A detailed deletion mapping of a series of human prostatic carcinomas, using restriction fragment length polymorphism (RFLP) analysis of all chromosomes, showed allelic losses on individual chromosomes at variable frequencies. Allelic losses occurred on chromosomal arms 8p, 10pq, 16q and 18q in more than 30% of the cases. Losses of genetic information from one or two of the chromosomes 8, 10, or 16 were always present in tumors showing allelic losses, indicating that genes on these chromosomes have a central role in prostatic cancer. A more extensive study of these chromosomes was thus carried out and showed the highest frequency of allelic deletions to occur on the short arm of chromosome 8 (65%) (where the minimally deleted region was between the PLAT locus and pter). The long arm of chromosome 16 had allelic deletions in 56% of informative cases, with three different break points (the most distal being between D16S4 and D16S7). Chromosome 10 exhibited a complex deletion pattern, showing allelic losses from both the short (p) and the long (q) arms, evidence of non-reciprocal translocations of the q arm, and monosomy in some cases. Our data indicate that tumor suppressor genes involved in the oncogenesis of prostatic carcinoma may be localized between 8pter and the PLAT locus and that additional/alternative tumor suppressor gamma are likely to be localized on chromosome 10 and on the long arm of chromosome 16. More aggressive tumors were accompanied by a high or frequency of allelic losses.

The chromosomal positions of three genes that are selectively expressed in mouse testis cells have been identified. These genes include (i) TAZ83, which codes for an early- to mid-pachytene germ cell stage-expressed, cysteine-rich transmembrane protein (cyritestin) with homologies to various snake toxins and guinea pig sperm-egg fusion proteins; (ii) TNZ1, which is expressed in neonatal Leydig cells; and (iii) TAZ4, a testis-specific gene isolated by immunoscreening with antiserum raised against Sertoli cell membranes. Our experimental data, derived from chromosomal in situ hybridizations and RFLP studies of genetic backcrosses, indicate that (i) the TAZ83 (cyritestin) gene maps to chromosome 8, band A2, near the Plat locus; (ii) TNZ1 is located in the proximal region of chromosome 11; and (iii) TAZ4 is located at band D in the distal portion of chromosome 11, near the Hlr1 locus, with a related sequence, TAZ4-rs1, in the proximal part of chromosome 1.

The aim of the present study was to explore the association between paraganglioma-like adenomas of the thyroid (PLAT) and chronic lymphocytic thyroiditis (CLT) or Hashimoto's thyroiditis, and to discuss its possible relationship to papillary thyroid carcinoma. Six cases were analyzed by standard histopathological and immunohistochemical techniques. All 6 cases (all females) had clinical and/or histological evidence of CLT. Only one patient had 2 PLATs. The PLATs were devoid of inflammation and sharply demarcated from the surrounding CLT. There is cyto-morphological overlap with papillary thyroid carcinoma (nuclear grooves and pseudo-inclusions). We conclude that PLAT is associated with CLT more frequently than any other thyroid lesion, and feel that this is more than merely a chance association. PLAT shares several cytological features with papillary carcinoma and cases have been seen where they have occurred in the same thyroid. PLAT could therefore represent an unusual variant of papillary carcinoma.

BACKGROUND

The serum kinetics of carcinoembryonic antigen (CEA) after resection of lung carcinoma are not well characterized. Its prognostic implications remain unclear. This study was designed to clarify the correlation between postoperative CEA time-course and patient prognosis.

METHODS

The authors analyzed early postoperative CEA time-course in 31 lung carcinoma patients using nonlinear least square analysis with the following three equations: Equation 1: C(t) = C(0); Equation 2: C(t) = C(0)exp(-kt); and Equation 3: C(t) (C(0)PLAT)exp(-kt) + PLAT, in which t: postoperative day; C(t): postoperative CEA; PLAT: postoperative CEA at plateau; C(0): CEA at postoperative Day zero; and k: rate constant of elimination. The equation that yielded the least Akaike's information criterion was adopted as the best fitting regression equation for each patient. When Equation 3 was adopted, postoperative CEA production (PROD) was calculated as PLAT multiplied by k.

RESULTS

Equations 1, 2, and 3 were adopted for 16 (Group 1), 0, and 15 (Group 2) patients, respectively. In Group 1, no decrease in serum CEA level after surgery was detected and CEA production appears to have been little or none. In Group 2, biologic CEA half-life was 1.1 +/- 0.7 days and was not useful for predicting patient prognosis. Tumor recurrences were observed in 9 of the Group 2 patients 19 +/- 9 months postoperatively and there was no significant difference in PLAT or PROD between patients with and without recurrence. Early recurrence within 6 months after surgery was recognized in 5 (early-REC) of the 15 Group 2 patients, in whom there was a tendency for PLAT to be higher than in the other 10 patients without recurrence (early-NON) (early-REC: 3.8 +/- 4.9 ng/mL; early-NON: 1.5 +/- 1.1 ng/mL; P = 0.08). PROD was significantly higher in early-REC than in early-NON (early-REC: 3.5 +/- 4.2 ng/mL/day; early-NON: 0.8 +/- 0.4 ng/mL/day; P < 0.05).

CONCLUSIONS

Although PROD is more sensitive than PLAT, both parameters appear to be useful as prognostic tools for predicting early recurrence after resection of lung carcinoma. This is probably because they represent the number of residual tumor cells immediately after surgery.

OBJECTIVE

To evaluate and compare coagulation variables following the administration of oxypolygelatin and dextran 70 to clinically healthy dogs.

METHODS

Randomized cross-over experimental study.

METHODS

A total of eight healthy adult female Beagles aged 2-4 years old and weighing 11.8 +/- 2.7 kg.

METHODS

The dogs received a 15-minute intravenous (IV) infusion of 5 mL kg-1 oxypolygelatin or 10 mL kg-1 6% dextran 70. Before (PRE) and at 2, 5, and 24 hours after administration, packed cell volume (PCV), total solids concentration (TS), prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen concentration (FIB), platelet numbers (Plat), factor VIII coagulant activity (VIII:C), von Willebrand factor antigen concentration (vWf:Ag) and platelet function and buccal mucosal bleeding time (BMBT) were measured. Platelet function was assessed using aggregation and by measuring ATP release from aggregating platelets over 6 minutes, with 20, 10, and 5 micro m ADP and 5 and 10 micro g of collagen mL-1 as platelet activation agonists.

RESULTS

All baseline values were within our normal ranges, except for one dog that had low vWf:Ag PRE values prior to both dextran and oxypolygelatin administration. Following dextran and oxypolygelatin administration, the PCV and TP were significantly (p < 0.05) decreased. Plat, FIB, and vWf:Ag decreased, while BMBT and VIII:C increased following dextran administration. Dextran also caused a significant decrease in platelet aggregation in response to ADP. Oxypolygelatin caused a significant decrease in vWf:Ag, Plat, and FIB compared to PRE values. The total amount of ATP released, standardized to platelet number, did not vary significantly for either group at any sampling time from PRE values. No significant changes from PRE values were noted at any time in either group for PT or APTT.

CONCLUSIONS

At the doses administered, both dextran and oxypolygelatin can interfere with hemostatic variables in healthy dogs, but dextran's effect is more profound and prolonged when compared to oxypolygelatin.

CONCLUSIONS

Oxypolygelatin causes fewer hemostatic abnormalities when compared to dextran, making it a superior colloid for administration at the doses tested.

Synchrotrons are opening new paths in innovative anti-cancer radiotherapy strategies. Indeed, the fluence of X-rays induced by synchrotrons is so high (10(6) times higher than standard medical irradiators) that it enables the production of X-ray beams tunable in energy (monochromatic beams) and in size (micrometric beams). Monochromatic synchrotron X-ray beams theoretically permit photoactivate high-Z elements to be introduced in or close to tumours in order to increase the yield of damage by enhanced energy photoabsorption. This is notably the case of attempts with iodinated contrast agents used in tumour imaging (the computed tomography therapy approach) and with platinated agents used in chemotherapy (the PAT-Plat approach). Micrometric synchrotron X-ray beams theoretically permit very high radiation doses to accumulate in tumours by using arrays of parallel microplanar beams that spare the surrounding tissues (the microbeam radiation therapy approach). These anti-cancer applications of synchrotron radiation have been developed at the European Synchrotron Radiation Facility to be applied to glioma, one of the tumour tissues most refractory to standard treatments. In the present paper the molecular and cellular mechanisms involved in these three approaches are reviewed, in the context of recent advances in radiobiology. Furthermore, by considering the unavoidable biases, an attempt to propose a comparison of the different results obtained in preclinical trials dealing with rats bearing tumours is given.

Although clinical observations have shown that estrogen receptor-positive (ER+) breast tumors are more responsive to hormonal therapy than ER-negative (ER-) tumors, it remains controversial whether ER status can predict chemotherapeutic response. To determine if there was any correlation between estrogen and progesterone values and in vitro chemosensitivity to various anticancer drugs, clonogenic (CA), estrogen (ERA), and progesterone (PRA) assays on breast cancers were performed on 100 patients. Clonogenic assays were performed using the double-layer soft agar technique with continuous drug exposures. ERAs and PRAs were performed using the charcoal-coated dextran method. Chemosensitivity was defined as 50% inhibition of colony formation. ERA was considered positive if greater than or equal to 5 fmole/mg cytosol and PRA positive if greater than or equal to 10 fmole/mg cytosol. Significant tumor growth (greater than 30 colonies/plate) was achieved in 81/100 assays. ERA and PRA values were not predictive of colony formation in vitro. Of all agents or combinations of agents tested (L-PAM, 5-FU, MTX, adriamycin, vinblastine, cis-plat, FAC, CMF), only the response to 5-FU correlated significantly with ERA. Eight of 11 (73%) of the ER- tumors were sensitive to 5-FU, whereas only 6/20 (30%) of ER+ tumors were sensitive (P less than 0.05). ER- tumors were also more likely to be sensitive to CMF (P = 0.09) and adriamycin (P = 0.07) than ER+ tumors. PRA values were not predictive of chemosensitivity, nor did combining PRA and ERA enhance the predictive value of ERA alone.

BACKGROUND

Oral candidiasis is an opportunistic infection of the oral cavity which usually occurs in the immunocompromised individuals. Candida albicans (C. albicans) is the most common species of yeast responsible for oral candidiasis. This study investigated the effects of Satureja hortensis L. essentiall oil (EO) on the planktonic, biofilm formation and mature biofilms of C. albicans isolates from buccal lesions of HIV(+) individuals.

METHODS

MTT reduction assay, broth micro-dilution method and scanning electron microscopy (SEM) were employed to determine the effect of mentioned EO on the C. albicans planktonic and biofilm forms. GC-GC/MS was used to detect the major active compounds of EO.

RESULTS

Thymol (45.9%), gamma-terpinen (16.71%), carvacrol (12.81%) and p-cymene (9.61%) were found as the most abundant constituents. MIC values ranged from 250 to 400 μg/ml and MFC values ranged from 350 to 500 μg/ml. All C. albicans isolates formed biofilm on polystyrene plats but the quantity of biofilm mass (optical density) was different for the isolates ranging from 0.850 to 0.559 nm. The mean of biofilm formation by C. albicans isolates was reduced by 87.1 ± 3.7%, 73.6 ± 5.1%, 69.4 ± 5.3% and 67 ± 4.2% at 4800, 3200, 2400 and 1600 μg/ml, respectively. In sub-MIC concentration, SEM analysis revealed loosening of cells, deformity of three dimensional structures of biofilms and shrinkage in cell membranes of sessile cells.

CONCLUSIONS

In conclusion, the substantial anti-fungal activity showed by S. hortensis L. EO suggests exploitation of this oil as potential natural anti-biofilm product to deal with the problem of buccal cavity lesion associated with C. albicans.

A low anaerobic power has been proposed as a factor that may be limiting the achievement of a plateau in VO2 of children who perform maximal aerobic power tests. This study examined the frequency of plateau achievement in pre-pubertal children and compared VO2max, peak (PP) and mean (MP) anaerobic power in subjects who either achieved a plateau (PLAT) or did not (NO PLAT). Eighteen healthy pre-pubertal (Tanner Stage, pubic hair = 1) males (age = 9.1 1.6 yrs, ht = 134.4 +/- 9.7cm, wt = 33.3 +/- 9.2kg, VO2max = 40.0 +/- 6.7 ml x kg(-1) min(-1)) were tested. All subjects completed a 30 sec Wingate Anaerobic Test and a McMaster aerobic protocol to volitional fatigue on a cycle ergometer. Only 33% of the subjects met the PLAT criterion. No differences were found for PP or MP between those who achieved a plateau and those who did not (PLAT: PP= 6.3 +/- 0.8W/kg and MP = 5.2 +/- 0.7W/kg; NO PLAT: PP= 6.3 +/- 1.2 W/kg and MP = 5.2 +/- 1.3 W/kg). We conclude that anaerobic power is not a factor limiting the achievement of a plateau in VO2 of pre-pubertal boys who perform maximal aerobic power tests.

Brucellosis is a zoonosis of great and worldwide public health concern that can cause a severe febrile illness in humans. In Pakistan, brucellosis is a critical problem in both animals and humans. This study aimed to gain insight into its prevalence and to analyze the potential risk factors of patients with acute febrile illness (AFI) of an unknown cause, at the hospitals of Rawalpindi and Islamabad in Pakistan. In total, 446 blood samples were collected from patients and screened for brucellosis using the Rose Bengal Plat Test (RBPT). All the serum samples were investigated for Brucella DNA using specific real-time PCR. Age, sex, occupation, urbanicity, socioeconomic status and history of animal contact were recorded and assessed as potential risk factors. The proportion of acute febrile illness patients for whom brucellosis could be suspected was 10.1% by the RBPT. Brucella DNA was detected in 26 (5.8%) cases and identified as B. abortus. Contact with infected animals, consumption of raw milk and socioeconomic status showed a highly significant (p ˂ 0.05) correlation with seropositivity. Elderly patients (19.7% RBPT and 12.1% PCR) and females (13% RBPT and 9.3% PCR) were of high risk of brucellosis. Patients suffering from brucellosis-related manifestations should be screened for brucellosis, especially those in contact with animals or those consuming their unprocessed products, given the increased risk. The results of this study, which highlight that Brucella abortus as an important cause of acute febrile illnesses in humans, aid the development of effective control strategies for human brucellosis in Pakistan.

Tyrosine kinase inhibitors (TKIs), such as gefitinib, are widely used as standard treatments for non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations. However, the subsequent inevitable drug resistance has become a major challenge in clinical treatment. The aim of this study was to investigate the role of tissue-type plasminogen activator (PLAT) in gefitinib resistance in NSCLC.

The function of PLAT was determined using gefitinib-resistant cells and a nude mouse model. The gene knockdown was achieved by Lentivirus based RNA silence technique. Expression of relevant genes and proteins, cell viability, proliferation, apoptosis, cell cycle, reactive oxygen species levels, mitochondrial membrane potential and differential gene expression was detected by RT-qPCR, western blot, cell counting kit-8 assay, EdU incorporation, flow cytometry, JC-1 dye assay and complementary DNA arrays. The effects of PLAT knockdown on tumorigenesis was analyzed in vivo.

Gefitinib-resistant cells expressed higher levels of PLAT and that knockdown of PLAT in resistant cells restored gefitinib sensitivity. Tumor proliferation was limited in vivo following PLAT knockdown. Moreover, PLAT knockdown affected mitochondrial function, caused caspase activation and cell cycle arrest, and activated TNF-α signaling, leading to apoptosis of gefitinib-resistant PC9 cells.

Our results suggest that PLAT reduces apoptosis of NSCLC cells and knockdown of PLAT enhances anticancer effect of gefitinib by upregulating TNF-α signaling.

Adrenal pheochromocytoma (PCC) is a very rare tumor that stems from chromaffin cells, which can develop into malignant tumor. During the operation, abundant blood vessels were often observed in PCC than other adrenal tumors, which increases the difficulty and risk of the surgery. Therefore, it is important to investigate the mechanism of PCC angiogenesis. Twelve surgical specimens of PCC from Ruijin Hospital, Shanghai Jiaotong University were grouped into high and low microvessel density (MVD) group. They were also divided into rich blood supply and nonenriched blood supply group, according to computed tomography (CT) manifestation. Comparative proteomic analysis based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatics analysis revealed that 206 proteins differentially regulated in the high MVD group compared with low MVD group (p < 0.05). Besides, 61 proteins were discovered to be significantly changed when the 12 samples were grouped according to CT manifestation. By intersecting the differentially changed protein from MVD and CT grouping, 25 proteins were filtered out, with pathological function. COX4I2 was verified to be increased gradually with angiogenesis with increasing severity, and PLAT was shown to be decreased with angiogenesis in PCC, by quantitative reverse-transcription polymerase chain reaction and immunohistochemistry. The quantitative proteomics result indicated that the tumor angiogenesis in PCC is associated with hypoxia. COX4I2 and PLAT were highly correlated with blood supply in PCC which contribute to angiogenesis in PCC, which could be used as biomarkers to better indicate tumor angiogenesis, or targets to regress tumor angiogenesis as treatment.

BACKGROUND

The pendulum test is commonly used to quantify knee extensor spasticity, but it is currently unknown to what extent common pendulum test metrics can detect spasticity in patients with neurological injury or disease, and if the presence of flexor spasticity influences the test outcomes.

METHODS

A retrospective analysis was conducted on 131 knees, from 93 patients, across four different patient cohorts. Clinical data included Modified Ashworth Scale (MAS) scores for knee extensors and flexors, and years since diagnosis. BioTone™ measures included extensor strength, passive and active range of motion, and pendulum tests of most affected or both knees. Pendulum test metrics included the relaxation index (RI), 1st flexion amplitude (F1amp) and plateau angle (Plat), where RI=F1amp/Plat. Two-way ANOVA tests were used to determine if pendulum test metrics were influenced by the degree of knee flexor spasticity graded by the MAS, and ANCOVA was used to test for confounding effects of age, years since injury, strength and range of motion (ROM). In order to identify the best pendulum test metrics, Receiver Operator Characteristic analysis and logistic regression (LR) analysis were used to classify knees by spasticity status (none or any) and severity (low/moderate or high/severe).

RESULTS

Pendulum test metrics for knee extensors were not influenced by degree of flexor spasticity, age, years since injury, strength or ROM of the limb. RI, F1amp and Plat were>> 70% accurate in classifying knees by presence of clinical spasticity (from the MAS), but were less accurate (< 70%) for grading spasticity level. The best classification accuracy was obtained using F1amp and Plat independently in the model rather than using RI alone.

CONCLUSIONS

We conclude that the pendulum test has good predictive value for detecting the presence of extensor spasticity, independent of the existence of flexor spasticity. However, the ability to grade spasticity level as measured by MAS using the RI and/or F1amp may be limited. Further study is warranted to explore if the pendulum test is suitable for quantifying more severe spasticity.

Essentials C57BL/6J-tissue plasminogen activator (tPA)-deficient mice are widely used to study tPA function. Congenic C57BL/6J-tPA-deficient mice harbor large 129-derived chromosomal segments. The 129-derived chromosomal segments contain gene mutations that may confound data interpretation. Passenger mutation-free isogenic tPA-deficient mice were generated for study of tPA function.

Background The ability to generate defined null mutations in mice revolutionized the analysis of gene function in mammals. However, gene-deficient mice generated by using 129-derived embryonic stem cells may carry large segments of 129 DNA, even when extensively backcrossed to reference strains, such as C57BL/6J, and this may confound interpretation of experiments performed in these mice. Tissue plasminogen activator (tPA), encoded by the PLAT gene, is a fibrinolytic serine protease that is widely expressed in the brain. A number of neurological abnormalities have been reported in tPA-deficient mice. Objectives To study genetic contamination of tPA-deficient mice. Materials and methods Whole genome expression array analysis, RNAseq expression profiling, low- and high-density single nucleotide polymorphism (SNP) analysis, bioinformatics and genome editing were used to analyze gene expression in tPA-deficient mouse brains. Results and conclusions Genes differentially expressed in the brain of Plat(-/-) mice from two independent colonies highly backcrossed onto the C57BL/6J strain clustered near Plat on chromosome 8. SNP analysis attributed this anomaly to about 20 Mbp of DNA flanking Plat being of 129 origin in both strains. Bioinformatic analysis of these 129-derived chromosomal segments identified a significant number of mutations in genes co-segregating with the targeted Plat allele, including several potential null mutations. Using zinc finger nuclease technology, we generated novel 'passenger mutation'-free isogenic C57BL/6J-Plat(-/-) and FVB/NJ-Plat(-/-) mouse strains by introducing an 11 bp deletion into the exon encoding the signal peptide. These novel mouse strains will be a useful community resource for further exploration of tPA function in physiological and pathological processes.

Eukaryotic cells have developed a diverse repertoire of Rab GTPases to regulate vesicle trafficking pathways. Together with their effector proteins, Rabs mediate various aspects of vesicle formation, tethering, docking and fusion, but details of the biological roles elicited by effectors are largely unknown. Human Rab6 is involved in the trafficking of vesicles at the level of Golgi via interactions with numerous effector proteins. We have previously determined the crystal structure of Rab6 in complex with DENND5, alternatively called Rab6IP1, which comprises two RUN domains (RUN1 and RUN2) separated by a PLAT domain. The structure of Rab6/RUN1-PLAT (Rab6/R1P) revealed the molecular basis for Golgi recruitment of DENND5 via the RUN1 domain, but the functional role of the RUN2 domain has not been well characterized. Here we show that a soluble DENND5 construct encompassing the RUN2 domain binds to the N-terminal region of sorting nexin 1 by surface plasmon resonance analyses.

OBJECTIVE

Increases in common carotid intima-media thickness (CC-IMT), as measured by B-mode ultrasonography, have been widely used in both population studies and clinical trials in the search for risk factors for early atherosclerosis progression and have been found to correlate with age and with high concentrations of low-density lipoprotein cholesterol, leukocytes, and hemoglobin. We have now investigated the relation between several baseline hemostatic and conventional risk factors and CC-IMT changes over 16 months in 64 patients with peripheral arterial disease randomly selected from the prospective PLAT study series.

METHODS

Samples from 24 patients (37.5%) who showed increases in CC-IMT during the follow-up period were compared with those from 40 (62.5%) in whom CC-IMT remained unchanged.

RESULTS

Baseline conventional risk factors and coagulation variables were similar in the two groups except for higher plasma concentrations of von Willebrand factor (vWF) (178.3 +/- 53.6% versus 141.2 +/- 53.7%, P=.01) and factor VII (FVII) (133.9 +/- 36.4% versus 107.0 +/- 27.3%, P=.001) in the patients with increased CC-IMT. CC-IMT increase correlated positively with plasma levels of FVII (r=.31, P<.01) and vWF (r=.31, P<.01). Multiple stepwise regression analysis identified FVII as the only independent variable associated with an increase in CC-IMT (beta=.83, P=.01).

CONCLUSIONS

High plasma concentration of FVII and vWF may be associated with the progression of early carotid atherosclerosis in patients with peripheral arterial disease.

BACKGROUND

Ovulation and luteinization of follicles are complex biological processes initiated by the preovulatory luteinizing hormone surge. The objective of this study was to identify genes that are differentially expressed in bovine granulosa cells (GC) of ovulatory follicles.

METHODS

Granulosa cells were collected during the first follicular wave of the bovine estrous cycle from dominant follicles (DF) and from ovulatory follicles (OF) obtained 24 h following injection of human chorionic gonadotropin (hCG). A granulosa cell subtracted cDNA library (OF-DF) was generated using suppression subtractive hybridization and screened.

RESULTS

Detection of genes known to be upregulated in bovine GC during ovulation, such as ADAMTS1, CAV1, EGR1, MMP1, PLAT, PLA2G4A, PTGES, PTGS2, RGS2, TIMP1, TNFAIP6 and VNN2 validated the physiological model and analytical techniques used. For a subset of genes that were identified for the first time, gene expression profiles were further compared by semiquantitative RT-PCR in follicles obtained at different developmental stages. Results confirmed an induction or upregulation of the respective mRNAs in GC of OF 24 h after hCG-injection compared with those of DF for the following genes: ADAMTS9, ARAF, CAPN2, CRISPLD2, FKBP5, GFPT2, KIT, KITLG, L3MBLT3, MRO, NUDT10, NUDT11, P4HA3, POSTN, PSAP, RBP1, SAT1, SDC4, TIMP2, TNC and USP53. In bovine GC, CRISPLD2 and POSTN mRNA were found as full-length transcript whereas L3MBLT3 mRNA was alternatively spliced resulting in a truncated protein missing the carboxy-terminal end amino acids, 774KNSHNEL780. Conversely, L3MBLT3 is expressed as a full-length mRNA in a bovine endometrial cell line. The 774KNSHNEL780 sequence is well conserved in all mammalian species and follows a SAM domain known to confer protein/protein interactions, which suggest a key function for these amino acids in the epigenetic control of gene expression.

CONCLUSIONS

We conclude that we have identified novel genes that are upregulated by hCG in bovine GC of OF, thereby providing novel insight into peri-ovulatory regulation of genes that contribute to ovulation and/or luteinization processes.

Recent studies suggest that the traditional case-control study design does not have sufficient power to discover rare risk variants. Two different methods-collapsing and family data-are suggested as alternatives for discovering these rare variants. Compared with common variants, rare variants have unique characteristics. In this paper, we assess the distribution of rare variants in family data. We notice that a large number of rare variants exist only in one or two families and that the association result is largely shaped by those families. Therefore we explore the possibility of integrating both the collapsing method and the family data method. This combinational approach offers a potential power boost for certain causal genes, including VEGFA, VEGFC, SIRT1, SREBF1, PIK3R3, VLDLR, PLAT, and FLT4, and thus deserves further investigation.

Effectors of the Rab small GTPases are large multi-domain proteins which have proved difficult to express in soluble form in Escherichia coli. Generally, effectors are recruited to a distinct subcellular compartment by active (GTP-bound) Rabs, which are linked to membranes by one or two prenylated Cys residues at their C-termini. Following recruitment via their Rab-binding domain (RBD), effectors carry out various aspects of vesicle formation, transport, tethering and fusion through their other domains. Previously, successful purification of the RUN-PLAT tandem domains (residues 683-1061) of the 1263-residue Rab6-interacting protein 1 (R6IP1) required co-expression with Rab6, as attempts to solubly express the effector alone were unsuccessful. R6IP1 is also known as DENN domain-containing protein 5 (DENND5) and is expressed as two isoforms, R6IP1A/B (DENND5A/B), which differ by 24 amino acids at the N-terminus. Here, a deletion in R6IP1 was engineered to enable soluble expression and to improve the quality of the crystals grown in complex with Rab6. A large 23-residue loop linking two α-helices in the RUN1 domain was removed and replaced with a short linker. This loop resides on the opposite face to the Rab6-binding site and is not conserved in the RUN-domain family. In contrast to wild-type R6IP1-Rab6 crystals, which took several weeks to grow to full size, the engineered R6IP1 (RPdel)-Rab6 crystals could be grown in a matter of days.

Polycystic Kidney Disease is an autosomal dominant disease common in some lines of Bull Terriers (BTPKD). The disease is linked to the canine orthologue of human PKD1 gene, Pkd1, located on CFA06, but no disease-associated mutation has been reported. This study sequenced genomic DNA from two Bull Terriers with BTPKD and two without the disease. A non-synonymous G>A transition mutation in exon 29 of Pkd1 was identified. A TaqMan® SNP Genotyping Assay was designed and demonstrated the heterozygous detection of the mutation in 47 Bull Terriers with BTPKD, but not in 102 Bull Terriers over one year of age and without BTPKD. This missense mutation replaces a glutamic acid residue with a lysine residue in the predicted protein, Polycystin 1. This region of Polycystin 1 is highly conserved between species, and is located in the first cytoplasmic loop of the predicted protein structure, close to the PLAT domain and the second transmembrane region. Thus, this change could alter Polycystin 1 binding or localization. Analytic programs PolyPhen 2, Align GVGD and SIFT predict this mutation to be pathogenic. Thus, BTPKD is associated with a missense mutation in Pkd1, and the application of this mutation specific assay could reduce disease transmission by allowing diagnosis of disease in young animals prior to breeding.

Here we report the insertion of a synthetic version of the cDNA encoding the jellyfish (Aequorea victoria) green fluorescent protein (gfph ) into the genome of pseudorabies (Aujeszky's disease) virus (PrV). A putative latency promoter (PLAT) located at the inverted repeat region of the PrV genome was chosen as the target site for the insertion. Recombinant viral DNA designated as vLAT-gfp was generated as a result of homologous recombination between the transfected viral DNA and a plasmid containing the GFP-expression cassette flanked by viral sequences homologous to the target region. Plaques containing recombinant virus were selected visually using a fluorescent microscope. We demonstrated a GFP-expression in infected neurons of rat brain which showed normal morphology at early stage of viral infection by monitoring fluorescent light emission.

Thrombocytopenia is defined as platelet count less than 150,000 plat/mm3. Etiologic factors involved include immunological (NAIT and ITP), fetal infectious disease, chromosomal and nonchromosomal, and miscellaneous causes. While the understanding of fetal thrombocytopenia is driven by reason to do fetal blood sampling, discovery of neonatal thrombocytopenia is driven by blood counts performed because of the risk of infections. The most serious consequence of thrombocytopenia in the fetus/neonate is intracranial hemorrhage which can occur in utero as early as 18 weeks gestation. The key factor in perinatal prevention of intracranial hemorrhage is early diagnosis and treatment, possibly in utero. Cordocentesis under direct ultrasound guidance and platelet transfusions have played a major role in the management of fetal/neonatal thrombocytopenia. Ongoing studies and high resolution ultrasound will continue to explore and hopefully clarify fetal and neonatal thrombocytopenia and facilitate recognition of primary and secondary thrombocytopenias.