Human lysosomal protective protein. Glycosylation, intracellular transport, and association with beta-galactosidase in the endoplasmic reticulum.

In lysosomes beta-galactosidase and neuraminidase acquire a stable and active conformation through their association with the protective protein. The latter is homologous to serine carboxypeptidases and has cathepsin A-like activity which is distinct from its protective function towards the two glycosidases. To define signals in the human protective protein important for its intracellular transport, and to determine the site of its association with beta-galactosidase, we have generated a set of mutated protective protein cDNAs carrying targeted base substitutions. These mutants were either singly transfected into COS-1 cells or cotransfected together with wild type human beta-galactosidase. We show that all point mutations cause either a complete or partial retention of the protective protein precursor in the endoplasmic reticulum. This abnormal accumulation leads to degradation of the mutant proteins probably in this compartment. Only the oligosaccharide chain on the 32-kDa subunit acquires the mannose 6-phosphate recognition marker, the one on the 20-kDa subunit seems to be merely essential for the stability of the mature protein. In cotransfection experiments, wild type beta-galactosidase and protective protein appear to assemble already as precursors, soon after synthesis, in the endoplasmic reticulum. Mutated protective protein precursors that are retained in the endoplasmic reticulum or pre-Golgi complex interact with and withhold normal beta-galactosidase molecules in the same compartments, thereby preventing their normal routing.     

In vivo cytotoxic T lymphocyte induction with soluble proteins administered in liposomes.

The in vivo induction of a CTL response usually requires that Ag be endogenously synthesized so that appropriate processing can occur. In most of the few examples where successful CTL induction was reported with proteins and peptides, unacceptable adjuvants or means of Ag formulation were used. In the present report, liposomes were used to incorporate the soluble proteins OVA and beta-galactosidase. This simple and convenient to use approach, which requires minimal amounts of Ag, results in priming for a CD8+ CTL response and the establishment of immunologic memory. The liposome approach may not only prove a convenient means of inducing CTL responses in vivo but may also be useful to study the mechanisms of Ag processing.     

Oleanane glycosides from Glycyrrhiza yunnanensis roots.

From the roots of Glycyrrhiza yunnanensis, collected in Yunnan, China, six new oleanane-type triterpene glycosides named yunganosides A1, B1, C1, D1, E2 and F2 were isolated together with hypaphorine. The structures of these glycosides were established by spectroscopic and chemical means.     

UNC-5, a transmembrane protein with immunoglobulin and thrombospondin type 1 domains, guides cell and pioneer axon migrations in C. elegans.

The unc-5 gene is required for guiding pioneering axons and migrating cells along the body wall in C. elegans. In mutants, dorsal migrations are disrupted, but ventral and longitudinal movements are largely unaffected. The gene was tagged for molecular cloning by transposon insertions. Based on genomic and cDNA sequencing, the gene encodes UNC-5, a transmembrane protein of 919 aa. The predicted extracellular N-terminus comprises two immunoglobulin and two thrombospondin type 1 domains. Except for an SH3-like motif, the large intracellular C-terminus is novel. Mosaic analysis shows that unc-5 acts in migrating cells and pioneering neurons. We propose that UNC-5 is a transmembrane receptor expressed on the surface of motile cells and growth cones to guide dorsal movements.     

Influence of dissolved oxygen concentration on the biosynthesis of cephalosporin C.

Cephalosporin C was produced by a highly productive strain of Cephalosporium acremonium under industrial production conditions by fed-batch cultivation in a 40-l stirred-tank reactor using a complex medium containing 50 g l-1 peanut flour. The influence of dissolved oxygen concentration (pO2, DOC), which was maintained at different constant levels between 5 and 40% of its saturation value, during the production phase by means of a parameter-adaptive pO2-controller, on the cephalosporin C biosynthesis, was investigated. The concentrations of cephalosporin C (CPC) and its precursors penicillin N (PEN N), deacetoxycephalosporin C (DAOC), and deacetylcephalosporin C (DAC) were monitored by on-line HPLC. The concentrations of amino acids, valine (VAL), cysteine (CYS), alpha-amino-adipic acid (alpha-AAA), the dipeptide alpha-amino-adipyl-cysteine (AC), and the tripeptide alpha-amino-adipyl-cysteinyl-valine (ACV) were determined by off-line HPLC. By reducing the pO2 in the production phase from 40 to 5% of its saturation value, the CPC concentration diminished from 7.2 to 1.1 g l-1 and the PEN N concentration increased from 2.57 to 7.65 g l-1. The DAC concentration also dropped from 3.13 to 0.42 g l-1; however, the DAOC concentration was less influenced. The concentrations of AC and ACV were also less affected. The small DOC did not lead to an accumulation of the intermediate AC and ACV during the production phase. With increasing DOC in the range of 5-20%, the maximal specific production rate, the cell mass concentration-based and the substrate-based yield coefficients for CPC increased almost linearly, and fell back for PEN N.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reductions of neural activities to upper airway muscles after elevations in static lung volume.

We evaluated the hypothesis that the tonic discharge of pulmonary stretch receptors significantly influences the respiratory-modulated activities of cranial nerves. Decerebrate and paralyzed cats were ventilated with a servo-respirator, which produced changes in lung volume in parallel with integrated phrenic activity. Activities of the facial, hypoglossal, and recurrent laryngeal nerves and nerves to the thyroarytenoid muscle and triangularis sterni were recorded. After a stereotyped pattern of lung inflation, tracheal pressure was held at 1, 2, 4, or 6 cmH2O during the subsequent ventilatory cycle. Increases in tracheal pressure caused progressive reductions in both inspiratory and expiratory cranial nerve activities and progressive elevations in triangularis sterni discharge; peak levels of phrenic activity declined modestly. Similar changes were observed in normocapnia and hypercapnia. We conclude that the tonic discharge of pulmonary stretch receptors is an important determinant of the presence and magnitude of respiratory-modulated cranial nerve activity. This reflex mechanism may maintain upper airway patency and also regulate expiratory airflow.     

Purification of Trypanosoma cruzi surface proteins involved in adhesion to host cells

We have identified four surface 83 kDa proteins of pI values 6.3, 6.4, 6.5 and 6.6 in T. cruzi trypomastigotes which specifically bind to rat heart myoblasts. These proteins were purified by isoelectric focusing and anion-exchange chromatography in an FPLC system. These 83 kDa proteins inhibit the attachment of trypomastigotes to myoblasts in a concentration-dependent manner, indicating that these trypomastigote proteins mediate the attachment of trypomastigotes to heart myoblasts.     

Formation of 2-methyltryptophan in the biosynthesis of thiostrepton: isolation of S-adenosylmethionine:tryptophan 2-methyltransferase

L-2-Methyltryptophan was found to be an intermediate in the biosynthesis of the antibiotic thiostrepton. It was isolated from growing cultures and resting cells of Streptomyces laurentii in trapping experiments after the application of labeled L-methionine or L-tryptophan. Its formation from L-tryptophan and S-adenosylmethionine was studied in a cell-free extract of S. laurentii. Although several attempts to purify the soluble methyltransferase by standard methods failed, some of its characteristics could be determined in the crude extract. The enzyme has a sharp pH optimum at pH 7.8. The apparent Km value for S-adenosylmethionine is 120 microM and the Ki value for S-adenosylhomocysteine is 480 microM. The enzyme is not stereoselective with respect to D- or L-tryptophan, but the D-isomer is converted at a slower rate than the L-isomer. Indolepyruvic acid is also methylated, while indole is not a substrate. The methyl group is transferred with retention of its configuration, contrary to most other methyltransferase reactions.     

Stabilization of Z-DNA by demethylation of thymine bases: 1.3-A single-crystal structure of d(m5CGUAm5CG)

Methylation of cytosine bases at the C5 position has been known to stabilize Z-DNA. We had previously predicted from calculations of solvent-accessible surfaces that the methyl group at the same position of thymine has a destabilizing effect on Z-DNA. In the current studies, the sequence d(m5CGUAm5CG) has been crystallized and its structure solved as Z-DNA to 1.3-A resolution. A well-defined octahedral hexaaquomagnesium complex was observed to bridge the O4 oxygens of the adjacent uridine bases at the major groove surface, and four well-structured water molecules were found in the minor groove crevice at the d(UA) dinucleotide. These solvent interactions were not observed in the previously published Z-DNA structure of the analogous d(m5CGTAm5CG) sequence. A comparison of the thymine and uridine structures supports our prediction that demethylation of thymine bases helps to stabilize Z-DNA. A comparison of this d(UA)-containing Z-DNA structure with the analogous d(TA) structure shows that access of the O4 position is hindered by the C5 methyl of thymine due to steric and hydrophobic inhibition. In the absence of the methyl group, a magnesium-water complex binds to and slightly affects the structure of the Z-DNA major groove surface. This perturbation of the solvent structure at the major groove surface is translated into a much larger 1.41-A widening of the minor groove crevice, thereby allowing the specific binding of two water molecules at well-defined sites of each internal d(UA) base pair. Possible mechanisms by which modifications at the major groove surface of Z-DNA can affect the solvent properties of the minor groove crevice are discussed.     

Polarized electronic spectra of Z-DNA single crystals

Polarized electronic absorption spectra of the (100) face of single crystals of the Z-form double helical duplex of d(m5CGUAm5CG) have been obtained from Kramers-Kronig analysis of reflection data. The c crystallographic axis is parallel to the helix axis and shows but weak absorption. The b axis is perpendicular to the helix axis and shows a structureless absorption band centered at 270 nm with an oscillator strength of 0.26. Calculations of the crystal spectra utilizing available transition moment data for the individual chromophores are carried through using the oriented gas model (no interbase interactions) and, again, employing all base-base interactions (point dipole) in the duplex. The calculated hypochromism of the 270 nm band is much less than the experimental value obtained from the crystal data. The crystal spectra appear to be representative of Z-form double helices of essentially infinite length and not of a collection of twelve base duplexes. No evidence for n pi* transitions polarized parallel to the helix axis is found.