Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo.

Clinical and experimental studies suggest that angiogenesis is a prerequisite for solid tumour growth. Several growth factors with mitogenic or chemotactic activity for endothelial cells in vitro have been described, but it is not known whether these mediate tumour vascularization in vivo. Glioblastoma, the most common and most malignant brain tumour in humans, is distinguished from astrocytoma by the presence of necroses and vascular proliferations. Here we show that expression of an endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF), is induced in astrocytoma cells but is dramatically upregulated in two apparently different subsets of glioblastoma cells. The high-affinity tyrosine kinase receptor for VEGF, flt, although not expressed in normal brain endothelium, is upregulated in tumour endothelial cells in vivo. These observations strongly support the concept that tumour angiogenesis is regulated by paracrine mechanisms and identify VEGF as a potential tumour angiogenesis factor in vivo.     

Rapid induction of mRNAs forPC3 by partial hepatectomy and epidermal growth factor

The expression of immediate early gene plays a pivotal role in rat hepatocyte proliferation from G₀ to G₁ phases and the progression through G₁ phase of the cell cycle within several hours after 2/3 hepatectomy. We investigated the different gene expressions within 1 h after 2/3 hepatectomy by representational difference analysis. Sequence analysis indicated thatPC3 induced by NGF was a kind of immediate early gene and might be correlated with liver regeneration. Moreover, we found that 2/3 hepatectomy could induce the expressing ofPC3 mRNA by Northern blot with a peak 1–2 h after surgery. In primary cultures of rat hepatocytes, addition of EGF resulted in rapid and transient induction ofPC3 mRNA. It was first reported thatPC3 gene belongs to immediate early gene associated with liver regeneration.     

Requirement for nerve growth factor in the development of myelinated nociceptors in vivo

In adult animals, sensory neurons innervating the skin are phenotypically diverse. We have now investigated whether nerve growth factor (NGF) has a physiological role in the development of this diversity. We gave antisera against NGF to rats from postnatal day 1 (PND 1) to adulthood (5 weeks). We found a virtually complete depletion of high threshold mechanoreceptors conducting in the A delta range (2-13 ms-1) in the sural nerve. This afferent type, normally present in large numbers, appeared to have been replaced by D-hair afferents, sensitive mechanoreceptors which normally are relatively rare. NGF deprivation had this effect only in early postnatal life; treatment from postnatal day 14 to adulthood had no effect. We conclude that the presence of NGF postnatally in skin is necessary for the proper phenotypic development of A delta cutaneous nociceptors.     

Apolipoprotein L-I is the trypanosome lytic factor of human serum

Human sleeping sickness in east Africa is caused by the parasite Trypanosoma brucei rhodesiense. The basis of this pathology is the resistance of these parasites to lysis by normal human serum (NHS). Resistance to NHS is conferred by a gene that encodes a truncated form of the variant surface glycoprotein termed serum resistance associated protein (SRA). We show that SRA is a lysosomal protein, and that the amino-terminal alpha-helix of SRA is responsible for resistance to NHS. This domain interacts strongly with a carboxy-terminal alpha-helix of the human-specific serum protein apolipoprotein L-I (apoL-I). Depleting NHS of apoL-I, by incubation with SRA or anti-apoL-I, led to the complete loss of trypanolytic activity. Addition of native or recombinant apoL-I either to apoL-I-depleted NHS or to fetal calf serum induced lysis of NHS-sensitive, but not NHS-resistant, trypanosomes. Confocal microscopy demonstrated that apoL-I is taken up through the endocytic pathway into the lysosome. We propose that apoL-I is the trypanosome lytic factor of NHS, and that SRA confers resistance to lysis by interaction with apoL-I in the lysosome.     

Basic fibroblast growth factor prevents death of lesioned cholinergic neurons in vivo

Cutting the axons of the cholinergic neurons that project to the hippocampal formation results in death of most of these cells. Previous studies have shown that administration of nerve growth factor before or at the same time as the lesion will prevent this cell death. Here we demonstrate that basic fibroblast growth factor (FGF) administered into the brain reduces the death of cholinergic neurons in the medial septum and diagonal band of Broca after transection of their axons, in both young adult and aged rats. Moreover, FGF can partially protect against death of cholinergic neurons even when administered two days after axonal transection. These results indicate a possible function for FGF in the normal support of basal forebrain cholinergic neurons, but its range of activity could be wider, for FGF also supports noncholinergic neurons in vitro, it is localized in many of the central nervous system neurons, and it is found in relatively high concentrations in the brain.     

Nucleosome positioning can affect the function of a cis-acting DNA element in vivo

Positioning of nucleosomes has been proposed as one mechanism whereby the activity of DNA is regulated: cis-acting elements located in linker DNA might be more accessible for interaction with trans-acting protein factors than they would be if they were directly associated with histones in nucleosome core particles. The eleven base pairs constituting the autonomously replicating sequence (ARS) of the high-copy-number TRP1ARS1 plasmid of Saccharomyces cerevisiae are located in a linker region near the edge of a positioned nucleosome and form an origin of replication. Could nucleosome positioning render the ARS accessible for interaction with the proteins necessary for its function? I have tested this hypothesis by making deletions and an insertion to move the ARS into the nucleosome DNA and then examining the effects on ARS function. There is a marked decrease in copy number when the ARS is moved into the central DNA region of the nucleosome core particle, a region known to differ in structure and stability from the peripheral segments of nucleosome DNA.     

Proteolytic turnover of the Gal4 transcription factor is not required for function in vivo

Transactivator-promoter complexes are essential intermediates in the activation of eukaryotic gene expression. Recent studies of these complexes have shown that some are quite dynamic in living cells owing to rapid and reversible disruption of activator-promoter complexes by molecular chaperones, or a slower, ubiquitin-proteasome-pathway-mediated turnover of DNA-bound activator. These mechanisms may act to ensure continued responsiveness of activators to signalling cascades by limiting the lifetime of the active protein-DNA complex. Furthermore, the potency of some activators is compromised by proteasome inhibition, leading to the suggestion that periodic clearance of activators from a promoter is essential for high-level expression. Here we describe a variant of the chromatin immunoprecipitation assay that has allowed direct observation of the kinetic stability of native Gal4-promoter complexes in yeast. Under non-inducing conditions, the complex is dynamic, but on induction the Gal4-promoter complexes 'lock in' and exhibit long half-lives. Inhibition of proteasome-mediated proteolysis had little or no effect on Gal4-mediated gene expression. These studies, combined with earlier data, show that the lifetimes of different transactivator-promoter complexes in vivo can vary widely and that proteasome-mediated turnover is not a general requirement for transactivator function.     

Local aggregation of hormone-receptor complexes is required for activation by epidermal growth factor.

An analogue of epidermal growth factor (EGF) which is virtually devoid of biological activity retains receptor binding activity but cannot form cell surface clusters or patches. Bivalent anti-EGF antibodies restore both bioactivity and patch formation. The sensitivity of fibroblasts to native EGF can also be enhanced greatly by these antibodies, especially in hormone-resistant cell lines.