Adeno-associated virus type 2-mediated transfer of ecotropic retrovirus receptor cDNA allows ecotropic retroviral transduction of established and primary human cells.

The cellular receptors that mediate binding and internalization of retroviruses have recently been identified. The concentration and accessibility of these receptors are critical determinants in accomplishing successful gene transfer with retrovirus-based vectors. Murine retroviruses containing ecotropic glycoproteins do not infect human cells since human cells do not express the receptor that binds the ecotropic glycoproteins. To enable human cells to become permissive for ecotropic retrovirus-mediated gene transfer, we have developed a recombinant adeno-associated virus type 2 (AAV) vector containing ecotropic retroviral receptor (ecoR) cDNA under the control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter (vRSVp-ecoR). Established human cell lines, such as HeLa and KB, known to be nonpermissive for murine ecotropic retroviruses, became permissive for infection by a retroviral vector containing a bacterial gene for resistance to neomycin (RV-Neo(r)), with a transduction efficiency of up to 47%, following transduction with vRSVp-ecoR, as determined by the development of colonies that were resistant to the drug G418, a neomycin analog. No G418-resistant colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. Southern and Northern blot analyses revealed stable integration and long-term expression, respectively, of the transduced murine ecoR gene in clonal isolates of HeLa and KB cells. Similarly, ecotropic retrovirus-mediated Neo(r) transduction of primary human CD34+ hematopoietic progenitor cells from normal bone marrow was also documented, but only following infection with vRSVp-ecoR. The retroviral transduction efficiency was approximately 7% without prestimulation and approximately 14% with prestimulation of CD34+ cells with cytokines, as determined by hematopoietic clonogenic assays. No G418-resistant progenitor cell colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. These results suggest that sequential transduction of primary human cells with two different viral vectors may overcome limitations encountered with a single vector. Thus, the combined use of AAV- and retrovirus-based vectors may have important clinical implications for ex vivo and in vivo human gene therapy.     

Symbiont community diversity is more variable in corals that respond poorly to stress

Coral reefs are declining globally as climate change and local water quality press environmental conditions beyond the physiological tolerances of holobionts—the collective of the host and its microbial symbionts. To assess the relationship between symbiont composition and holobiont stress tolerance, community diversity metrics were quantified for dinoflagellate endosymbionts (Family: Symbiodiniaceae) from eight Acropora millepora genets that thrived under or responded poorly to various stressors. These eight selected genets represent the upper and lower tails of the response distribution of 40 coral genets that were exposed to four stress treatments (and control conditions) in a 10‐day experiment. Specifically, four ‘best performer’ coral genets were analyzed at the end of the experiment because they survived high temperature, high pCO₂, bacterial exposure, or combined stressors, whereas four ‘worst performer’ genets were characterized because they experienced substantial mortality under these stressors. At the end of the experiment, seven of eight coral genets mainly hosted Cladocopium symbionts, whereas the eighth genet was dominated by both Cladocopium and Durusdinium symbionts. Symbiodiniaceae alpha and beta diversity were higher in worst performing genets than in best performing genets. Symbiont communities in worst performers also differed more after stress exposure relative to their controls (based on normalized proportional differences in beta diversity), than did best performers. A generalized joint attribute model estimated the influence of host genet and treatment on Symbiodiniaceae community composition and identified strong associations among particular symbionts and host genet performance, as well as weaker associations with treatment. Although dominant symbiont physiology and function contribute to host performance, these findings emphasize the importance of symbiont community diversity and stochasticity as components of host performance. Our findings also suggest that symbiont community diversity metrics may function as indicators of resilience and have potential applications in diverse disciplines from climate change adaptation to agriculture and medicine.     

Inheritable forms of medullary thyroid carcinoma

Medullary thyroid carcinoma (MTC) arises from parafollicular or C cells of the thyroid that produce calcitonin. It accounts for 5-10% of all thyroid cancers. Hereditary MTC represents 20-30% of all MTCs. It can be transmitted with an autosomal dominant pattern, either as a single entity, familial MTC, or it can arise as part of a multiple endocrine neoplasia (MEN) syndrome type 2A or 2B. The identification of hereditary MTC has been facilitated in recent years by the direct analysis of the ret proto-oncogene.     

Effects of deletion of the prolactin receptor on ovarian gene expression

Prolactin (PRL) exerts pleiotropic physiological effects in various cells and tissues, and is mainly considered as a regulator of reproduction and cell growth. Null mutation of the PRL receptor (R) gene leads to female sterility due to a complete failure of embryo implantation. Pre-implantatory egg development, implantation and decidualization in the mouse appear to be dependent on ovarian rather than uterine PRLR expression, since progesterone replacement permits the rescue of normal implantation and early pregnancy. To better understand PRL receptor deficiency, we analyzed in detail ovarian and corpora lutea development of PRLR-/- females. The present study demonstrates that the ovulation rate is not different between PRLR+/+ and PRLR-/- mice. The corpus luteum is formed but an elevated level of apoptosis and extensive inhibition of angiogenesis occur during the luteal transition in the absence of prolactin signaling. These modifications lead to the decrease of LH receptor expression and consequently to a loss of the enzymatic cascades necessary to produce adequate levels of progesterone which are required for the maintenance of pregnancy.     

Inhibition of αIIbβ3 Ligand Binding by an αIIb Peptide that Clasps the Hybrid Domain to the βI Domain of β3

Agonist-stimulated platelet activation triggers conformational changes of integrin αIIbβ3, allowing fibrinogen binding and platelet aggregation. We have previously shown that an octapeptide, ^p1YMESRADR⁸, corresponding to amino acids 313–320 of the β-ribbon extending from the β-propeller domain of αIIb, acts as a potent inhibitor of platelet aggregation. Here we have performed in silico modelling analysis of the interaction of this peptide with αIIbβ3 in its bent and closed (not swing-out) conformation and show that the peptide is able to act as a substitute for the β-ribbon by forming a clasp restraining the β3 hybrid and βI domains in a closed conformation. The involvement of species-specific residues of the β3 hybrid domain (E356 and K384) and the β1 domain (E297) as well as an intrapeptide bond (^pE315-^pR317) were confirmed as important for this interaction by mutagenesis studies of αIIbβ3 expressed in CHO cells and native or substituted peptide inhibitory studies on platelet functions. Furthermore, NMR data corroborate the above results. Our findings provide insight into the important functional role of the αIIb β-ribbon in preventing integrin αIIbβ3 head piece opening, and highlight a potential new therapeutic approach to prevent integrin ligand binding.     

Mechanisms involved in platelet activation induced by a monoclonal antibodye anti glycoprotein IIb–IIIa: Inositol phosphate production is not the primary event

The mechanisnms involved in platele aggregation by a monoclonal antibody (mAb) P256 specific for the GPIIb-IIIa complex was investigated following metabolic ³²P labelling of platelets. When compared with thrombin, inositol phosphates (InsP) production during P256-induced activation was delayed and no apparent peak, but a small and sustained production of [³²P]-Ins(1,4,5)P₃ and [³²P]-Ins(1,4,5)P₄, was observed between 20 and 90 s. [³²P]-Ins(1,3,4)P₃ was aslo produced with a manimumafter 90 s. Addition of ADP scavenger creatine phosphate/creatine phosphokinase (CP/CPK) and of the cycloxygenase inhibitor aspirin together with P256 almost totally abolished InsPP formation, whereas platelet aggregation and protein phosphorylation were partially inhibited. F(ab′)₂ fragments of P256 also aggregated platelets but to a smaller extent than IgG, and without any measurable InsPs. To characterize further P-256-induced activation, the phosphorylation of p43, the main substrate of protein kinase C (PKC) and the phosphorylation of tyrosine protein (P-Tyr) was also studied. PKC activation was smaller with P256-IgG than with thrombin but both thrombin and P265-IgG induced a similar profile of P-Tyr involving seven major bands, whereas P265-F(ab′)₂ only occasionally activated PKC but always significantly phosphorylated a 64,000 molecular P-Tyr. The data indicate that the binding of P256 to HPIIb,-IIIa, in contrast with thrombin, does not initially lead directly to the activation of the phosphoinositide phospholipase C to produce InsP's but rather involves the activation of protein kinases and also both fragments F(ab′)₂ and Fc play a specific role in the platelet responses to the mAb. Only the crosstalk between the two pathways evoked by F(ab′)₂ Fc respectively allows the activation of all platelet activation systems.     

Linkage analysis in spinal muscular atrophy, by six closely flanking markers on chromosome 5

The proximal spinal muscular atrophies (SMA) represent the second most common autosomal recessive disorder, after cystic fibrosis. The gene responsible for chronic SMA has recently been mapped to chromosome 5q by using genetic linkage studies. Among six markers mapping to this region, five were shown to be linked with the SMA locus in 39 chronic SMA families each containing at least two affected individuals. Multilocus analysis by the method of location score was used to establish the best estimate of the SMA gene location. Our data suggest that the most likely location for SMA is between loci D5S6 and D5S39. The genetic distances between these two markers are estimated to be 6.4 cM in males and 11.9 cM in females. Since meiosis were informative with D5S39 and D5S6 in 92% and 87% of SMA families, respectively, it is hoped that the present study will contribute to the calculation of genetic risk in SMA families.     

Long-term cure of the photosensitivity of murine erythropoietic protoporphyria by preselective gene therapy.

Definitive cure of an animal model of a human disease by gene transfer into hematopoietic stem cells has not yet been accomplished in the absence of spontaneous in vivo selection for transduced cells. Erythropoietic protoporphyria is a genetic disease in which ferrochelatase is defective. Protoporphyrin accumulates in erythrocytes, leaks into the plasma and results in severe skin photosensitivity. Using a mouse model of erythropoietic protoporphyria, we demonstrate here that ex vivo preselection of hematopoietic stem cells transduced with a polycistronic retrovirus expressing both human ferrochelatase and green fluorescent protein results in complete and long-term correction of skin photosensitivity in all transplanted mice.     

Altered climate leads to positive density‐dependent feedbacks in a tropical wet forest

Climate change is predicted to result in warmer and drier Neotropical forests relative to current conditions. Negative density‐dependent feedbacks, mediated by natural enemies, are key to maintaining the high diversity of tree species found in the tropics, yet we have little understanding of how projected changes in climate are likely to affect these critical controls. Over 3 years, we evaluated the effects of a natural drought and in situ experimental warming on density‐dependent feedbacks on seedling demography in a wet tropical forest in Puerto Rico. In the +4°C warming treatment, we found that seedling survival increased with increasing density of the same species (conspecific). These positive density‐dependent feedbacks were not associated with a decrease in aboveground natural enemy pressure. If positive density‐dependent feedbacks are not transient, the diversity of tropical wet forests, which may rely on negative density dependence to drive diversity, could decline in a future warmer, drier world.     

Long-term outcome of patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency

AIMS: Conflicting results exist regarding bone mineral density (BMD), metabolism and reproductive function of adult patients with congenital adrenal hyperplasia (CAH). We evaluated the long-term outcome and the impact of chronic glucocorticoid replacement in these patients. METHODS: Physical characteristics, serum hormone concentrations, BMD and metabolism were studied in 45 consecutive CAH adult patients. RESULTS: Among the 36 women, only 14 (39%) had regular menses. Among the 27 women with classical CAH, the mean number of surgical reconstructions of virilized genitalia was 2.1 +/- 0.2. Twenty of them (74%) were sexually active. Three men presented with testicular adrenal rest tumors. Twenty-five patients (55%) had decreased BMD at the femoral neck and/or at the lumbar spine. BMI was correlated with the BMD T-score at the femoral neck (p < 0.001) and at the lumbar spine (p < 0.01). Hydrocortisone dose was negatively correlated with the BMD T-score at the femoral neck (p = 0.04). Subjects with osteopenia had a significantly lower BMI and received higher hydrocortisone dose than those with normal BMD. Overweight was found in 21 patients (47%). There was a significantly positive correlation between HOMA and BMI (p < 0.001), and between HOMA and 17-OHP levels (p = 0.016). CONCLUSIONS: Adult patients with CAH treated with long-term glucocorticoids are at risk for decreased BMD, increased BMI, and disturbed reproductive function.