes 8 and 19. A conserved linkage group was identified between mouse chromosome 8 and human chromosome 16. The map will be useful for establishing linkage of other markers to mouse chromosome 8.

Human platelet 12-lipoxygenase (hp-12LOX, 662 residues+iron nonheme cofactor) and its major metabolite 12S-hydroxyeicosatetraenoic acid have been implicated in cardiovascular and renal diseases, many types of cancer and inflammatory responses. However, drug development is slow due to a lack of structural information. The major hurdle in obtaining a high-resolution X-ray structure is growing crystals, a process that requires the preparation of highly homogenous, reproducible and stable protein samples. To understand the properties of hp-12LOX, we have expressed and studied the behavior, function and low-resolution structure of the hp-12LOX His-tagged recombinant enzyme and its mutants in solution. We have found that it is a dimer easily converted into bigger aggregates, which are soluble/covalent-noncovalent/reversible. The heavier oligomers show a higher activity at pH 8, in contrast to dimers with lower activity showing two maxima at pH 7 and pH 8, indicating the existence of two different conformers. In the seven-point C->>S mutant, aggregation is diminished, activity has one broad peak at pH 8 and there is no change in specificity. Truncation of the N(t)-beta-barrel domain (PLAT, residues 1-116) reduces activity to approximately 20% of that shown by the whole enzyme, does not affect regio- or stereospecificity and lowers membrane binding by a factor of approximately 2. "NoPLAT" mutants show strong aggregation into oligomers containing six or more catalytic domains regardless of the status of the seven cysteine residues tested. Time-of-flight mass spectrometry suggests two arachidonic acid molecules bound to one molecule of enzyme. Small angle X-ray scattering studies (16 A resolution, chi approximately 1) suggest that two hp-12LOX monomers are joined by the catalytic domains, with the PLAT domains floating on the flexible linkers away from the main body of the dimer.

Anemia associated with advanced cancer is common. Contributing factors include the anemia of chronic disease, chemotherapy, radiation therapy, and bone marrow invasion with tumor. Based on the observation that endogenous erythropoietin (EPO) levels in anemic patients with cancer are inadequate for the degree of anemia, three randomized double-blind, placebo-controlled trials of recombinant human erythropoietin (rHuEPO) treatment in anemic patients with cancer were performed in patients (1) not receiving concomitant chemotherapy (NO CTX), (2) receiving myelosuppressive chemotherapy that did not include cisplatin (CTX-NO PLAT), and (3) receiving myelosuppressive cisplatin-containing chemotherapy (CTX-PLAT). In the NO CTX trial, patients were treated with rHuEPO 100 U/kg or placebo subcutaneously (SQ) three times per week for up to 8 weeks. In the CTX trials, patients were treated with rHuEPO 150 U/kg or placebo SQ three times per week for 12 weeks. Four hundred thirteen patients were enrolled (124, NO CTX; 157, CTX-NO PLAT; and 132, CTX-PLAT). In all three trials, patients receiving rHuEPO had a significantly (P < .004) greater increase in hematocrit (HCT) than placebo-treated patients. In the two CTX trials combined, rHuEPO-treated patients also had a significantly (P < or = .009) lower transfusion requirement than placebo-treated patients after the first month of therapy. Quality of life improved significantly (P < .05) in responding >> or = 6%-point HCT increase without transfusion) rHuEPO-treated patients compared with placebo-treated patients. Overall, no adverse events occurred more frequently in rHuEPO-treated patients compared with placebo-treated patients. Following completion of the double-blind phase, patients received rHuEPO on an open-label basis as needed for correction of anemia with the dose titrated to a maximum of 900 U/kg/wk. During total rHuEPO exposure (either started at the beginning of double-blind therapy for patients initially randomized to rHuEPO or at the beginning of open-label therapy for patients initially randomized to placebo; 363 treated/347 evaluable for efficacy), 40.0%, 56.1%, and 58.3% of the NO-CTX, CTX-NO PLAT, and CTX-PLAT patients, respectively, responded to rHuEPO therapy with an increase of HCT>> or = 6% unrelated to transfusion.(ABSTRACT TRUNCATED AT 400 WORDS)

Mutations in two genes, PKD1 and PKD2, account for most cases of autosomal dominant polycystic kidney disease, one of the most common monogenetic disorders. Here we report the 3.6-angstrom cryo-electron microscopy structure of truncated human PKD1-PKD2 complex assembled in a 1:3 ratio. PKD1 contains a voltage-gated ion channel (VGIC) fold that interacts with PKD2 to form the domain-swapped, yet noncanonical, transient receptor potential (TRP) channel architecture. The S6 helix in PKD1 is broken in the middle, with the extracellular half, S6a, resembling pore helix 1 in a typical TRP channel. Three positively charged, cavity-facing residues on S6b may block cation permeation. In addition to the VGIC, a five-transmembrane helix domain and a cytosolic PLAT domain were resolved in PKD1. The PKD1-PKD2 complex structure establishes a framework for dissecting the function and disease mechanisms of the PKD proteins.