Oncogenic activation of the neu-encoded receptor protein by point mutation and deletion.

The rat neu gene, which encodes a receptor-like protein homologous to the epidermal growth factor receptor, is frequently activated by a point mutation altering a valine residue to a glutamic acid residue in its predicted transmembrane domain. Additional point mutations have been constructed in a normal neu cDNA at and around amino acid position 664, the site of the naturally arising mutation. A mutation which causes a substitution of a glutamine residue for the normal valine at residue 664 leads to full oncogenic activation of the neu gene, but five other substitutions do not. Substituted glutamic acid residues at amino acid positions 663 or 665 do not activate the neu gene. Thus only a few specific residues at amino acid residue 664 can activate the oncogenic potential of the neu gene. Deletion of sequences of the transforming neu gene demonstrates that no more than 420 amino acids of the 1260 encoded by the gene are required for full transforming function. Mutagenesis of the transforming clone demonstrates a correlation between transforming activity and tyrosine kinase activity. These data indicate that the activating point mutation induces transformation through (or together with) the activities of the tyrosine kinase.     

Characterization of T helper 1 and 2 cell subsets in normal mice. Helper T cells responsible for IL-4 and IL-5 production are present as precursors that require priming before they develop into lymphokine-secreting cells

We have shown that the requirements for the production of IL-4 and IL-5 by normal L3T4+T cells from murine spleen are very different from those for the production of IL-2. Secretion of detectable quantities of IL-4 and IL-5 and induction of the mRNA for each lymphokine occurs in vitro only after cells are primed and re-stimulated. This priming can be achieved by mitogens (Con A), by antibodies to the TCR (anti-T3) or by stimulation with alloantigen. In contrast, requirements for induction of lymphokine production after priming resemble those for initial production of IL-2. Thus the majority of T cells of helper phenotype that have the potential to become IL-4- and IL-5-secreting T cells, exist in the form of precursors requiring stimulation and several days of culture as well as re-stimulation with mitogen or Ag before they become detectable as lymphokine-secreting cells. In contrast, among fresh CD4+T cells, secretion of IL-2, IL-3, granulocyte/macrophage CSF, and IFN-gamma is easily detected within 24 h of stimulation with mitogen or Ag. These observations establish that distinct phenotypes of Th cells are found at different times after stimulation and support the concept that synthesis and secretion of different lymphokines or groups of lymphokines are regulated independently. Furthermore the patterns of lymphokines secreted by fresh vs primed Th cells, which largely correspond to the patterns that have been used to define the Th1 and Th2 subsets among Th cell lines, provides evidence that different subsets of normal T cells exist that may correspond to these designations. Secretion of different lymphokines by two subsets of Th cells at different times in an immune response, and perhaps in different places, suggests a model in which the ratio of the two T cell subsets (Th1 vs Th2) and state of differentiation of each (precursor vs effector), influence or determine the direction of the response, with variations in these parameters leading to differing responses.     

Effects of a medical team coordinator on length of hospital stay.

OBJECTIVE: To determine the effect of a medical team coordinator (MTC) on the length of stay in a teaching hospital. DESIGN: Randomized controlled trial. SETTING: Two of four general medical clinical teaching units (CTUs). PATIENTS: Patients admitted to the CTUs between July and October 1990 except those who were admitted directly to an intensive care unit or whose death was expected within 48 hours. The 267 patients were randomly assigned to receive either standard medical care or standard medical care plus MTC services. INTERVENTION: The MTC was a baccalaureate nurse whose role was to facilitate administrative tasks such as discharge planning, to coordinate tests and procedures, and to collect and collate patient information. MAIN OUTCOME MEASURES: Length of hospital stay. A subgroup of 40 patients was asked to complete a brief survey on medical care information and satisfaction. RESULTS: The MTC intervention reduced the mean length of stay by 1.97 days (p less than or equal to 0.04, 95% confidence interval [CI] 1.02 to 2.92 days). Subanalysis by diagnostic group revealed that most of this effect was in an ill-defined group of disorders. In the survey more patients in the MTC group than in the other group reported being satisfied with their medical care (89% v. 62%; p less than or equal to 0.05, 95% CI 2% to 52%). CONCLUSIONS: The services of an MTC help to reduce the length of hospital stay for some groups of patients. Further research is necessary to examine which components of the MTC intervention are most effective and in what conditions.     

Negative regulators of growth.

The proliferation of cells is regulated by countervailing positively- and negatively-acting signaling networks. The anti-proliferative signals, the study of which has been much neglected until recently, are often conveyed by growth-inhibitory peptides. Elements that mediate the cellular response to growth inhibitors are encoded by tumor suppressor genes that if lost may lead to the runaway growth of the cancer cell.     

Lipoproteins of Haemophilus influenzae type b.

Haemophilus influenzae type b Minn A produced 12 lipoproteins with apparent molecular weights of between 14,000 and 67,000. The lipoproteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of delipidated extracts of cells grown in [3H]palmitate. When the delipidated cell extracts were subjected to acid methanolysis, tritium was quantitatively recovered as palmitate and methyl palmitate, indicating that the [3H]palmitate had not been degraded and reincorporated into nonlipid material during cell growth. One of the lipoproteins comigrated with outer membrane protein (OMP) P6. OMP P6 was purified from [3H]palmitate-labeled cells. The purified protein preparation contained both amide- and ester-linked fatty acids. We conclude that (i) H. influenzae type b produces several lipoproteins, and (ii) one of these lipoproteins is OMP P6, a protein under consideration as a vaccine component.     

Exposure and electronic interaction of tyrosine and tryptophan residues in human apolipoprotein A-IV

We have investigated the exposure and electronic interaction of tyrosine and tryptophan residues in human apolipoprotein A-IV (apo A-IV). Differential absorption spectroscopy and chemical titration demonstrated that human apo A-IV contains six tyrosine residues, four of which are buried in the hydrophobic interior of the protein and two of which are exposed on the protein surface. Denaturation of the protein by guanidinium chloride caused progressive exposure of the buried tyrosines. The fluorescence emission spectra of apo A-IV were characterized by a blue-shifted tryptophan emission with a low relative quantum yield of 0.37 and a tyrosine emission with a relative quantum yield of 0.62. Fluorescence quenching studies demonstrated a low fractional exposure of tryptophan in the native state. Denaturation of apo A-IV was accompanied by an increase in the relative quantum yield which peaked at the denaturation midpoint. Fluorescence excitation techniques demonstrated energy transfer from tyrosine residues with a transfer efficiency of 0.40 in the native state; the efficiency was conformation dependent and decreased with protein unfolding. Fluorescence studies of tetranitromethane-modified apo A-IV suggested that a significant fraction of energy transfer proceeds from the exposed tyrosine residues. These data demonstrate the existence of intramolecular fluorescence energy transfer and tryptophan quenching in human apolipoprotein A-IV and suggest that the amino terminus of this protein is situated in a hydrophobic domain within energy-transfer range of nonvicinal tyrosine residues.     

Regulation of proliferation of bovine aortic endothelial cells, smooth muscle cells, and adventitial fibroblasts in collagen lattices

We compared the proliferation of bovine aortic cells grown in collagen lattices. Smooth muscle cells continued to divide for 2 weeks while adventitial fibroblasts ceased to divide after 4-5 days. Endothelial cells did not proliferate within an untreated collagen lattice; however, if the lattice was covered with culture medium, endothelial cells populated its surface and proliferated to form a monolayer. We also found that both smooth muscle cells and endothelial cells, like fibroblasts, are able to contract a collagen lattice to a small fraction of its original volume, although endothelial cells are able to do so only if the lattice is covered with culture medium.     

Simultaneous expression of early and late histone messenger RNAs in individual cells during development of the sea urchin embryo

The transition from early (E) to late (L) histone gene expression in developing sea urchin (Strongylocentrotus purpuratus) embryos was examined for H2B, H3, and H4 mRNAs by in situ hybridization of class-specific probes. Hybridization patterns indicate that the shift from E to L mRNAs occurs gradually and simultaneously in all blastomeres. Thus, during the transition the ratio of L to E mRNAs is similar in most cells. This suggests that no sudden changes in histone composition occur in individual cells which might be related to alterations in gene expression associated with differentiation of cell lineages. Around the midpoint of the transition, clusters of cells progressively appear which contain little, if any, E or L histone mRNA. This modulation of expression is coordinated for the three late genes examined because most individual cells contain either high or low levels of all three mRNAs. At blastula stage these clusters of unlabeled cells appear to be randomly distributed throughout the embryo. Subsequently the unlabeled regions expand and are found predominantly in aboral ectoderm as these cells cease to divide. Thus, the L/E histone mRNA ratio is not differentially regulated in diverse cell lineages, and the major differences in total histone mRNA content among individual cells may be related to cell cycle and/or the cessation of division.     

Dynamics of orange peel fermentation during ensilage

The dynamics of fermentation during ensilage were studied on orange peel (variety Shamouti) ensiled in 50 plastic containers, 10 kg in each, with an outlet for seepage. At predetermined intervals the containers were weighed and samples were taken from three of them for chemical and microbial analysis. Fermentation losses amounted to up to ca one-third of the fresh peel dry matter (DM) content. Most losses occurred within 10 d of commencement of fermentation, and were attributable to gas release. The major fermentation products were ethanol, lactic and acetic acids (16, 3 and 3% of DM, respectively). The dominant microbial populations were lactobacilli and yeasts. Tests are in progress to inhibit the yeasts and thereby reduce fermentation losses.