Disruption of the low affinity receptor-binding site in NGF allows neuronal survival and differentiation by binding to the trk gene product.

Nerve growth factor (NGF), like many other growth factors and hormones, binds to two different receptor molecules on responsive cells. The product of the proto-oncogene trk, p140trk, is a tyrosine kinase receptor that has been identified as a signal-transducing receptor for NGF, while the role of the low affinity NGF receptor, p75NGFR, in signal transduction is less clear. The crystal structure of NGF has recently been determined, although structures involved in receptor binding and biological activity are unknown. Here we show that Lys-32, Lys-34, and Lys-95 form a positively charged interface involved in binding to p75NGFR. Simultaneous modification of Lys-32 with either of the two other lysines resulted in loss of binding to p75NGFR. Despite the lack of binding to p75NGFR, these mutants retained binding to p140trk and biological activity, demonstrating a functional dissociation between the two NGF receptors.     

Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans.

The presence of nitric oxide (NO) in the exhaled air of humans and of anaesthetized rabbits and guinea pigs was demonstrated by chemiluminescence, diazotization and mass spectrometry. This NO is endogenously produced in the lung by an NO synthase, since its generation in guinea pigs and rabbits was inhibited by N omega-nitro-L-arginine methyl ester and NG-monomethyl-L-arginine, inhibitors of this enzyme. The effect of the inhibitors was reversed by the precursor of NO synthesis, L-arginine. Since NO is produced by normal vascular endothelium for the physiological regulation of blood flow and pressure and also by activated macrophages to contribute to non-specific immunity, our experiments suggest that NO may play both vascular regulatory and host defence roles in pulmonary physiology and pathophysiology.     

Feedback regulation of ornithine decarboxylase expression. Studies using a polysomal run-off system

The rate-controlling enzyme in polyamine synthesis, ornithine decarboxylase (ODC), is subject to feedback regulation by the polyamines at the level of translation. In the present study we used a cell-free translation system to further investigate the mechanism by which this regulation occurs. Lysates of ODC-overproducing cells were capable of synthesizing large amounts of ODC. The degree of initiation was poor in the lysates and the synthesis of ODC was mainly a result of continued elongation of peptide chains on pre-initiated ribosomes. By determining the amount of ODC produced in the lysate, we obtained an estimate of the number of ribosomes that were actively translating ODC mRNA at the moment of lysis. Using this polysomal run-off assay we demonstrated that the polyamine-mediated regulation of ODC synthesis occurs without any change in the number of ribosomes associated with the message. This finding indicates that the polyamines exert a coordinate effect on initiation and elongation.     

Biosurfactant yields and nutrient consumption of Pseudomonas fluorescens 378 studied in a microcomputer controlled multifermentation system

Production of biosurfactant AP-6 and consumption of carbon (succinic acid) and nitrogen (ammonium ions) by Pseudomonas fluorescens 378 were studied under different growth conditions. The study was performed in a microcomputer controlled multibatch fermentation system which enabled simultaneous running of 10 fermentors. The fermentors were mantled glass vessels, temperature controlled by circulated water, and mixing was arranged by magnetic stirrers. They were connected to the computer system (pH measurement and control) via signal conditioning cards. The microcomputer had a 128 kbytes RAM, two 800-kbyte floppy disc drives, a graphic terminal, and expansion cards. Biosurfactant production was independent of the carbon-to-nitrogen ratio and the phosphorus content in the medium. Omitting the Fe(III) supplement to the medium increased the product yield by 120%. Changes in oxygen transfer rate and pH in the iron deficient cultures did not have any effect on the product yield. Iron deficiency increased the cell consumption of carbon source. Consumption of carbon source in relation to nitrogen uptake (carbon/nitrogen quotient) increased with increasing quotient in the growth medium. The uptake of carbon and nitrogen changed in the intervals of 1.2-1.5 g/g biomass and 0.09-0.16 g/g biomass, respectively. The consumption of carbon increased from 1.5 g/g biomass to 2.0 g/g biomass when the medium concentration of phosphorus was decreased from 0.18 to 0.027 g/L.     

Variability of measurements of cranial growth in the rabbit

This study concerns techniques used in experimental cranial growth research: roentgen cephalometry, roentgen stereophotogrammetry, and gross measurements (osteometry). A comparison of the precision of these methods has not been found in the literature. Computation of technical errors is fundamental to the sound evaluation of registered findings, and such a presentation must be obligatory in all biometric reports. We compared the measurement error of roentgen cephalometric and osteometric data with that obtained by roentgen stereophotogrammetry (RSA). RSA demonstrates a superior replicability, and this technique gives possibilities for kinematic and volumetric determinations simultaneously with distance evaluation. Roentgen cephalometry has the advantage of enabling distance and angular measurements between any well-defined skeletal points or lines. This technique, preferably after implantation of bone markers, is a reliable alternative, but optimal results necessitates calculations of the magnification factor for each bone segment involved. Direct osteometry does not contribute additional information, but problems of image magnification are omitted. Preferably, one individual should perform all measurements regardless of the method used. Growth rates and values calculated by one technique cannot be directly transformed to some other approach. In all probability, assessments of distance changes would gain substantially by using one technical approach consistently throughout actual age intervals. The least variable measurements of sutural growth are made for sutures growing primarily in one plane and with substantial growth rates. One must realize that differences among studies may be due to the limitations of, in particular, the cephalometric and osteometric techniques.     

Gene and mRNA for precursor polypeptide VI from adenovirus type 2.

We present a 1,040-base-pair-long sequences of adenoviruses type 2 DNA which encodes the complete gene for precursor polypeptide VI (pVI). pVI consists of 250 amino acids amounting to a molecular weight of 26,990. The proteolytic cleavage maturing pVI to virion polypeptide VI removes 33 amino acids from the amino-terminal end of the polypeptide, thus giving the mature polypeptide VI a molecular weight of 23,400. The UAA stop codon terminating pVI translation is separated by 84 nucleotides from the initiator triplet for the hexon gene. Both polypeptides are encoded by the same translational reading frame, suggesting the evolution of pVI and hexon as separate proteins by the introduction of a termination codon and selection of a new splice acceptor site in an ancestral fused polypeptide chain. The splice site where the common tripartite leader is attached to the pVI mRNA precedes the initiator codon for pVI translation by one nucleotide and forms, together with other late splice acceptor sites, a late adenovirus consensus acceptor site. We also demonstrate that the 3' end of the mRNA's belonging to the L2 3'-cotermination family is located only 31 nucleotides upstream from the splice junction of the pVI mRNA. Furthermore, we show that four novel polypeptides of molecular weights 80,000, 39,000, 36,000, and 10,500 are encoded by region L2.     

Virus-receptor interaction in the adenovirus system I. Identification of virion attachment proteins of the HeLa cell plasma membrane.

Plasma membranes from HeLa cells were isolated in a two-phase polymer system. To compare the efficiency of attachment protein extraction, a normalized assay for the assessment of adenovirus type 2 (Ad2) receptor-active components interfering with the attachment of Ad2 to HeLa cells was developed. An optimized detergent extraction procedure, 0.5% Triton X-100, was used, and solubilized membrane proteins were radioisotope labeled in vitro. Proteins with affinity for Ad2 virions were quantified and identified in a sucrose gradient sedimentation assay and by affinity chromatography with cross-linked Ad2 virions immobilized to AH-Sepharose 4B. From virions recovered in the sucrose gradient system, one major membrane component of high affinity was identified with a polypeptide molecular weight of around 40,000. Glycosylated proteins isolated by wheat germ lectin chromatography with high affinity for immobilized virus particles were isolated, and two major components with apparent molecular weights of 40,000 and 42,000 were identified. We suggest that a glycosylated protein with high affinity for Ad2 virions and a polypeptide molecular weight of 40,000 to 42,000 is one component of the Ad2 attachment site on HeLa cells.