Adverse Phenotype Research Articles

Behavioral analysis of Ste20 kinase SPAK knockout mice

SPAK/STK39 is a mammalian protein kinase involved in the regulation of inorganic ion transport mechanisms known to modulate GABAergic neurotransmission in the both central and the peripheral nervous systems. We have previously shown that disruption of the gene encoding SPAK by homologous recombination in mouse embryonic stem cells results in viable mice that lack expression of the kinase [16]. With the exception of reduced fertility, these mice do not exhibit an overt adverse phenotype. In the present study, we examine the neurological phenotype of these mice by subjecting them to an array of behavioral tests. We show that SPAK knockout mice displayed a higher nociceptive threshold than their wild-type counterparts on the hot plate and tail flick assays. SPAK knockout mice also exhibited a strong locomotor phenotype evidenced by significant deficits on the rotarod and decreased activity in open-field tests. In contrast, balance and proprioception was not affected. Finally, they demonstrated an increased anxiety-like phenotype, spending significantly longer periods of time in the dark area of the light/dark box and increased thigmotaxis in the open-field chamber. These results suggest that the kinase plays an important role in CNS function, consistent with SPAK regulating ion transport mechanisms directly involved in inhibitory neurotransmission.

ADAM8/MS2/CD156, an Emerging Drug Target in the Treatment of Inflammatory and Invasive Pathologies

While it is highly accepted that ADAM family members with ubiquitous expression patterns, such as ADAM10 and ADAM17 have major roles in homoeostasis and pathology, ADAM8 was initially considered as an immune-specific ADAM with a cell-specific expression pattern. Therefore, ADAM8 had a "sleeping beauty" existence for many years, and has recently come back into focus as it was detected under several pathological conditions. These were found to typically involve inflammation and remodelling of the extracellular matrix, including cancers and serious respiratory diseases such as asthma. In these diseases, induced expression of ADAM8 by different stimuli results in cleavage of various substrates, including cell adhesion molecules, cytokine receptors, and ECM components. Involvement of ADAM8 in individual diseases indicates its usefulness as both a diagnostic and prognostic marker. Even more strikingly, as ADAM8 progressively emerges as a key effector in pathological processes, so does its attractiveness as a therapeutic target rather than being a mere indicator of disease and its progression. This is encouraged by analysis of ADAM8 null mice, identifying no adverse phenotype in the absence of functional ADAM8. Thus, ADAM8 potentially is an attractive drug target in a variety of diseases. In this review, the current knowledge on ADAM8 in diseases and avenues for specific inhibition based on unique biochemical features of ADAM8 will be presented.

Absence of α-synuclein affects dopamine metabolism and synaptic markers in the striatum of aging mice

Despite numerous evidences for neurotoxicity of overexpressed α-synuclein, a protective function was suggested for endogenous α-synuclein and other members of the synuclein family. This protective role is most important for and evident in presynaptic terminals, where synucleins are normally accumulated. However, mice lacking synucleins display no adverse phenotype. In particular, no significant changes in striatal dopamine metabolism and only subtle deficit of dopaminergic neurons in the substantia nigra were found in juvenile or adult mice. To assess whether aging and synuclein deficiency may have additive detrimental effect on the nigrostriatal system, we studied dopaminergic neurons of the substantia nigra and their striatal synapses in 24–26-month-old α-synuclein and γ-synuclein null mutant mice. Significant ∼36% reduction of the striatal dopamine was found in aging α-synuclein, but not γ-synuclein null mutant mice when compared to age-matching wild type mice. This was accompanied by the reduction of TH-positive fibers in the striatum and decrease of striatal levels of TH and DAT. However, no progressive loss of TH-positive neurons was revealed in the substantia nigra of synuclein-deficient aging animals. Our results are consistent with a hypothesis that α-synuclein is important for normal function and integrity of synapses, and suggest that in the aging nervous system dysfunction of this protein could become a predisposition factor for the development of nigrostriatal pathology.

Anti-oestrogens but not oestrogen deprivation promote cellular invasion in intercellular adhesion-deficient breast cancer cells

IntroductionAnti-oestrogens have been the mainstay of therapy in patients with oestrogen-receptor (ER) positive breast cancer and have provided significant improvements in survival. However, their benefits are limited by tumour recurrence in a significant proportion of initially drug-responsive breast cancer patients because of acquired anti-oestrogen resistance. Relapse on such therapies clinically presents as local and/or regional recurrences, frequently with distant metastases, and the prognosis for these patients is poor. The selective ER modulator, tamoxifen, classically exerts gene inhibitory effects during the drug-responsive phase in ER-positive breast cancer cells. Paradoxically, this drug is also able to induce the expression of genes, which in the appropriate cell context may contribute to an adverse cell phenotype. Here we have investigated the effects of tamoxifen and fulvestrant treatment on invasive signalling and compared this with the direct effects of oestrogen withdrawal to mimic the action of aromatase inhibitors.MethodsThe effect of oestrogen and 4-hydroxy-tamoxifen on the invasive capacity of endocrine-sensitive MCF-7 cells, in the presence or absence of functional E-cadherin, was determined by Matrigel invasion assays. Studies also monitored the impact of oestrogen withdrawal or treatment with fulvestrant on cell invasion. Western blotting using phospho-specific antibodies was performed to ascertain changes in invasive signalling in response to the two anti-oestrogens versus both oestradiol treatment and withdrawal.ResultsTo the best of our knowledge, we report for the first time that tamoxifen can promote an invasive phenotype in ER-positive breast cancer cells under conditions of poor cell-cell contact and suggest a role for Src kinase and associated pro-invasive genes in this process. Our studies revealed that although this adverse effect is also apparent for further classes of anti-oestrogens, exemplified by the steroidal agent fulvestrant, it is absent during oestrogen withdrawal.ConclusionsThese data highlight a previously unreported effect of tamoxifen (and potentially further anti-oestrogens), that such agents appear able to induce breast cancer cell invasion in a specific context (absence of good cell-cell contacts), where these findings may have major clinical implications for those patients with tumours that have inherently poor intercellular adhesion. In such patients oestrogen deprivation with aromatase inhibitors may be more appropriate.

The influence of leptin on early life programming of obesity

Epidemiological evidence together with experimental models shows a direct relationship between fetal and early postnatal growth patterns and an increased risk of adult metabolic disease. Maternal health and nutrition are key determinants in influencing infant growth but the precise molecular mechanisms underlying this relationship are unclear, although it is evident that there are critical time windows when these effects are important. Animal models show mechanistic parallels with human populations and highlight that the early environment represents a therapeutic window for protection from obesity and metabolic disease. The observation that developmental programming can be reversed has been demonstrated in studies in which both maternal and neonatal leptin treatment prevents the induction of the adverse metabolic phenotype. Given that orally administered peptides are absorbed intact by the new born, the prospect of providing supplemental leptin either as drops or in milk deserves serious consideration as a means of reducing or reversing the obesity and type 2 diabetes epidemic.

9.25 Evaluation of Appropriateness of Left Ventricular Mass in a Paediatric Population with Obesity

Introduction. Metabolic syndrome (MetS) definition is based on either abnormal values, or ongoing therapy demonstrating history of exposure to risk factors. This classification implies that therapy does not modify the MetS status, whereas therapy-modified components might modify attribution to, and prognosis of MetS. We tested the hypothesis that definition of MetS based on history rather than on direct evidence of abnormal risk factors is associated with a more benign cardiovascular (CV) phenotype. Methods. We studied 1642 African-American (1045 women) and 1289 Caucasian (681 women) HyperGEN participants, without prevalent CV disease or renal failure (serum creatinine >2.5mg/dL). MetS was defined by ATPIII, using cut-offs of blood tests, waist girth and measured blood pressure (BP), irrespective of on-going therapy (M1). M1 was compared with a subgroup of participants classified as MetS, based on history of exposure and on-going therapy to normalize risk factors (M2). We used LV mass index (LVMi), relative wall thickness (RWT) and a the ratio of stroke volume to pulse pressure (SV/PP) adjusted by heart rate, body weight and age (prognostically validated as a raw estimate of arterial compliance), as measures of end-organ damage. Results. Among 1102 participants with MetS (38%), 780 were confirmed by M1, whereas 322 (15%) had MetS by M2; 1892 were free of MetS by both criteria (no-MetS). Among M2 participants, 67% had normal fasting glucose, 71% normal triglycerides, 32% normal HDL-cholesterol, 16% normal waist girth and only 4% had normal BP. After adjusting for age, sex and ethnicity, mean LVMi was higher with in M1 (41.9 g/m 2.7) than in M2 (39.9 g/m 2.7) or no-Mets participants (37.6g/m2.7; all p<0.01). Similarly, RWT was higher in M1 (0.35) than in both M2 and no-MetS (both 0.34; both p<0.003). Also SV/PP was lower in MetS by M1 than in either no-MetS or MetS by M2 (both p<0.0001). Conclusions. Diagnosis of MetS based on real-time abnormal risk factors is associated with more adverse CV phenotype than found in subjects with history of exposure corrected by treatment. The correction of MetS in some subjects is due to normalization of metabolic abnormalities, possibly due to concomitant therapy, with BP frequently suboptimal, and is associated with a more benign CV phenotype, intermediate between MetS and no-MetS.

9.17 Influence of Non-Haemodynamic Factors on Left Ventricular Mass in Adolescents with High Prevalence of Obesity: the Strong Heart Study

Introduction. Metabolic syndrome (MetS) definition is based on either abnormal values, or ongoing therapy demonstrating history of exposure to risk factors. This classification implies that therapy does not modify the MetS status, whereas therapy-modified components might modify attribution to, and prognosis of MetS. We tested the hypothesis that definition of MetS based on history rather than on direct evidence of abnormal risk factors is associated with a more benign cardiovascular (CV) phenotype. Methods. We studied 1642 African-American (1045 women) and 1289 Caucasian (681 women) HyperGEN participants, without prevalent CV disease or renal failure (serum creatinine >2.5mg/dL). MetS was defined by ATPIII, using cut-offs of blood tests, waist girth and measured blood pressure (BP), irrespective of on-going therapy (M1). M1 was compared with a subgroup of participants classified as MetS, based on history of exposure and on-going therapy to normalize risk factors (M2). We used LV mass index (LVMi), relative wall thickness (RWT) and a the ratio of stroke volume to pulse pressure (SV/PP) adjusted by heart rate, body weight and age (prognostically validated as a raw estimate of arterial compliance), as measures of end-organ damage. Results. Among 1102 participants with MetS (38%), 780 were confirmed by M1, whereas 322 (15%) had MetS by M2; 1892 were free of MetS by both criteria (no-MetS). Among M2 participants, 67% had normal fasting glucose, 71% normal triglycerides, 32% normal HDL-cholesterol, 16% normal waist girth and only 4% had normal BP. After adjusting for age, sex and ethnicity, mean LVMi was higher with in M1 (41.9 g/m 2.7) than in M2 (39.9 g/m 2.7) or no-Mets participants (37.6g/m2.7; all p<0.01). Similarly, RWT was higher in M1 (0.35) than in both M2 and no-MetS (both 0.34; both p<0.003). Also SV/PP was lower in MetS by M1 than in either no-MetS or MetS by M2 (both p<0.0001). Conclusions. Diagnosis of MetS based on real-time abnormal risk factors is associated with more adverse CV phenotype than found in subjects with history of exposure corrected by treatment. The correction of MetS in some subjects is due to normalization of metabolic abnormalities, possibly due to concomitant therapy, with BP frequently suboptimal, and is associated with a more benign CV phenotype, intermediate between MetS and no-MetS.

Metabolic phenotypes, vascular complications, and premature deaths in a population of 4,197 patients with type 1 diabetes.

OBJECTIVE—Poor glycemic control, elevated triglycerides, and albuminuria are associated with vascular complications in diabetes. However, few studies have investigated combined associations between metabolic markers, diabetic kidney disease, retinopathy, hypertension, obesity, and mortality. Here, the goal was to reveal previously undetected association patterns between clinical diagnoses and biochemistry in the FinnDiane dataset.RESEARCH DESIGN AND METHODS—At baseline, clinical records, serum, and 24-h urine samples of 2,173 men and 2,024 women with type 1 diabetes were collected. The data were analyzed by the self-organizing map, which is an unsupervised pattern recognition algorithm that produces a two-dimensional layout of the patients based on their multivariate biochemical profiles. At follow-up, the results were compared against all-cause mortality during 6.5 years (295 deaths).RESULTS—The highest mortality was associated with advanced kidney disease. Other risk factors included 1) a profile of insulin resistance, abdominal obesity, high cholesterol, triglycerides, and low HDL2 cholesterol, and 2) high adiponectin and high LDL cholesterol for older patients. The highest population-adjusted risk of death was 10.1-fold (95% CI 7.3–13.1) for men and 10.7-fold (7.9–13.7) for women. Nonsignificant risk was observed for a profile with good glycemic control and high HDL2 cholesterol and for a low cholesterol profile with a short diabetes duration.CONCLUSIONS—The self-organizing map analysis enabled detailed risk estimates, described the associations between known risk factors and complications, and uncovered statistical patterns difficult to detect by classical methods. The results also suggest that diabetes per se, without an adverse metabolic phenotype, does not contribute to increased mortality.

The adverse cardiopulmonary phenotype of caveolin-1 deficient mice is mediated by a dysfunctional endothelium

Recently generated caveolin-1 deficient mice ( cav-1 −/− ) display several physiological alterations such as severe heart failure and lung fibrosis. The molecular mechanisms how the loss of caveolin-1 (cav-1) mediates these alterations are currently under debate. A plethora of studies support a role of cav-1 as a negative regulator of endothelial nitric oxide synthase (eNOS). Accordingly, constitutive eNOS hyperactivation was observed in cav-1 −/− . Given the hyperactivated eNOS enzyme we hypothesized that disturbed eNOS function is involved in the development of the cardiopulmonary pathologies in cav-1 −/− . The present study argues that loss of cav-1 results in enhanced eNOS activity but not in increased vascular tetrahydrobiopterin (BH 4) levels (which acts as an essential eNOS cofactor) thereby causing a stoichiometric discordance between eNOS activity and BH 4 sufficient to cause dysfunctional eNOS signaling. The resultant oxidative stress is largely responsible for major cardiac and pulmonary defects observed in cav-1 −/− . BH 4 donation to cav-1 −/− led to a normalized BH 4/BH 2 ratio, to reduced oxidant stress, to substantial improvements of both systolic and diastolic heart function and to marked amelioration of the impaired lung phenotype. Notably, the antioxidant tetrahydroneopterin which is not essential for eNOS function showed no relevant effect. Taken together these novel findings indicate that dysfunctional eNOS is of central importance in the genesis of the cardiopulmonary phenotype of cav-1 −/− . Additionally, these findings are generally of paramount importance since they underline the deleterious role of an uncoupled eNOS in cardiovascular pathology and they additionally suggest BH 4 as an effective cure.

The mTOR target 4E‐BP1 contributes to differential protein expression during normoxia and hypoxia through changes in mRNA translation efficiency

Hypoxia causes a rapid and sustained inhibition in mRNA translation that is characterized by both a transient phosphorylation of eukaryotic initiation factor 2-alpha (eIF2alpha) and by inhibition of the mRNA cap binding protein eIF4E via activation of two distinct inhibitory proteins, the mammalian target of rapamycin (mTOR) target 4E-BP1 and the eIF4E transporter 4E-T. Although the importance of eIF2alpha phosphorylation during hypoxia has been clearly demonstrated, there is little information on the potential relevance of eIF4E regulation. We generated HeLa cells stably expressing a short hairpin interfering RNA (shRNA) against 4E-BP1 and found that despite efficient knockdown, no significant changes occurred in the overall inhibition of mRNA translation during hypoxia. However, using a proteomics approach we identified seven proteins that were exclusively expressed in the 4E-BP1 knockdown cells during both normoxic and hypoxic conditions. Further investigation of the transcriptional and translational regulation of these genes by quantitative RT-PCR indicated that the loss of 4E-BP1 causes a significant increase in the rate of protein synthesis of S100 calcium-binding protein A4 (S100A4) and transgelin 2. These 4E-BP1 regulated proteins have previously been associated with tumor cell motility, invasion and metastasis and may thus contribute to an adverse tumor phenotype.

Chronic NOS inhibition prevents adverse lung remodeling and pulmonary arterial hypertension in caveolin-1 knockout mice

Recently generated caveolin-1 deficient mice (cav-1 ko) suffer from severe lung fibrosis with marked pulmonary hypertension and arterial hypoxemia and may therefore serve as an useful animal model of this devastating human disorder. Accumulating evidence strongly supports the negative regulatory influence of caveolin-1 on endothelial nitric oxide synthase resulting in a constitutive hyperactivation of the nitric oxide (NO) pathway in cav-1 ko. We therefore hypothesized that a disturbed NO signaling is implicated in the evolution of the adverse lung phenotype of cav-1 ko. For this purpose, cav-1 ko of 2 months age were compared with knockout counterparts experiencing 2-month postnatal NO synthase inhibition by N G-nitro- l-arginine methyl ester ( l-NAME) treatment. Chronic l-NAME administration prevented adverse lung remodeling in cav-1 ko. Furthermore, l-NAME donation led to a normalized oxygen saturation (91.5±1.8% vs. 98.5±2.3%, P<0.01, n=10–12), a marked decrease in right ventricular hypertrophy (LV/RV ratio: 4.0±0.3 vs. 2.7±0.3, P<0.01, n=10–12) and reductions of the elevated pulmonary artery pressure (40.2±3.1 mmHg vs. 26.3±4.6 mmHg, P<0.01, n=6). Collectively, these improvements resulted in an enhanced exercise capacity of l-NAME-treated cav-1 ko. Finally, we found evidence for enhanced oxidative stress in untreated cav-1 ko which was substantially reduced by chronic l-NAME administration to cav-1 ko. In view of these data, we speculate that a perturbation of NO signaling, together with enhanced O 2 − production originating from NO synthases, may play a pivotal role in the pathogenesis of the adverse pulmonary phenotype seen in cav-1 ko.

Respiratory Virus-Induced Regulation of Asthma-Like Responses in Mice Depends upon CD8 T Cells and Interferon-γ Production

Respiratory virus infections can significantly influence the development of airway disease by both predisposing and exacerbating the developing lung immune environment. In contrast, the initiation of a more desirable anti-viral response may better prepare the local environment and protect it from developing an adverse long-term disease phenotype. BALB/c or C57BL/6 mice exposed to respiratory syncytial virus (RSV) infection at the same time as allergen sensitization were assessed for airway function, cytokine responses, and inflammatory parameters. Depending on the genetic strain of mouse used, BALB/c versus C57BL/6, RSV could differentially protect against the development of airway allergen responses. Although RSV was able to block allergen sensitization and induction of airway hyperresponsiveness and eosinophilic inflammation in C57BL/6 mice, the infection did not reduce the allergic responses in BALB/c mice. The alteration of airway responsiveness did not depend on the timing of RSV infection in C57BL/6 mice in conjunction to the allergen sensitization protocol. Neutralization experiments demonstrated that interferon-gamma contributed significantly to the RSV-induced airway attenuation of the allergic responses, whereas transfer of CD8 T cells from RSV-infected animals suggested that they were partially responsible for the altered environment. These data suggest that a respiratory viral infection impacts on the local lung environment and may reflect specific aspects of the hygiene hypothesis. However, the outcome of this interaction depends on the immunological response of the host.

Mitochondria Damage Leading to High Frequency of Lymphoma and Myeloproliferative Disorder in Transgenic Mice Overexpressing a Human 8-Oxoguanine DNA-Glycosylase Gene.

Alterations of nuclear genes in human lymphoma and leukemias have been well investigated in past several decades and established a predominant role in the pathogenesis. However, the relationship of mitochondrial genome alteration or dysfunction and human lymphoma and leukemias remains large unknown. Mitochondria are dynamic organelles that play critical roles in oxidative phosphorylation, energy metabolism, cell growth and apoptosis. We have successfully generated a novel transgenic (TG) mouse model of mitochondrial disorder by overexpressing human hOGG1, a base excision DNA repair gene, in the mitochondria of a wide variety of tissues in mice. We reported here the high frequency of essential thrombocythemia (ET)-like myeloproliferative disorder and lymphoma in the TG mice. TG mice were generated via pronuclear microinjection of a mammalian expression construct of a mitochondrial isoform of hOGG under the control of a mouse metallothionein-1 promoter. Peripheral blood smears were prepared from TG and non-TG control mice for platelet counts and morphologic evaluation. TG mice were sacrificed and various organs were harvested for histologic, biochemical and molecular studies. Two of the male founder mice successfully transmitted the transgene to their offspring at an expected frequency of 50%. All the female mice failed to reproduction. All TG mice expressed high levels of hOGG mRNA in their liver by RT-PCR and direct DNA sequencing. Over-expression of this gene produced a wide range of adverse biological phenotype, manifesting early-onset obesity, metabolic disturbance, female infertility and high frequency of ET-like myeloproliferative disorder and lymphoma (>90%) in nodal and extranodal sites. Eight TG mice (ranging from 1 to 2 years) become moribund and subsequently sacrificed. Upon histologic examination, the TG mice displayed moderate to severe splenomegaly. The spleen weights were 2-, 4- and 10 times respectively in the 3 TG founder mice as compared to wild type, age-matched mice. Extensive abdominal lymphadenopathy and numerous enlarged nodules involving liver, spleen, peritoneum, lung and diaphragm were identified. Microscopically, the red pulp in the enlarged spleens is markedly expanded with aggregates of large, mature but dysplastic-appearing megakaryocytes, focally disrupting the normal splenic structure. Various lymphomas ranged from low-grade lymphoma to high-grade Burkitt-like or lymphoblastic lymphomas were seen. Peripheral blood from these aged TG mice showed marked thrombocytosis and platelet clumping. In bone marrow, the aged TG mice displayed marked myeloid and megakaryocytic hyperplasia with relative erythroid hypoplasia in the absence of marrow fibrosis, indicative of myeloid and megakaryocytic proliferation. Bone marrow involvement by B-cell lymphoma was also seen. Of note, none of the above phenotype was seen in younger TG mice (4 months), indicating that the full development of this pathologic process is age-dependent. The molecular basis for this process is currently under investigation. Our preliminary data showed that mitochondrial DNA deletion or decrease in DNA copy numbers due to overexpressed hOGG1 and imbalance of base excision repair resulted in defects in mitochondrial respiration and increased ROS production. We thus hypothesize that oxidative stress caused by mitochondrial malfunction may play important part in the development of hematopoietic malignancies.

Toxicogenomics and Cross-Species Biomarker Discovery: Applications in Drug Discovery and Safety Assessment

Toxicogenomics has evolved into a useful technique for providing greater mechanistic insights into adverse effects that will spur the development of novel approaches for identifying and understanding toxicity issues. The ability to capture a snapshot of the transcriptome at any given time during the development of an adverse phenotype allows unprecedented molecular views into the dynamic physiological changes that are occurring on either time or dose continuum for a toxicology study of interest advancing our basic knowledge of adverse events, and providing the necessary scientific framework for developing new strategies and tools for safety assessment programs. The development of an effective subset of cost effective devices for identifying toxicity earlier in the drug development process will help identify the most promising candidate compounds to move forward leading to a reduction in compound attrition due to toxicity. In addition, there is a need in the pharmaceutical industry to develop safety and efficacy biomarkers that are relevant to multiple species such as rat, dog, and human. Genomics provides an opportunity to discover novel cross-species biomarkers for identifying phenotypes such as liver fibrosis, especially if the biomarkers are tissue specific secreted proteins that can be monitored in the serum. This review includes an example of how databases from multiple species, in this case rat and human tissues, can be utilized to identify candidate cross-species diagnostic markers of hepatitis and fibrosis. This study illustrates a genomic approach for identifying candidate cross-species biomarkers of cirrhosis/fibrosis for humans and rats, and a previously known biomarker of fibrosis (APOA1) and a novel candidate biomarker of fibrosis, FETUB were identified. As more “omic” databases are built, a reservoir of molecular information will become available for toxicologist to gain more extensive views on the physiological alterations induced by adverse events, which will inevitably lead to the development of better tools for predicting, identifying, categorizing, and determining cross-species impact of the toxicity and ultimate provide a novel scientific scaffold for improving safety assessment protocols.

The role of fetal inherited thrombophilia in the development of adverse pregnancy outcomes

ADVERSE PREGNANCY OUTCOMES CATHERINE GIBSON, NARD JANSSEN, WILLEM KIST, ALASTAIR MACLENNAN, BILL HAGUE, ERIC HAAN, PAUL GOLDWATER, KEVIN PRIEST, GUSTAAF DEKKER, Adelaide University, Obstetrics and Gynaecology, Adelaide, South Australia, Australia, VU University Medical Center, Obstetrics and Gynaecology, Amsterdam, Netherlands, Women’s and Children’s Hospital, Adelaide, North Adelaide, South Australia, Australia, Women’s andChildren’sHospital,DepartmentofGeneticMedicine, Adelaide, South Australia, Australia, Women’s and Children’s Hospital, Microbiology and Infectious Diseases, Adelaide, South Australia, Australia, Department of Health, Epidemiology Branch, Adelaide, South Australia, Australia, AdelaideUniversity,MaternalMedicine, Adelaide, SouthAustralia, Australia OBJECTIVE: To investigate the role of fetal inherited thrombophilia in the development of a range of adverse pregnancy outcomes, including pregnancyinduced hypertensive disorders (PIHD), antepartum haemorrhage (APH), intrauterine growth restriction!10th percentile (IUGR) andpretermbirth (PTB). STUDY DESIGN: 717 cases and 609 controls were genotyped for Factor V Leiden (FVL, G1691A), Prothrombin gene mutation (PGM, G20210A), and Methylenetetrahydrofolate reductase (MTHFR) C677T and MTHFR A1298C using genomic DNA extracted from newborn screening cards. RESULTS: For babies born!28 weeks gestation, PGM was associated with an increased risk of IUGR(OR6.40, 1.66-24.71) andAPHwith IUGR(OR6.35, 1.63-24.75). Homozygous PGM also increased the risk of PIHD with IUGR for term-born babies (OR 50.81, 1.75-1476.90). Homozygosity for MTHFR A1298C was associated with an increased risk of IUGR for babies born 28-31 weeks gestation (OR 4.00, 1.04-15.37), and with APH and IUGR for babies born !32 weeks gestation (OR 3.57, 1.09-11.66). MTHFR C677T was associated with a reduced risk of PTB and IUGR (OR 0.52, 0.28-0.96) for babies born 32-36 weeks gestation. Homozygous FVL was associated with an increased risk of PIHD with IUGR for term-born babies (OR 37.15, 1.33-1041.30), but decreased the risk of PTB!32 weeks gestation (OR 0.55, 0.31-0.98). There were no associations with any thrombophilic polymorphism and APH alone. CONCLUSION: These results suggest that some fetal thrombophilic polymorphisms may be related to adverse pregnancy outcomes, in particular IUGR, but this may not be the only association. Further studies matching maternal and fetal genotypes are required to investigate if both are needed for the adverse pregnancy outcome phenotype to be expressed. S14 SMFM Abstracts

Abbreviated action potential kinetics in a mouse model of potassium channel overexpression during hippocampal development.

Loss or "gain" of function mutations in voltage-gated ion channels often results in an adverse neurological phenotype. We have examined the electrical characteristics of hippocampal pyramidal cells in a transgenic mouse model to determine how overexpression of a Shaker-type potassium channel subunit during early postnatal development might alter excitability properties of developing neurons. We found that in CA3 neurons potassium channel overexpression led to a transient shortening in duration of single action potentials during the first two postnatal weeks. There was an increase in maximal repolarization rate, without significant effect on the rate of rise. Transgenic CA3 neurons also showed a decrease of firing frequency in response to sustained depolarizing current injection. In contrast, repolarization of action potentials in CA1 neurons was not significantly altered by trangene expression. Western Blot Analysis of membrane-associated transgene protein indicated that transgene protein levels decreased during development, in agreement with functional measures of spike width. Our data indicate that the functional consequences of potassium channel transgene expression are dependent on cellular environment and developmental stage. A transient period of hypoexcitability during a critical period of development for CA3 neurons may contribute to the hyperexcitable phenotype observed in adult animals.

Transgene-induced lesion mimic.

Lesion mimic, i.e., the spontaneous formation of lesions resembling hypersensitive response (HR) lesions in the absence of a pathogen, is a dramatic phenotype occasionally found to accompany the expression of different, mostly unrelated, transgenes in plants. Recent studies indicated that transgene-induced lesion formation is not a simple case of necrosis, i.e., direct killing of cells by the transgene product, but results from the activation of a programmed cell death (PCD) pathway. Moreover, activation of HR-like cell death by transgene expression is viewed as an important evidence for the existence of a PCD pathway in plants. The study of lesion mimic transgenes is important to our understanding of PCD and the signals that control it in plants. PCD-inducing transgenes may provide clues regarding the different entry points into the cell death pathway, the relationships between the different branches of the pathway (e.g., developmental or environmental), or the different mechanisms involved in its induction or execution. Cell death-inducing transgenes may also be useful in biotechnology. Some lesion mimic transgenes were found to be induced in plants a state of systemic acquired resistance (SAR). These genes can be used in the development of pathogen-resistant crops. Other cell death-inducing transgenes may be used as specific cell ablation tools. Although mainly revealed unintentionally, and at times considered 'an adverse phenotype', lesion mimic transgenes should not be ignored because they may prove valuable for studying PCD as well as developing useful traits in different plants and crops.

The unusual translational initiation codon AUU limits the expression of the infC (initiation factor IF3) gene of Escherichia coli.

The expression of infC, the structural gene for translational initiation factor IF3, has been studied in different constructs under the control of the lambda PL and tac promoters. The amount of synthesized IF3 has been determined by a quantitative functional test and the levels of IF3-specific mRNA have been estimated. The synthesis of IF3 is strongly enhanced when the unusual AUU initiation codon is changed to AUG by site-directed mutagenesis. Removal of the sequence upstream from the start codon including most of the Shine-Dalgarno sequence, as well as part of a 10 bp region with potential complementarity to an internal region of the 16S rRNA, which is unique to the IF3 mRNA, reduced but did not completely abolish the high expression of infC obtained after introduction of the AUG initiation codon. The level of IF3 mRNA was found to be positively influenced by the presence of the rplT gene in the plasmid downstream from the infC gene. In vivo accumulation of a large excess of IF3, obtained when the infC gene was placed under the control of an incompletely repressed tac promoter, was not accompanied by any noticeable adverse phenotype.

Description of a large pedigree with an adverse lipoprotein cholesterol phenotype: the Bogalusa Heart Study.

A large pedigree (N = 356) with a high prevalence of heart disease and associated adverse lipoprotein phenotype was studied. The adverse lipoprotein phenotype is characterized by both low levels of high-density-lipoprotein cholesterol (HDL-C) alone (16.3%) and in combination with other adverse lipoprotein levels (12.8%). In all, 44.2% of all pedigree members had at least one adverse lipoprotein level. Analysis of mating types showed that all lipids and lipoproteins possess familial clustering with 25-36% of offspring above median levels when both parents had levels below the median, while 67-83% had levels above the median when both parents had levels above the median. Using adjusted lipid and lipoprotein levels, a statistically significant linear trend was found between the degree of relationship to pedigree members with heart disease, and both the low-density-lipoprotein cholesterol/high-density-lipoprotein cholesterol (LDL-C/HDL-C) ratio (P less than .05), and the very-low-density-lipoprotein cholesterol (VLDL-C; P less than .01) level. A similar analysis using the prevalence of adverse lipoprotein levels as the dependent variable and degree of relationship to heart diseased pedigree numbers as the independent variable showed significant (P less than .05) relationships with VLDL-C and the LDL-C/HDL-C ratio. Further genetic analyses of this pedigree may reveal genetic mechanisms responsible for the familiality of lipoprotein levels in this pedigree.