Behavioural characterisation of the alpha-mannosidosis guinea pig.

alpha-Mannosidosis is a lysosomal storage disorder resulting from a functional deficiency of the lysosomal enzyme alpha-mannosidase. This deficiency results in the accumulation of various oligosaccharides in the lysosomes of affected individuals, causing somatic pathology and progressive neurological degeneration that results in cognitive deficits, ataxia, and other neurological symptoms. We have a naturally occurring guinea pig model of this disease which exhibits a deficiency of lysosomal alpha-mannosidase and has a similar clinical presentation to human alpha-mannosidosis. Various tests were developed in the present study to characterise and quantitate the loss of neurological function in alpha-mannosidosis guinea pigs and to follow closely the progression of the disease. General neurological examinations showed progressive differences in alpha-mannosidosis animals from approximately 1 month of age. Significant differences were observed in hind limb gait width from 2 months of age and significant cognitive (memory and learning) deficits were observed from 3 months of age. Evoked response tests showed an increase in somatosensory P1 peak latency in alpha-mannosidosis guinea pigs from approximately 2 months of age, as well as progressive hearing loss using auditory brainstem evoked responses. The alpha-mannosidosis guinea pig therefore appears to exhibit many of the characteristics of the human disease, and will be useful in evaluating therapies for treatment of central nervous system pathology.

Antitumor activity of sorafenib in FLT3-driven leukemic cells.

Activating internal tandem duplication (ITD) insertions in the juxtamembrane domain of the FLT3 tyrosine kinase are found in about one fourth of patients with acute myeloid leukemia and have been shown to be an independent negative prognostic factor for survival. We show that sorafenib (BAY 43-9006, Nexavar) potently inhibits FLT3 enzymatic and signaling activities. In HEK293 cells stably transfected with FLT3-WT or FLT3-ITD, sorafenib blocked basal and ligand dependent FLT3-mediated tyrosine autophosphorylation as well as extracellular signal-regulated kinase1/2 and Stat5 phosphorylation. In leukemia cell lines MV4-11 and EOL-1, sorafenib treatment resulted in decreased cell proliferation and inhibition of FLT3 signaling. The growth of the FLT3-independent RS4-11 cell line was only weakly inhibited by sorafenib. Cell cycle arrest and induction of apoptosis were observed upon treatment with sorafenib in MV4-11 and EOL-1 cells. The antitumor efficacy of sorafenib was evaluated against the MV4-11 leukemia grown subcutaneously in NCr nu/nu mice. Doses of 3 and 10 mg/kg administered orally for 14 days resulted in six and nine out of 10 animals with complete responses, respectively. The demonstration that sorafenib exhibits potent target inhibition and efficacy in FLT3-driven models suggests that this compound may have a therapeutic benefit for patients with FLT3-driven leukemias.

Control of source fertility on the eruptive activity of Piton de la Fournaise volcano, La Réunion.

The eruptive activity of basaltic hotspot volcanoes displays major fluctuations on times scales of years to decades. Theses fluctuations are thought to reflect changes in the rate of mantle melt supply. However, the crustal filter generally masks the mantle processes involved. Here, we show that the cyclic and generally increasing activity of the Piton de la Fournaise volcano (La Réunion) since the mid 20th century is tightly linked to the fertility of its source, as recorded by 87Sr/86Sr and incompatible trace elements ratios of lavas. We identify a twofold control of source fertility on eruptive activity: melt extraction from fertile, incompatible element-enriched veins initiates decadal-scale eruptive sequences, so that vein distribution in the plume source directly controls the cyclic activity. Indirectly, reactive flow of enriched melts increases mantle porosity and promotes melts extraction from the peridotite matrix. This process is thought to have caused a fourfold increase in magma supply between 1998 and 2014 at Piton de la Fournaise, and could also explain magma surges at other frequently active hotspot volcanoes, such as Kilauea, Hawaii. The short-term eruptive activity of hotspot volcanoes appears to be ultimately linked to the distribution and size of lithological heterogeneities in mantle plumes.

Integrative network analysis identifies novel drivers of pathogenesis and progression in newly diagnosed multiple myeloma.

Multiple myeloma (MM) is an incurable malignancy of bone marrow plasma cells characterized by wide clinical and molecular heterogeneity. In this study we applied an integrative network biology approach to molecular and clinical data measured from 450 patients with newly diagnosed MM from the MMRF (Multiple Myeloma Research Foundation) CoMMpass study. A novel network model of myeloma (MMNet) was constructed, revealing complex molecular disease patterns and novel associations between clinical traits and genomic markers. Genomic alterations and groups of coexpressed genes correlate with disease stage, tumor clonality and early progression. We validated CDC42BPA and CLEC11A as novel regulators and candidate therapeutic targets of MMSET-related myeloma. We then used MMNet to discover novel genes associated with high-risk myeloma and identified a novel four-gene prognostic signature. We identified new patient classes defined by network features and enriched for clinically relevant genetic events, pathways and deregulated genes. Finally, we demonstrated the ability of deep sequencing techniques to detect relevant structural rearrangements, providing evidence that encourages wider use of such technologies in clinical practice. An integrative network analysis of CoMMpass data identified new insights into multiple myeloma disease biology and provided improved molecular features for diagnosing and stratifying patients, as well as additional molecular targets for therapeutic alternatives.

High somatic mutation and neoantigen burden are correlated with decreased progression-free survival in multiple myeloma.

Tumor-specific mutations can result in immunogenic neoantigens, both of which have been correlated with responsiveness to immune checkpoint inhibitors in highly mutagenic cancers. However, early results of single-agent checkpoint inhibitors in multiple myeloma (MM) have been underwhelming. Therefore, we sought to understand the relationship between mutation and neoantigen landscape of MM patients and responsiveness to therapies. Somatic mutation burden, neoantigen load, and response to therapy were determined using interim data from the MMRF CoMMpass study (NCT01454297) on 664 MM patients. In this population, the mean somatic and missense mutation loads were 405.84(s=608.55) and 63.90(s=95.88) mutations per patient, respectively. There was a positive linear relationship between mutation and neoantigen burdens (R2=0.862). The average predicted neoantigen load was 23.52(s=52.14) neoantigens with an average of 9.40(s=26.97) expressed neoantigens. Survival analysis revealed significantly shorter progression-free survival (PFS) in patients with greater than average somatic missense mutation load (N=163, 0.493 vs 0.726 2-year PFS, P=0.0023) and predicted expressed neoantigen load (N=214, 0.555 vs 0.729 2-year PFS, P=0.0028). This pattern is maintained when stratified by disease stage and cytogenetic abnormalities. Therefore, high mutation and neoantigen load are clinically relevant risk factors that negatively impact survival of MM patients under current standards of care.

HRad17, a human homologue of the Schizosaccharomyces pombe checkpoint gene rad17, is overexpressed in colon carcinoma.

Using the palindromic PCR-cDNA display method, we have cloned a novel gene overexpressed by human colon carcinoma relative to normal colon. Among normal tissues examined, only testis expresses it at a high level. Sequence analysis revealed its extensive homology with checkpoint genes rad17 of Schizosaccharomyces pombe and RAD24 of Saccharomyces cerevisiae. This novel gene designated as hRad17 is localized to chromosome 5q12,13.1, a region known to be deleted in a variety of human cancers. Promoter region and one pseudogene of hRad17 have been identified. Whereas the increased expression of hRad17 by human colon carcinomas may be related to the known resistance of these cells to DNA-damaging agents during therapy, the deletion of hRad17 in a variety of cancers may predispose them to increased rate of mutation and heightened sensitivity to DNA-damaging agents, including radiation and anticancer drugs.

A novel actin cDNA is expressed in the neurons of Aplysia californica.

A novel actin cDNA has been isolated from an abdominal ganglion cDNA library of Aplysia californica by differential screening. This cDNA of 1596 nucleotides in length encodes a putative actin protein of 41.8 kDa. This protein shows 95.2% identity with another Aplysia actin gene previously shown to be expressed in the muscular sheath of the ganglion (DesGroseillers et al. (1990) Nucleic Acids Res. 18, 3654). However, the 5' and 3' untranslated regions of these cDNAs are completely different. PCR experiments performed with mRNA isolated from dissected neurons, ovotestis or kidney reveal that the gene is expressed in the neurons of the ganglia and in other tissues as well. Southern blot analysis reveals that the neuronal actin gene is a member of a large gene family.

Survival and engraftment of mouse embryonic stem cell-derived implants in the guinea pig brain.

alpha-Mannosidosis is a lysosomal storage disease resulting from a deficiency of the enzyme alpha-D-mannosidase. A major feature of alpha-mannosidosis is progressive neurological decline, for which there is no safe and effective treatment available. We have a guinea pig model of alpha-mannosidosis that models the human condition. This study investigates the feasibility of implanting differentiated mouse embryonic stem cells in the neonatal guinea pig brain in order to provide a source of alpha-mannosidase to the affected central nervous system. Cells implanted at a low dose (1.5 x 10(3)cells per hemisphere) at 1 week of age were found to survive in very low numbers in some immunosuppressed animals out to 8 weeks. Four weeks post-implantation, cells implanted in high numbers (10(5) cells per hemisphere) formed teratomas in the majority of the animals implanted. Although implanted cells were found to migrate extensively within the brain and differentiate into mature cells of neural (and other) lineages, the safety issue related to uncontrolled cell proliferation precluded the use of this cell type for longer-term implantation studies. We conclude that the pluripotent cell type used in this study is unsuitable for achieving safe engraftment in the guinea pig brain.

Overexpression of HRad17 mRNA in human breast cancer: correlation with lymph node metastasis.

PURPOSE: A novel human gene, designated HRad17, was identified as the human homologue of the Rad17 of Schizosaccharomyces pombe and Rad24 of Saccharomyces cerevisiae. In yeast, these genes play a critical role in maintaining genomic stability. The aim of this study was to evaluate the expression of HRad17 in human breast cancer. EXPERIMENTAL DESIGN: We investigated HRad17 mRNA expression in 64 cases of human breast cancer by means of reverse-transcription-PCR, in situ hybridization, and immunohistochemistry. RESULTS: The HRad17 mRNA was overexpressed in 35 cases (54.7%). Twenty-four (68.6%) of 35 cases with HRad17 overexpression in cancer tissues were node-positive, whereas only 8 (27.6%) of 29 cases without HRad17 overexpressions were node-positive. The expression of HRad17 mRNA correlated with both lymph node metastasis (P = 0.001) and high Ki67 labeling index (P = 0.006). Although not significantly different, expression of HRad17 mRNA tended to correlate with tumor size (P = 0.06) and expression of mutant p53 protein (P = 0.10). Furthermore, expression of HRad17 mRNA was an independent predictor of axillary lymph node metastasis as well as of lymphatic permeation by multivariate analysis (P < 0.0001). CONCLUSIONS: Our study demonstrates that HRad17 might be related to the development of lymph node metastasis in human breast cancers. Although its function still remains unclear, the expression of HRad17 mRNA could open up a new window for the diagnostic staging and treatment of human breast cancers.

Serum level of the periostin, a homologue of an insect cell adhesion molecule, in thymoma patients.

Periostin protein shares structural and sequence homology with fasciclin I, which is an insect adhesion molecule. Periostin has a typical signal peptide at the N-terminal end suggesting it is a secreted protein. Recently, we developed a novel sandwich chemiluminescence assay to determine serum concentrations of periostin. We investigated the serum periostin level in thymoma patients, and attempted to determine the correlation between serum periostin level and clinicopathological factors of thymoma patients who had undergone surgery between January 1994 and July 1996. Serum periostin levels were not significantly different between the thymoma patients (1264.4+/-122.9 ng/ml) and the normal control (962.0+/-118.6 ng/ml) (P=0.0877). There was no relationship between serum periostin level and age, gender or pathological subtype. However the serum periostin level of stage IV patients (1497.0+/-285.8 ng/ml) was significantly higher than normal control (P=0.0460). These data suggest that serum periostin level may indicate tumor invasion and progression of thymoma.

Additive effects of norepinephrine and cyclic AMP on the activation of the protein kinase from adipose tissue.

In this work the kinetics of activation of the cyclic AMP-dependent protein kinase by several catecholamines and ACTH, have been studied in rat epididymal fat pads and isolated fat cells. The method of Soderling and co-workers which permits the measurement of the state of activation of the protein kinase after hormonal stimulation in adipose tissue, has been used. Kinetics experiments where norepinephrine was used showed that the results obtained with isolated cells conform to the models of Sutherland and Brostrom and co-workers. Wtih intact tissue, norepinephrine not only stimulates the protein kinase activity measured without exogenous cyclic AMP but also the total activity measured in the presence of cyclic AMP (5 X 10(-6) M); thus the effect of norepinephrine, obtained during incubation of the tissue, and that of cyclic AMP, added to the soluble fraction after incubation, were additive. This effect seems to be of the beta type because it is blocked completely by propranolol. A weak, additive but significant effect was also obtained with epinephrine and isoproterenol but not with ACTH. Neither cyclic GMP nor cyclic IMP seems implicated in this effect. It was shown that stroma vascular cells which are present in the fat pads are not involved. These results suggest that the effects of norepinephrine on the protein kinase of the fat pads cannot be completely explained by the model of Brostrom and colleagues.

Overexpression of Hrad17 gene in non-small cell lung cancers correlated with lymph node metastasis.

We used palindromic PCR-driven cDNA differential display technique to identify and isolate a gene, human homologue of the Schizosaccharomyces pombe checkpoint gene rad17, from colon cancer tissues. The loss of checkpoint control in mammalian cells results in genomic instability, leading to the amplification, rearrangement, or loss of chromosomes, events associated with tumor progression. We hypothesized that the Hrad17 may be expressed in non-small cell lung cancer (NSCLC). We attempted to determine the influence of Hrad17 expression on clinicopathological features for patients with NSCLC who had undergone surgery. Expression of Hrad17 messenger RNA was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) in 102 non-small cell lung carcinomas and adjacent histologically normal lung samples from patients for whom follow up data were available. Hrad17 transcripts were detected in 26 (25.5%) of the tumor samples, although some of the paired normal lung samples showed weak expression. There was no relationship between Hrad17 gene expression and age, gender or T-status. About 13 of 31 (41.9%) NSCLC patients with Hrad17 overexpressions were node positive, on the other hand, 13 of 76 (18.3%) cases without Hrad17 overexpressions were node positive. Thus the expression of Hrad17 mRNA correlated with lymph node metastasis (P=0.0231) from NSCLC. Hrad17 protein was highly expressed at the advancing margin of the tumor of lung cancer tissue but not within the normal lung tissue by immunohistochemistry. Thus the expression of Hrad17 might correlate with more advanced NSCLC.

Optimizing dosing of oncology drugs.

The purpose of this article is to acknowledge the challenges in optimizing the dosing of oncology drugs and to propose potential approaches to address these challenges in order to optimize effectiveness, minimize toxicity, and promote adherence in patients. These approaches could provide better opportunities to understand the sources of variability in drug exposure and clinical outcomes during the development and premarketing evaluation of investigational new drugs.

Morphopathological features in tissues of alpha-mannosidosis guinea pigs at different gestational ages.

Alpha-mannosidosis is an inherited metabolic disorder characterized by a reduction in alpha-D-mannosidase and intralysosomal accumulation of undegraded mannose-containing oligosaccharides. The alpha-mannosidosis guinea pig exhibits pathological similarities to its human counterpart, which make it a valuable animal model. To trace the progression of alpha-mannosidosis during foetal development, brain and visceral organs from affected and unaffected guinea pigs at 30, 36, 38, 51 and 65 days of gestation (dg) were examined by light and electron microscopy (term: approximately 68 dg). In the affected brain, distended lysosomes (vacuoles) were scarce up to 38 dg and were seen in few differentiating neuronal cells but mostly in macrophages, pericytes and endothelial cells. At 51 and 65 dg, several vacuoles were observed in some neurones, in many Purkinje cells, pericytes, endothelial and microglial cells, and in few cerebellar internal granule cells. Myelination had started by 51 dg. Non-myelinated axonal spheroids were detected in the brainstem at 65 dg. In the kidney cortex and liver, an increase in vacuolation was noticed between 36 and 65 dg. Some vacuolated cells were also noticed in the lungs and spleen at 51 and 65 dg. Altogether, these histological observations suggest that alpha-mannosidosis is unlikely to affect ontogenesis before the second half of gestation in guinea pigs; however, the morphopathological features recorded during the last quarter of gestation (which may roughly correspond to the period covering near term to 1-2 years of age in human) were clearly noticeable and may have had some impact.

Analysis of genes encoding highly conserved lysine-rich proteins in Aplysia californica and Saccharomyces cerevisiae.

To isolate a gene that can be used as an internal control in studies on gene expression in Aplysia californica neurons, we have characterized a cDNA clone (pKRP-A) isolated on the basis of its high expression in A. californica neurons. This cDNA is of 850 nucleotides and codes for a putative 29-kDa lysine-rich protein. Blotting experiments revealed that the gene is expressed in all tested A. californica tissues, and in individually identified neurons of the abdominal ganglion, suggesting that this gene can be efficiently used as internal control in studies of gene expression. We have also isolated one cDNA and two different genomic clones from yeast libraries that show 59% identity with pKRP-A. Sequence comparison of genomic clones, as well as PCR and Southern blotting experiments, revealed that at least two homologous genes are present in yeast. Northern blotting experiments revealed that the expression of the gene is strongly repressed at 39 degrees C.

Effect of active immunization against testosterone on plasma gonadotrophin concentrations, spermatogenic function, testicular blood flow, epididymis mass and mating behaviour in adult rams.

The long-term effects of active immunization against testosterone were studied in rams, with particular reference to blood concentrations of gonadotrophin and testosterone, spermatogenesis, testis blood flow and mating behaviour. Ten 18-month-old Merino rams, kept on pasture, were studied for 1 year. Every 2 months, five rams received injections of BSA in Freund's adjuvant and five other rams were treated with testosterone-3(o-carboxymethyl)oxime-BSA as immunogen. Anti-testosterone antibodies (mean titre: 1:4484 +/- 582, after boosters) were maintained in the circulation, with the help of regular booster injections. In time, immunization reduced live mass in testosterone-immunized rams; however, there was no effect on testicular volume throughout the whole study. In testosterone-immunized rams, significantly higher concentrations of gonadotrophins were found in jugular venous plasma, as well as increased concentrations of total plasma testosterone. LH pulse frequency, amplitude and nadir were increased significantly in testosterone-immunized rams. After 12 months of immunization, no differences were found in the number of spermatozoa per ejaculate, in daily sperm production or in testis mass between the two groups of rams; however, testicular blood flow (per testis) and epididymis mass were significantly reduced in testosterone-immunized rams. Testosterone immuno-neutralization also resulted in a significant reduction in the number of mounts culminating in ejaculation performed during a 10 min trial carried out on a number of occasions during the experiment. Additional information on these rams was collected 3 months after castration. However, there were no significant differences in mean plasma LH and FSH concentrations, either before, or after, a single GnRH injection between the two groups of rams at this time.

Effect of active immunization against oestradiol in developing ram lambs on plasma gonadotrophin and testosterone concentrations, time of onset of puberty and testicular blood flow.

Merino ram lambs were actively immunized against oestradiol-6 (o-carboxy methyl) oxime-BSA conjugate at 14 weeks of age and received a booster injection 4 weeks later. This treatment led to an increase in plasma concentrations of gonadotrophin and tended to enhance the increase in testicular volume until 26 weeks of age; however, testis size and mass at time of castration (30 weeks of age) were similar to values in BSA-immunized lambs. Detrimental effects were observed in some oestradiol-immunized ram lambs, for example a steep decline in testicular volume towards the end of the experiment, the presence of large vacuoles within the seminiferous epithelium and, in one lamb, few germ cell at 30 weeks of age. Testicular blood plasma flow was significantly reduced in oestradiol-immunized lambs (P < 0.01). The steroidogenic function of the testis was markedly enhanced in oestradiol-immunized lambs as reflected by high plasma concentrations of testosterone measured at 22, 26 and 30 weeks of age and by high testosterone production calculated from blood flow and venous-arterial differences at 30 weeks of age. Nevertheless, total live mass gain over the 16 week study was not increased in oestradiol-immunized lambs. Testicular biopsies were taken at 22 and 26 weeks of age in half of the lambs in each treatment group. Testicular volume measured at castration was decreased in control lambs in which biopsies were taken (P < 0.05), and plasma concentrations of testosterone measured at 30 weeks of age were significantly lower in oestradiol-immunized lambs in which biopsies were taken (P < 0.02) compared with lambs in which no biopsy had been taken. It is concluded that active immunization against oestradiol in ram lambs does not advance the time of onset of puberty and does not confer any reproductive or maturational advantages.

Activation of the ubiquitin pathway in rat skeletal muscle by catabolic doses of glucocorticoids.

To evaluate whether catabolic levels of glucocorticoids activate the ubiquitin pathway in conjunction with their known proteolytic effect in skeletal muscle, rats were injected daily with corticosterone (CTC; 10 mg/100 g body wt) for 7 days. Two peaks of urinary excretion of 3-methylhistidine (3-MH), a specific marker of myofibrillar proteolysis, were observed at days 1 and 3 (165 and 295% of controls, respectively). Levels of ubiquitin pathway mRNAs in skeletal muscle were assessed around the 3-MH peaks. In the extensor digitorum longus, a first rise of two polyubiquitin (pUb) mRNAs was seen at day 1 (183 and 162% of control for the UbB and UbC transcripts, respectively, P < 0.01). An accumulation of both E2-14k mRNAs (140%, P < 0.02, and 157% of controls, P < 0.01) and proteasome C8 subunit mRNA (222% of control, P < 0.05) was seen at day 2. A second more important peak of induction of pUb mRNA was seen at day 3 (251 and 217% of controls for the UbB and UbC transcripts, respectively, P < 0.001). All transcripts returned to near control levels by day 4. In the soleus, induction of E2-14k mRNA started at day 3 and reached 216 and 208% of controls at day 4 (P < 0.001), whereas an increase of pUb mRNA was observed at days 3 (213 and 241%, P < 0.05) and 4 (211 and 221%, P < 0.001). A rise of proteasome C8 subunit mRNA accumulation was also seen in the soleus at days 3 (217%, P < 0.05) and 4 (157%, P < 0.05). Reduced ubiquitin conjugate levels, possibly due to their rapid degradation through increased proteasome activity, were observed in both muscle types at day 3. The parallel between the catabolic effects of CTC and activation of the ubiquitin pathway in muscles of CTC-treated rats strongly suggests the involvement of this system in glucocorticoid-induced muscular atrophy.

Serum level of the periostin, a homologue of an insect cell adhesion molecule, as a prognostic marker in nonsmall cell lung carcinomas.

BACKGROUND: Periostin protein shares structural and sequence homology with fasciclin I, which is an insect adhesion molecule. Periostin has a typical signal peptide at the N-terminal end, which suggests that it is a secreted protein. Recently, the authors developed a novel sandwich chemiluminescence assay to determine serum concentrations of periostin. METHODS: The authors investigated the serum periostin level in lung carcinoma patients and attempted to determine the influence of serum periostin level on clinical outcome for patients with nonsmall cell lung carcinoma (NSCLC) who had undergone surgery between January 1994 and July 1996. Expression of periostin messenger RNA was also examined by in situ RNA hybridization for lung carcinomas. RESULTS: The periostin gene was shown to be highly expressed at the tumor periphery of lung carcinoma tissue but not within the tumor by in situ RNA hybridization. Serum periostin levels were not significantly different between the NSCLC patients (1142.1 +/- 89.2 ng/mL) and the normal control (962.0 +/- 118.6 ng/mL) (P = 0.2985). There was no relation between serum periostin level and gender, stage, bone metastasis, N status, or T status. However, the serum periostin levels of NSCLC patients had decreased significantly by 4 weeks after the resection of the tumor. The NSCLC patients with high periostin level (> 962 ng/mL) had significantly poorer survival than the patients with normal periostin level (P = 0.0406). Using the Cox proportional hazards regression model, the authors found that stage (P < 0.0001) and serum periostin level (P = 0.0226) were independent prognostic factors. CONCLUSIONS: The in situ RNA hybridization data from the current study suggested that expression of periostin may be involved in tumor invasionand that the serum periostin level may serve as a prognostic marker for NSCLC.