Dominant-negative mutants of platelet-derived growth factor revert the transformed phenotype of human astrocytoma cells.

Malignant astrocytoma is the most common primary human brain tumor. Most astrocytomas express a combination of platelet-derived growth factor (PDGF) and PDGF receptor which could close an autocrine loop. It is not known whether these autocrine loops contribute to the transformed phenotype of astrocytoma cells or are incidental to that phenotype. Here we show that dominant-negative mutants of the PDGF ligand break the autocrine loop and revert the phenotype of BALB/c 3T3 cells transformed by the PDGF-A or PDGF-B (c-sis) gene. Then, we show that these mutants are selective in that they do not alter the phenotype of 3T3 cells transformed by an activated Ha-ras or v-src gene or by simian virus 40. Finally, we show that these mutants revert the transformed phenotype of two independent human astrocytoma cell lines. They have no effect on the growth of human medulloblastoma, bladder carcinoma, or colon carcinoma cell lines. These observations are consistent with the view that PDGF autocrine loops contribute to the transformed phenotype of at least some human astrocytomas.     

Roosting Requirements of Two Frugivorous Bats (Sturnira lilium and Arbiteus intermedius) in Fragmented Neotropical Forest1

As tropical forest fragmentation accelerates, scientists are concerned with the loss of species, particularly those that play important ecological roles. Because bats play a vital role as the primary seed dispersers in cleared areas, maintaining healthy bat populations is critical to natural forest regeneration. Observations of foraging bats suggest that many Neotropical fruit‐eating species have fairly general habitat requirements and can forage in many different kinds of disturbed vegetation; however, their roosting requirements may be quite different. To test whether or not general foraging requirements are matched by equally broad roosting requirements, we used radiotelemetry to locate roost sites of two common frugivorous bat species (Sturnira lilium and Artibeus intermedius) in a fragmented forest in southeastern Mexico. Sturnira lilium roosted inside tree cavities and selected large‐diameter roost trees in remnant patches of mature forest. Fewer than 2 percent of trees surveyed had a mean diameter equal to or greater than roost trees used by . S. lilium, Artibeus intermedius roosted externally on branches and vines and under palm leaves and selected roost trees of much smaller diameter. Compared to random trees, roost trees chosen by A. intermedius were closer to neighboring taller trees and also closer in height to these trees. Such trees likely provide cryptic roosts beneath multiple overlapping crowns, with sufficient shelter from predators and the elements. While males of A. intermedius generally roosted alone in small trees within secondary forest, females roosted in small groups in larger trees within mature forest and commuted more than three times farther than males to reach their roost sites. Loss of mature forest could impair the ability of frugivorous bats to locate suitable roost sites. This could have a negative impact on bat populations, which in turn could decrease forest regeneration in impacted areas.     

Endogenous proteinases modulate the function of the β-adrenergic receptor-adenylate cyclase system

Photoaffinity labeling techniques have recently demonstrated that mammalian β₁- and β₂-adrenergic receptors reside on peptides of M_r 62 000–64 000. These receptor peptides are susceptible to endogenous metalloproteinases which produce peptides of M_r 30 000–55 000. Several proteinase inhibitors markedly attenuate this process, specifically EDTA and EGTA. In this study we investigated the functional significance of this proteolysis (and its inhibition) in the β₂-adrenergic receptor-adenylate cyclase system derived from rat lung membranes. Membrane preparations containing proteolytically derived fragments of the receptor of M_r 40000–55 000 are fully functional with respect to their ability to bind β-adrenergic antagonist radioligands such as [³H]dihydroalprenolol and β-adrenergic antagonist photoaffinity reagents such as p-azido-m-[^{125I]iodobenzylcarazolol}. They retain the ability to form a high-affinity, agonist-promoted, guanine nucleotide-sensitive complex thought to represent a ternary complex of agonist, receptor and guanine nucleotide regulatory protein. Nonetheless, after proteolysis, GTP is less able to revert this high-affinity receptor complex to one of lower affinity, and all aspects of adenylate cyclase stimulation are reduced. In addition, the functional integrity of the N protein in membranes prepared without proteinase inhibitors is reduced as assessed by reconstitution studies with the cyc[su− variant of S49 lymphoma cell membranes. These results suggest that endogenous proteolysis does not directly impair the ability of β-adrenergic receptors to either bind ligands or interact with the guanine nucleotide regulatory protein. However, they imply that endogenous proteolysis likely impairs the functionality of other components of the adenylate cyclase system, such as the nucleotide regulatory protein.     

Myocardial Revascularization in Patients 70 Years of Age and Older

Myocardial revascularization has been carried out by us in 67 patients 70 years of age or older. Advanced coronary artery disease was found at angiography in more than two thirds of the patients. The postoperative morbidity and mortality compare very favorably with those in younger patients. The early and late mortality in the 67 patients was 4.5 percent and 6.0 percent, respectively. Fifty-seven survivors have been followed an average of 21 months; for most patients there has been a pronounced improvement in clinical classification. Properly selected, patients of advanced age can undergo successful revascularization surgical procedures. The adequacy of function of the left ventricle, proper timing of the surgical operation and an aggressive yet realistic approach seem to be major determinants for a good result.     

Characterization of leucocin A-UAL 187 and cloning of the bacteriocin gene from Leuconostoc gelidum.

Leucocin A-UAL 187 is a bacteriocin produced by Leuconostoc gelidum UAL 187, a lactic acid bacterium isolated from vacuum-packaged meat. The bacteriocin was purified by ammonium sulfate or acid (pH 2.5) precipitation, hydrophobic interaction chromatography, gel filtration, and reversed-phase high-performance liquid chromatography with a yield of 58% of the original activity. Leucocin A is stable at low pH and heat resistant, and the activity of the pure form is enhanced by the addition of bovine serum albumin. It is inactivated by a range of proteolytic enzymes. The molecular weight was determined by mass spectrometry to be 3,930.3 +/- 0.4. Leucocin A-UAL 187 contains 37 amino acids with a calculated molecular weight of 3,932.3. A mixed oligonucleotide (24-mer) homologous to the sequence of the already known N terminus of the bacteriocin hybridized to a 2.9-kb HpaII fragment of a 7.6-MDa plasmid from the producer strain. The fragment was cloned into pUC118 and then subcloned into a lactococcal shuttle vector, pNZ19. DNA sequencing revealed an operon consisting of a putative upstream promoter, a downstream terminator, and two open reading frames flanked by a putative upstream promoter and a downstream terminator. The first open reading frame downstream of the promoter contains 61 amino acids and is identified as the leucocin structural gene, consisting of a 37-amino-acid bacteriocin and a 24-residue N-terminal extension. No phenotypic expression of the bacteriocin was evident in several lactic acid bacteria that were electrotransformed with pNZ19 containing the 2.9-kb cloned fragment of the leucocin A plasmid.     

Epitope mapping of snake venom phospholipases A2 with pseudexin monoclonal antibodies

Fifteen different monoclonal antibodies, developed against a pseudexin A, B, and C mixture, were screened for linear epitope recognition. Peptides (9-mers) spanning pseudexin B were synthesized on alanine-derivatized polyethylene pins and subsequently probed with antibody. Four antibodies recognized linear epitopes of pseudexin A, pseudexin B, and also nonidentical sequences found in other phospholipases A₂ (PLA₂s) as determined by enzyme-linked immunosorbent assays. Three antibodies recognized a highly conserved site important in calcium binding and the interlocking of dimeric forms of PLA₂. Antibodies neutralizing lethal or enzymatic effects of PLA₂ did not recognize linear epitopes.     

Extracellular calcium mimics the actions of platelet-derived growth factor on mouse fibroblasts.

Microprecipitates of calcium phosphate (CaPO4) can substitute for platelet-derived growth factor (PDGF) to stimulate the growth of cultured 3T3 cells. In two-part complementation assays, CaPO4 behaves as a PDGF-like "competence factor"--that is, the mitogenic response to CaPO4 is enhanced synergistically by "progression factors" contained in platelet-poor plasma. In studies described here, we show that early cytoplasmic and intranuclear events in the mitogenic response to CaPO4 are equivalent to those induced by PDGF. However, no net increase in tyrosine kinase activity of either the PDGF-alpha or PDGF-beta receptor is seen following exposure to CaPO4. Our data suggest that calcium acts within the cell, regulating events which normally proceed from activation of PDGF receptors. Alternatively, microprecipitates of CaPO4 could act externally by activating a growth factor receptor which escapes detection with available reagents.     

Post-transcriptional control of protein synthesis in Balb/c-3T3 cells by platelet-derived growth factor and platelet-poor plasma

Platelet-derived growth factor (PDGF) and platelet-poor plasma, which lacks PDGF, both induce a rapid increase in the rate of total protein synthesis within quiescent, density-arrested Balb/c-3T3 cells. This stimulation of protein synthesis is associated with an increased aggregation of ribosomes into polyribosomes. Nuclear functions are not required for this response, as demonstrated by the observation that this stimulation of protein synthesis occurs in cells pretreated with actinomycin D and in enucleated cells (cytoplasts). The response to PDGF persists even after PDGF has been removed from the culture medium, but in contrast, when plasma is removed from the medium, polysomes disaggregate and protein synthesis declines. PDGF and plasma do not function synergistically to increase protein synthesis, whereas they do to induce optimum DNA synthesis. Thus stimulation of the translational apparatus may be necessary for the mitogenic response of Balb/c-3T3 cells to growth factors, but it is not by itself sufficient.     

Transposition of the gene cluster CYC1-OSM1-RAD7 in yeast

A mutant of the yeast Saccharomyces cerevisiae contains an increased amount of iso-1-cytochrome c because two copies of a segment, denoted COR, were transposed to a new position on chromosome VII, while the original COR region was retained at the normal position on chromosome X; this COR segment encompasses the CYC1, OSM1 and RAD7 loci which determine, respectively, iso-1-cytochrome c, osmotic sensitivity and ultraviolet light sensitivity. The analysis of genomic DNA with cloned probes indicates that the length of the COR segment is approximately 12,000 base-pairs. We suggest that certain normal strains of yeast, which possibly may contain reiterated sequences, can produce extended transpositions similar to prokaryotes.