A new tubulin-binding protein

A new brain protein is described which forms an insoluble complex with tubulin, with concomitant stoichiometric hydrolysis of GTP. The complex contains a maximum of one tubulin-binding protein (MW 52,500) per two tubulin dimers. The tubulin-binding protein (TBP) does not compete with colchicine, but in the presence of microtubule-associated proteins tubulin appeared less accessible to it. Proteins such as TBP might sequester tubulin and thereby function either to inhibit indiscriminate polymerization, or to promote ordered nucleation by maintaining high local concentrations.     

Genomic Consequences of Ecological Speciation in Astyanax Cavefish

The cave environment is consistently radically different than the surface environment because it lacks light, and animals adapting to cave life are subject to strong selective forces much different than those experienced by their ancestors who evolved in the presence of light. As such, their divergence from surface ancestors and eventual speciation is likely to be driven by the shift in ecology. We report here that hybrids between cave and surface Astyanax mexicanus fishes produce offspring with allelic frequencies that differ significantly from Mendelian expectations both for transmission ratios and for independent assortment of unlinked markers. Comparison of allelic content of DNA from fin clips and sperm pools show that the transmission ratio distortion likely occurs during spermatogenesis. Departures from expectations of independent assortment are essentially epistatic phenomena generating linkage disequilibrium. A novel analysis of the epistatic interactions reveals an apparent network of interactions among genes known or suspected to be involved in cave adaptation, implying that the epistasis arose as a “by product” of the divergence due to cave adaptation.     

<Superscript>15</Superscript>N natural abundance of fossil peat reflects the influence of animal-derived nitrogen on vegetation

'¹⁵N signatures of fossil peat were used to interpret past ecosystem processes on tectonically active subantarctic Macquarie Island. By comparing past vegetation reconstructed from the fossil record with present-day vegetation analogues, our evidence strongly suggests that changes in the '¹⁵N signatures of fossil peat at this location reflect mainly past changes in the proportion of plant nitrogen derived from animal sources. Associated with uplift above sea level over the past 8,500 years, fossil records in two peat deposits on the island chronicle a change from coastal vegetation with fur and elephant seal disturbance to the existing inland herbfield. Coupled with this change are synchronous changes in the '¹⁵N signatures of peat layers. At two sites ¹⁵N-enriched peat '¹⁵N signatures of up to +17‰ were associated with a high abundance of pollen of the nitrophile Callitriche antarctica (Callitrichaceae). At one site fossil seal hair was also associated with enriched peat '¹⁵N. Less ¹⁵N enriched '¹⁵N signatures (e.g. -1.9‰ to +3.9‰) were measured in peat layers which lacked animal associated C. antarctica and Acaena spp. Interpretation of a third peat profile indicates continual occupation of a ridge site by burrowing petrels for most of the Holocene. We suggest that ¹⁵N signatures of fossil peat remained relatively stable with time once deposited, providing a significant new tool for interpreting the palaeoecology.     

Hormonal regulation of estradiol biosynthesis, aromatase activity, and aromatase mRNA in rat ovarian follicles and corpora lutea

To determine the molecular basis for changes in aromatase (P450arom) activity in rat ovarian follicles and corpora lutea, seven clones for rat P450arom cDNA have been identified and isolated from a rat granulosa cell λgtll cDNA expression library using a 62 mer deoxyoligonucleotide probe (derived from an amino acid sequence of purified human placental aromatase) and a human placental P450arom cDNA probe. One of the rat P450arom cDNA clones contained an insert 1.2 kb in size. Both the human 1.8 kb cDNA and the rat 1.2 kb cDNA probes hybridized to a single species of P450arom mRNA that was 2.6 kb in size. Northern blot analysis revealed that corpora lutea isolated on day 15 of pregnancy contained high amounts of P450arom mRNA, whereas granulosa cells of antral follicles of hormonally primed, hypophysectomized rats (i.e., those from which mRNA was isolated to construct the cDNA library) contained only low amounts of P450arom mRNA. The lower amounts of P450arom in granulosa cells of preovulatory follicles in the estradiol-follicle-stimulating hormone primed hypophysectomized rats were unexpected because follicles incubated in medium containing testosterone substrate produce more estradiol than do corpora lutea isolated on day 15 of pregnancy and incubated under similar conditions. Additional studies will determine the hormonal events responsible for the elevated amounts and constitutive maintenance of P450arom mRNA and aromatase activity in luteal cells in vivo and in vitro.     

Calcitonin gene-related peptide immunoreactivity in airway epithelial cells of the human fetus and infant

Summary Calcitonin gene-related peptide-immunoreactive cells were identified within the epithelium of distal conducting airways in the human fetus and infant. Several peptides and amines, including calcitonin, have been identified previously within a specific population of airway epithelial cells. These cells, referred to as pulmonary neuroendocrine cells, are postulated to be airway chemoreceptors responsible for changes in ventilation and perfusion in response to changes in airway gas composition. Calcitonin gene-related peptide immunoreactive cells could be identified throughout the period of development studies (20 weeks gestation to 3 months of age), but were present in only limited numbers in less than 50% of individuals (n=23). In contrast, large numbers of calcitonin gene-related peptide immunoreactive cells were identified in 100% of infants (1–3 months, n=5) with bronchopulmonary dysplasia. The differential processing of mRNA transcribed from the calcitonin gene in neural and non-neural tissue suggests that calcitonin, rather than calcitonin gene-related peptide, is the primary product of translation in pulmonary neuroendocrine cells. However, considering the potent vasodilatory and bronchoconstrictive effects of calcitonin gene-related peptide, its presence in pulmonary neuroendocrine cells, even in small amounts, may be important in controlling pulmonary vaso- and/or bronchomotor tone. The presence of large numbers of calcitonin gene-related peptide immunoreactive cells in infants with bronchopulmonary dysplasia suggests that calcitonin gene-related peptide may be one further agent contributing to the pulmonary pathophysiology seen in this disease.     

Isolation and characterization of a variant myoblast cell line that is temperature sensitive for differentiation.

A new variant rat myogenic cell line, ts485, was isolated by subcloning the cell line ts3b2 (H. T. Nguyen, R. M. Medford, and B. Nadal-Ginard, Cell 34:281-293, 1983). Unlike the progenitor cell line, ts485 was thermosensitive for differentiation. Experiments with conditioned medium suggested that diffusible extracellular factors were not involved in dictating the differential phenotypes of ts485 cells cultured at the permissive and nonpermissive temperatures. Temperature shift experiments performed on cultures of ts485 cells indicated that the temperature-sensitive lesion was in a factor active during the growth phase and required to trigger a cascade of events leading to terminal differentiation.     

Activating and 'anxiogenic' effects of corticotropin releasing factor are not inhibited by blockade of the pituitary-adrenal system with dexamethasone

Central administration of corticotropin releasing factor (CRF) in rats produces pituitary-adrenal activation and a variety of "anxiogenic-like" effects. The present study was designed to explore the contribution of the peripheral pituitary-adrenocortical axis in mediating these CRF responses. Intraventricularly administered CRF produced suppression of responding in the conflict test and a marked locomotor activation. Neither behavioral effect was altered by the prior administration of dexamethasone in a dose that blocked pituitary-adrenal activation to CRF. These results support the hypothesis that behavioral effects of CRF are mediated by its action at central sites and not via an action on the pituitary-adrenocortical system.     

Calcium/phospholipid-dependent kinase recognizes sites in microtubule-associated protein 2 which are phosphorylated in living brain and are not accessible to other kinases

Microtubule-associated protein 2 (MAP-2) purified after microtubule assembly cycles from bovine brain had been shown to contain about 10 esterified phosphates (mol/mol), which were relatively phosphatase resistant and essentially confined to the projection domain which contributes to the visible arms on microtubules. The kinase responsible for phosphorylating these sites had not been identified. We have approached this question by using a phosphatase that releases the bulk of these residues and then determining which kinase can now add additional residues corresponding to those released. Three kinases were chosen because of their abundance in brain and/or proximity to microtubules. Of these only Ca/phospholipid-dependent kinase was able to recognize the previously occupied sites. We also found that MAP-2 isolated from rat brain without assembly cycles contained more phosphate than previously recognized, greater than 30 mol/mol, suggesting that 20 of these had been inadvertently released by phosphatase during assembly cycles. All 3 kinases (Ca/phospholipid-dependent, cAMP-dependent, and Ca/calmodulin-dependent kinase II) recognized more sites in the bovine than in the rat MAP-2.     

Cloning and sequencing of mRNAs coding for two adult alpha globin chains of the salamander Pleurodeles waltlii

A cDNA library of erythrocyte mRNAs was established from immature red blood cells of the adult amphibian, Pleurodeles waltlii (urodele; salamander). The cDNA clones corresponding to the four adult globin chains were first identified and characterized by positive selection and the cDNAs derived from the two (major and minor) alpha-globin chains sequenced. The sequences presented contain both the complete 3'-noncoding region and the coding region of both chains, with the exception of the first nine codons of the minor alpha-chain, and a portion of the 5'-noncoding region of the major chain. The amino acid sequences of the encoded alpha-globin polypeptides have been deduced and compared with those of Xenopus laevis and of man. These comparative studies suggest that the alpha-globins of Pleurodeles waltlii and Xenopus laevis may have diverged from a common ancestral gene at the time when mammalian and amphibian lines diverged, and that they then evolved separately. Duplication of the alpha-gene, which is responsible for the polypeptide heterogeneity, appears to have occurred earlier in Pleurodeles waltlii than in Xenopus laevis.