Adaptation and mal-adaptation to ambient hypoxia; Andean, Ethiopian and Himalayan patterns

The study of the biology of evolution has been confined to laboratories and model organisms. However, controlled laboratory conditions are unlikely to model variations in environments that influence selection in wild populations. Thus, the study of "fitness" for survival and the genetics that influence this are best carried out in the field and in matching environments. Therefore, we studied highland populations in their native environments, to learn how they cope with ambient hypoxia. The Andeans, African highlanders and Himalayans have adapted differently to their hostile environment. Chronic mountain sickness (CMS), a loss of adaptation to altitude, is common in the Andes, occasionally found in the Himalayas; and absent from the East African altitude plateau. We compared molecular signatures (distinct patterns of gene expression) of hypoxia-related genes, in white blood cells (WBC) from Andeans with (n = 10), without CMS (n = 10) and sea-level controls from Lima (n = 20) with those obtained from CMS (n = 8) and controls (n = 5) Ladakhi subjects from the Tibetan altitude plateau. We further analyzed the expression of a subset of these genes in Ethiopian highlanders (n = 8). In all subjects, we performed the studies at their native altitude and after they were rendered normoxic. We identified a gene that predicted CMS in Andeans and Himalayans (PDP2). After achieving normoxia, WBC gene expression still distinguished Andean and Himalayan CMS subjects. Remarkably, analysis of the small subset of genes (n = 8) studied in all 3 highland populations showed normoxia induced gene expression changes in Andeans, but not in Ethiopians nor Himalayan controls. This is consistent with physiologic studies in which Ethiopians and Himalayans show a lack of responsiveness to hypoxia of the cerebral circulation and of the hypoxic ventilatory drive, and with the absence of CMS on the East African altitude plateau.     

A UV Tolerant Mutant of Bacillus thuringiensis subsp. kurstaki Producing Melanin

A UV-resistant mutant (Bt-m) of Bacillus thuringiensis subsp. kurstaki, producing a dark brown pigment, identified as melanin, was studied. Bt-m had higher larvicidity against Heliothis armigera than its parent. Survival of Bt-m spores and their insecticidal activity to irradiation at 254 nm and 366 nm were higher than those of the parent. The only toxic polypeptide produced by Bt-m was Cry1Ac (130 kDa); it lost cry1Aa, cry2Aa, and cry2Ab. Received: 2 April 2001 / Accepted: 14 May 2001     

Interactions of beta and gamma ENaC with Nedd4 can be facilitated by an ERK-mediated phosphorylation.

Phosphorylation of the epithelial Na(+) channel (ENaC) has been suggested to play a role in its regulation. Here we demonstrate that phosphorylating the carboxyl termini of the beta and gamma subunits facilitates their interactions with the ubiquitin ligase Nedd4 and inhibits channel activity. Three protein kinases, which phosphorylate the carboxyl termini of beta and gammaENaC, have been identified by an in vitro assay. One of these phosphorylates betaThr-613 and gammaThr-623, well-conserved C-tail threonines in the immediate vicinity of the PY motifs. Phosphorylation of gammaThr-623 has also been demonstrated in vivo in channels expressed in Xenopus oocytes, and mutating betaThr-613 and gammaThr-623 into alanine increased the channel activity by 3.5-fold. Effects of the above phosphorylations on interactions between ENaC and Nedd4 have been studied using surface plasmon resonance. Peptides having phospho-threonine at positions beta613 or gamma623 bind the WW domains of Nedd4 two to three times better than the non-phosphorylated analogues, due to higher association rate constants. Using a number of different approaches it was demonstrated that the protein kinase acting on betaThr-613 and gammaThr-623 is the extracellular regulated kinase (ERK). It is suggested that an ERK-mediated phosphorylation of betaThr-613 and gammaThr-623 down-regulates the channel by facilitating its interaction with Nedd4.     

Evidence for coupling of membrane targeting and function of the signal recognition particle (SRP) receptor FtsY

Recent studies have indicated that FtsY, the signal recognition particle receptor of Escherichia coli, plays a central role in membrane protein biogenesis. For proper function, FtsY must be targeted to the membrane, but its membrane-targeting pathway is unknown. We investigated the relationship between targeting and function of FtsY in vivo, by separating its catalytic domain (NG) from its putative targeting domain (A) by three means: expression of split ftsY, insertion of various spacers between A and NG, and separation of A and NG by in vivo proteolysis. Proteolytic separation of A and NG does not abolish function, whereas separation by long linkers or expression of split ftsY is detrimental. We propose that proteolytic cleavage of FtsY occurs after completion of co-translational targeting and assembly of NG. In contrast, separation by other means may interrupt proper synchronization of co-translational targeting and membrane assembly of NG. The co-translational interaction of FtsY with the membrane was confirmed by in vitro experiments.     

Suppressor and reactive lymphocytes in radiation leukemia virus (RadLV)-induced leukemogenesis

Summary Suppressor cells capable of enhancing tumor growth in vivo and of abrogating a potential anti-tumor immunity in vitro are generated in C57BL/6 mice inoculated with the high-leukemogenic A-RadLV. Mice inoculated with low-leukemogenic D-RadLV do not develop suppressor cells but contain anti-tumor reactive lymphocytes that can inhibit in vivo tumor growth. Cyclophosphamide (CyF) treatment of mice inoculated with A-RadLV hampered suppressor cell function and rendered the animals' lymphocytes responsive to A-RadLV induced tumor cells in vitro. Administration of CyF also reduced leukemia incidence in mice inoculated with A-RadLV, but had no effect on leukemia induction by D-RadLV in irradiated mice. It is suggested that the high leukemogenic activity of A-RadLV depends on the virus' ability to recruit CyF-sensitive suppressor cells early in latency and that tumor progression in mice inoculated with D-RadLV is arrested due to the host immune response.     

Residue-level determinants of angiopoietin-2 interactions with its receptor Tie2

We combined computational and experimental methods to interrogate the binding determinants of angiopoietin-2 (Ang2) to its receptor tyrosine kinase (RTK) Tie2—a central signaling system in angiogenesis, inflammation, and tumorigenesis. We used physics-based electrostatic and surface-area calculations to identify the subset of interfacial Ang2 and Tie2 residues that can affect binding directly. Using random and site-directed mutagenesis and yeast surface display (YSD), we validated these predictions and identified additional Ang2 positions that affected receptor binding. We then used burial-based calculations to classify the larger set of Ang2 residues that are buried in the Ang2 core, whose mutations can perturb the Ang2 structure and thereby affect interactions with Tie2 indirectly. Our analysis showed that the Ang2-Tie2 interface is dominated by nonpolar contributions, with only three Ang2 and two Tie2 residues that contribute electrostatically to intermolecular interactions. Individual interfacial residues contributed only moderately to binding, suggesting that engineering of this interface will require multiple mutations to reach major effects. Conversely, substitutions in substantially buried Ang2 residues were more prevalent in our experimental screen, reduced binding substantially, and are therefore more likely to have a deleterious effect that might contribute to oncogenesis. Computational analysis of additional RTK-ligand complexes, c-Kit-SCF and M-CSF-c-FMS, and comparison to previous YSD results, further show the utility of our combined methodology.     

Pectin,a second inducer for laccase production by Botrytis cinerea

Pectin acts as a second inducer of extracellular laccase formation by Botrytis cinerea, in the presence of a phenolic substance as a first inducer, but pectin alone fails to induce enzyme formation. The possible advantages of this mechanism for the fungus during the process of infection and overcoming host resistance are discussed.     

Self-renewal and commitment to differentiation of human leukemic promyelocytic cells (HL-60)

More than 80% of cells from a human promyelocytic leukemic cell line (HL-60) possess the capacity for self-renewal as evidenced by their ability to form large primary colonies in semisolid medium and the presence within these colonies of cells capable of subsequent colony formation. Colony development is independent of the normal regulator-the myeloid colony stimulating factor. The observed autostimulation suggests the production of specific growth promoters by the cells. Differentiation either to mature granulocytes or macrophages, induced by various agents, was associated with reduced cloning potential. Nevertheless, colonies containing differentiated cells could be developed either by cloning cells in the presence of suboptimal concentrations of inducer or by adding inducers over colonies developed in its absence. Upon differentiation, there was a morphological change from compact to diffused colony morphology due to cell mobility in the semisolid medium. Even at suboptimal concentrations of inducer more than 95% of the colonies became diffused, indicating clonaI homogeneity of the population with respect to differentiation capacity. The loss of self-renewal was found to be one of the early properties which changed following the initiation of differentiation. The loss preceded not only the overt expression of maturation-specific functions but also cellular commitment to terminal differentiation; shorter contact with the inducer was required to cause loss of self-renewal than to induce an irreversible transition to differentiation. This resulted in cells that lost their self-renewal potential without being able to complete their program of differentiation.     

Effects of Conformationally Restrained Analogues of Serotonin on Its Uptake and Binding in Rat Brain

Abstract: Two series of serotonin analogues, in which the side chain amino group is constrained in the gauche or trans conformation, were utilized to study the preferred conformation of serotonin for interaction with two different neuronal sites. 6-Hydroxytetrahydro-β-carboline and 6-hydroxy-3-aminotetrahydrocarbazole were found to be potent inhibitors of serotonin uptake into hypothalamic synaptosomes, with IC₅₀ values of 0.13 μM for each analogue. The type of inhibition, as determined by Dixon plots, was found to be competitive, with K_i's of 3.0 × 10⁻⁸ M and 4.6 × 10⁻⁸ M for the β-carboline and carbazole derivatives, respectively. Methoxylation or lack of a hydroxy group at the 6 position of the carbazole derivative did not alter inhibitory potency, while methoxy or benzyloxy substitution decreased potency 22- to 326-fold. The serotonin analogues were 20 to 30 times less potent in inhibiting the synaptosomal transport of the catecholamines. With regard to [³H]serotonin binding to membranes obtained from brain homogenates, both analogues exhibited poor affinity compared with the transmitter. However, the β-carboline derivative was three times as potent as the carbazole analogue. These findings and earlier ones with regard to the effect of the serotonin analogues on brain monoamine oxidase activity support the idea that serotonin analogues interact differentially with the three different serotonergic sites examined.