A vascular gene trap screen defines RasGRP3 as an angiogenesis-regulated gene required for the endothelial response to phorbol esters

We identified Ras guanine-releasing protein 3 (RasGRP3) as a guanine exchange factor expressed in blood vessels via an embryonic stem (ES) cell-based gene trap screen to identify novel vascular genes. RasGRP3 is expressed in embryonic blood vessels, down-regulated in mature adult vessels, and reexpressed in newly formed vessels during pregnancy and tumorigenesis. This expression pattern is consistent with an angiogenic function for RasGRP3. Although a loss-of-function mutation in RasGRP3 did not affect viability, RasGRP3 was up-regulated in response to vascular endothelial growth factor (VEGF) stimulation of human umbilical vein endothelial cells, placing RasGRP3 regulation downstream of VEGF signaling. Phorbol esters mimic the second messenger diacylglycerol (DAG) in activating both protein kinase C (PKC) and non-PKC phorbol ester receptors such as RasGRP3. ES cell-derived wild-type blood vessels exposed to phorbol myristate acetate (PMA) underwent extensive aberrant morphogenesis that resulted in the formation of large endothelial sheets rather than properly branched vessels. This response to PMA was completely dependent on the presence of RasGRP3, as mutant vessels were refractory to the treatment. Taken together, these findings show that endothelial RasGRP3 is up-regulated in response to VEGF stimulation and that RasGRP3 functions as an endothelial cell phorbol ester receptor in a pathway whose stimulation perturbs normal angiogenesis. This suggests that RasGRP3 activity may exacerbate vascular complications in diseases characterized by excess DAG, such as diabetes.     

Alpha-actinin-4-mediated FSGS: an inherited kidney disease caused by an aggregated and rapidly degraded cytoskeletal protein

Focal segmental glomerulosclerosis (FSGS) is a common pattern of renal injury, seen as both a primary disorder and as a consequence of underlying insults such as diabetes, HIV infection, and hypertension. Point mutations in the alpha-actinin-4 gene ACTN4 cause an autosomal dominant form of human FSGS. We characterized the biological effect of these mutations by biochemical assays, cell-based studies, and the development of a new mouse model. We found that a fraction of the mutant protein forms large aggregates with a high sedimentation coefficient. Localization of mutant alpha-actinin-4 in transfected and injected cells, as well as in situ glomeruli, showed aggregates of the mutant protein. Video microscopy showed the mutant alpha-actinin-4 to be markedly less dynamic than the wild-type protein. We developed a "knockin" mouse model by replacing Actn4 with a copy of the gene bearing an FSGS-associated point mutation. We used cells from these mice to show increased degradation of mutant alpha-actinin-4, mediated, at least in part, by the ubiquitin-proteasome pathway. We correlate these findings with studies of alpha-actinin-4 expression in human samples. "Knockin" mice with a disease-associated Actn4 mutation develop a phenotype similar to that observed in humans. Comparison of the phenotype in wild-type, heterozygous, and homozygous Actn4 "knockin" and "knockout" mice, together with our in vitro data, suggests that the phenotypes in mice and humans involve both gain-of-function and loss-of-function mechanisms.     

Influence of food availability on demography and local population dynamics in a long-lived seabird

Few studies have addressed the effects of food availability as a proximate factor affecting local adult survival in long-lived organisms and their consequences at local population dynamics. We used capture-recapture analysis of resightings of 10 birth cohorts of ringed Audouin's gulls, Larus audouinii, to estimate adult survival and dispersal (both emigration and immigration). For the first time, permanent emigration (the transient effect in capture-recapture analysis) was modelled for the whole population and not only for the newly marked birds. Gulls exploit to a large extent fishes discarded from trawlers, and a trawling moratorium established since 1991 has decreased food supply for the colony. This was used as a natural experiment of food availability to assess its effects on adult survival and emigration. These and other demographic parameters were used in a projection modelling to assess the probabilities of extinction of the colony under two scenarios of lower and higher food availability. Food availability (together with the age of individuals) influenced emigration probabilities, but not adult survival, which was estimated at 0.91 (s.e. = 0.02). When food was in shorter supply during the chick-rearing period, emigration was very high (ca. 65%) for younger breeders, although this rate decreased sharply with age. Probabilities of extinction were very high when food availability was low, and when environmental stochasticity was introduced, and only stochastic immigration from the outside seemed to prevent extinction. The results highlight the importance of dispersal processes in the population dynamics of long-lived organisms.     

Magnetic fields and the melatonin hypothesis: a study of workers chronically exposed to 50-Hz magnetic fields

Because epidemiological studies report clinical disorders (mainly neurobehavioral alterations and/or cancer) that may be related to diminished melatonin secretion or to changes in its circadian rhythm in subjects living or working in environments exposed to magnetic fields, research on the effects of these fields in humans is particularly important. In this study, we examine the circadian rhythm of melatonin in 15 men exposed chronically and daily for a period of 1-20 yr, in the workplace and at home, to a 50-Hz magnetic field in search of any cumulative effect from those chronic conditions of exposure. The weekly geometric mean of individual exposures ranged from 0.1 to 2.6 microT. The results are compared with those for 15 unexposed men who served as controls (individual exposures ranged from 0.004 to 0.092 microT). Blood samples were taken hourly from 2000 to 0800. Nighttime urine was also collected and analyzed. This work shows that subjects exposed over a long period (up to 20 yr) and on a daily basis to magnetic fields experienced no changes in their plasma melatonin level, their urinary 6-sulfatoxymelatonin level, or the circadian rhythm of melatonin. Our data strongly suggest that magnetic fields do not have cumulative effects on melatonin secretion in humans and thus clearly rebut the "melatonin hypothesis" that a decrease in plasma melatonin concentration (or a disruption in its secretion) explains the occurrence of clinical disorders or cancers possibly related to magnetic fields.     

The cationic amphipathic alpha-helix of HIV-1 viral protein R (Vpr) binds to nucleic acids,permeabilizes membranes,and efficiently transfects cells

Viral protein R (Vpr) is a small protein of 96 amino acids that is conserved among the lentiviruses human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus. We recently sought to determine whether the karyophilic properties of Vpr, as well as its ability to bind nucleic acids, could be used to deliver DNA into cells. We have found that the C-terminal domain of Vpr-(52-96) is able to efficiently transfect various cell lines. Here, we show that the shortest active sequence for gene transfer corresponds to the domain that adopts a alpha-helix conformation. DNA binding studies and permeabilization assays performed on cells demonstrated that the peptides that are efficient in transfection condense plasmid DNA and are membranolytic. Electron microscopy studies and transfection experiments performed in the presence of inhibitors of the endocytic processes indicated that the major entry pathway of Vpr-DNA complexes is through endocytosis. Taken together, the results show that the cationic C-terminal alpha-helix of Vpr has DNA-condensing as well as membrane-destabilizing capabilities, both properties that are indispensable for efficient DNA transfection.     

Reproducibility of the circadian rhythms of serum cortisol and melatonin in healthy subjects: a study of three different 24-h cycles over six weeks

Plasma melatonin and cortisol are characterized by a marked circadian rhythm, but little information is available about the reproducibility and stability of these rhythms over several weeks in the same subjects. This study examined the characteristics of these rhythms in 31 healthy human subjects 20 to 30 years of age. They were synchronized with a diurnal activity from 0800 to 2300 and nocturnal rest. They participated in three 24-hour sessions (S1, S2, and S3): S2 took place two weeks after S1 and S3 4 weeks after S2. Blood samples were taken during each session at 3-hour intervals from 1100 to 2000 and hourly from 2200 to 0800. Comparison of the circadian rhythms between groups used repeated measures 2-way ANOVA, the cosinor method, and Bingham's test. Intraindividual variations were compared by the cosinor method and Bingham's test. The groups did not differ, but a slight difference in the amplitude or acrophase of individual circadian rhythms was observed in 5 of 31 subjects for melatonin and 1 of 31 for cortisol. The circadian means did not differ over the three sessions. These results show that the circadian profile of cortisol and melatonin are highly reproducible over a six-week period, in both individuals and groups. Our study clearly shows that these hormones can be considered to be stable markers of the circadian time structure and therefore useful tools to validate rhythms' synchronisation of human subjects.     

Assessment of thin-layer breast aspirates for immunocytochemical evaluation of HER2 status

OBJECTIVE: To determine whether immunocytochemistry (ICC) for HER2 on ThinPrep (TP)-processed breast fine needle aspiration biopsies (Cytyc Corp., Boxborough, Massachusetts, U.S.A.) is comparable to the findings of immunohistochemistry on corresponding surgically removed tissue. STUDY DESIGN: Immunostaining was performed on 63 malignant breast fine needle aspirates and compared to immunostaining on paraffin sections (PSs) from the subsequent biopsies. The HercepTest (Dako, Carpinteria, California, U.S.A.) and TAB250 antibodies were utilized. Cases in which the TP and paraffin HER2 results did not correlate were further assessed for gene amplification by differential polymerase chain reaction (dPCR). RESULTS: HER2 overexpression was found in 9 of the 63 cases (14%). TAB250 had higher specificity on PS versus TP (P = .008), and TAB250 had higher specificity on PS versus the HercepTest on PS and TP (P = .004 and .0001, respectively). CONCLUSION: HER2 immunostaining with both the HercepTest and TAB250 on TP is unreliable due to low specificity (72% and 83% for HercepTest and TAB250, respectively). However, both antibodies have high sensitivity (89% and 100%, respectively); suggesting that this method may have some utility as a preliminary screening test for HER2 status. Negative HER2 staining by ICC is highly predictive of the absence of HER2 overexpression, whereas positive HER2 staining on TP would require further validation by either dPCR of fluorescence in situ hybridization.     

Adsorption of DNA to mica mediated by divalent counterions: a theoretical and experimental study

The adsorption of DNA molecules onto a flat mica surface is a necessary step to perform atomic force microscopy studies of DNA conformation and observe DNA-protein interactions in physiological environment. However, the phenomenon that pulls DNA molecules onto the surface is still not understood. This is a crucial issue because the DNA/surface interactions could affect the DNA biological functions. In this paper we develop a model that can explain the mechanism of the DNA adsorption onto mica. This model suggests that DNA attraction is due to the sharing of the DNA and mica counterions. The correlations between divalent counterions on both the negatively charged DNA and the mica surface can generate a net attraction force whereas the correlations between monovalent counterions are ineffective in the DNA attraction. DNA binding is then dependent on the fractional surface densities of the divalent and monovalent cations, which can compete for the mica surface and DNA neutralizations. In addition, the attraction can be enhanced when the mica has been pretreated by transition metal cations (Ni(2+), Zn(2+)). Mica pretreatment simultaneously enhances the DNA attraction and reduces the repulsive contribution due to the electrical double-layer force. We also perform end-to-end distance measurement of DNA chains to study the binding strength. The DNA binding strength appears to be constant for a fixed fractional surface density of the divalent cations at low ionic strength (I < 0.1 M) as predicted by the model. However, at higher ionic strength, the binding is weakened by the screening effect of the ions. Then, some equations were derived to describe the binding of a polyelectrolyte onto a charged surface. The electrostatic attraction due to the sharing of counterions is particularly effective if the polyelectrolyte and the surface have nearly the same surface charge density. This characteristic of the attraction force can explain the success of mica for performing single DNA molecule observation by AFM. In addition, we explain how a reversible binding of the DNA molecules can be obtained with a pretreated mica surface.