Incomplete process of recombinant human platelet-derived growth factor produced in yeast and its effect on the biological activity.

Partially purified recombinant human Platelet-derived Growth Factor BB homodimer isolated from yeast culture media contains variable amounts of unprocessed PDGF-BB. This unprocessed PDGF-BB is found as a result of incomplete cleavage of the precursor to form the mature protein. Although the signal peptide is efficiently removed, a fraction of the PDGF secreted has an extended sequence corresponding to the truncated yeast alpha-factor leader. The data suggest that it is the amino acid chain from the truncated a-factor leader and not the sugar moiety attached to it that is responsible for the higher mitogenic activity found in this unprocessed molecule compared to highly purified PDGF-BB.     

Interfacial pH modulation of membrane protein function in vivo. Effect of anionic phospholipids.

In yeast cells, the magnitude of the membrane surface potential (phi) is determined to a large extent by the relative amount of anionic phospholipids (Cerbón and Calderón (1990) Biochim. Biophys. Acta 1028, 261-267). When a significant surface potential exists, the pH at the membrane surface (interfacial pH) will be different to that in the bulk suspending medium. We now report that: (1) In cells with higher phi (phosphatidylinositol-rich cells (PI-rich) and phosphatidylserine-rich cells (PS-rich) a 10-times lower proton concentration in the bulk was enough to achieve the maximum transport activity of H(+)-linked transport systems when compared to normal cells. (2) When the phi was reduced by increasing the concentration of cations in the medium, more protons were required to achieve maximum transport, that is, the pH activity curves shifted downwards to a more acidic pH. (3) The magnitude of the downward pH shift was around 2.5-times higher for the more charged membranes. (4) Around 10-times more KCl than MgCl2 was necessary to give an equivalent pH shift, in agreement with their capacity to reduce the phi of artificial bilayers. The interfacial pH calculated from the values of phi indicates that it was 0.4 pH units lower in the anionic phospholipid rich cells as compared to normal cells. The results indicate that membrane surface potential may explain the complex relationship between pH, ionic strength and membrane protein function. Maximum transport activities were found for glutamate at interfacial pH of 4.2-4.8 and were inhibited at interfacial pH = 3.2-3.4, suggesting that surface groups of the carrier proteins with pK values in the region 3.8-4.2 (aspartyl and glutamyl) are involved in binding and/or release of charged substrates.     

Modulation of microvascular growth and morphogenesis by reconstituted basement membrane gel in three-dimensional cultures of rat aorta: A comparative study of angiogenesis in Matrigel,collagen, fibrin,and plasma clot

Summary Rings of rat aorta cultured in Matrigel, a reconstituted gel composed of basement membrane molecules, gave rise to three-dimensional networks composed of solid cellular cords and occasional microvessels with slitlike lumina. Immunohistochemical and ultrastructural studies showed that the solid cords were composed of endothelial sprouts surrounded by nonendothelial mesenchymal cells. The angiogenic response of the aortic rings in Matrigel was compared to that obtained in interstitial collagen, fibrin, or plasma clot. Morphometric analysis demonstrated that the mean luminal area of the microvascular sprouts and channels was significantly smaller in Matrigel than in collagen, fibrin, or plasma clot. The percentage of patent microvessels in Matrigel was also markedly reduced. Autoradiographic studies of³H-thymidine-labeled cultures showed reduced DNA synthesis by developing microvessels in Matrigel. The overall number of solid endothelial cords and microvessels was lower in Matrigel than in fibrin or plasma clot. A mixed cell population isolated from Matrigel cultures formed a monolayer in collagen or fibrin-coated dishes but rapidly reorganized into a polygonal network when plated on Matrigel. The observation that gels composed of basement membrane molecules modulate the canalization, proliferation, and organization into networks of vasoformative endothelial cells in three-dimensional cultures supports the hypothesis that the basement membrane is a potent regulator of microvascular growth and morphogenesis. This work was supported by grants from the W. W. Smith Charitable Trust and grants CA14137 and HL43392 from the National Institutes of Health, Bethesda, MD.     

GROWTH PATTERN,REPRODUCTION, AND SELF-THINNING IN SEAWEEDS1

In unispecific plant stands, the logarithm of mean individual weight (w) depends on the logarithm of density (d) by the -3/4 power law (a slope of -1.5 and an intercept ranging from 2.3 to 5.0). The analysis of the w and d relationships in whole cohorts of two seaweed species from the Strait of Gibraltar shows deviations from the canonical equation. The kelp Phyllariopsis purpurascens (C. Agardh) Henry et South (Phaeophyta) growing at a 30-m depth has the lowest intercept value (0.6) recorded for any plant species and a slope not significantly different from -1.5. The slope value is in accordance with those found in species whose growth is not stopped by reproduction. Irradiance under a single layer of blades was lower than the photosynthetic light compensation point, and this could be due to overdispersion of the shoots and, in consequence, to the low intercept value of the self-thinning equation. The w to d relationships in Asparagopsis armata Harvey (Rhodophyta) show two different components: no dependence between these two variables (slope not significantly different from 0) at densities < 500 shoots·m^?2, and a slope more negative (-2.1) than proposed by the -3/2 power law at densities > 500 shoots·m^?2. The pattern at high densities could be due to intraspecific competition for light, whereas the slope ～0 at low densities could be related to inhibition of growth by reproduction (cystocarp and carpospore production). Therefore, rather than being considered exceptional, we suggest that a gradient of variability could be expected in the dependence of w on d when specific growth patterns and reproduction are considered.     

Biflavonoid differentiation in sixBartramia species (Bartramiaceae,Musci)

The occurrence of eight biflavones belonging to the biluteolin series, the apigenin-luteolin series and the 2,3-dihydro-biluteolin series, and one monomeric flavone with an acid group, was investigated in six taxa ofBartramia with emphasis on sect.Ithyphyllae. The variation of biflavone profiles contributes to the characterization of theBartramia species studied; substitution patterns signalize relevance also on the sectional level. Based upon the flavonoid composition,Bartramia afro-ithyphylla is suggested to be transferred from sect.Ithyphyllae to sect.Bartramia of the genusBartramia.     

Minor pollinosis fromCryptomeria japonica in Varese,northern Italy. Aerobiological monitoring and main clinical features

In the last 2 years we have conducted an aerobiological monitoring program ofCryptomeria japonica, a plant belonging to the family of Taxodiaceae that sometimes causes pollinosis in the period from February to April. Throughout 1994, we checked the incidence of its sensitization and the clinical effects in 85 subjects with correlated seasonal symptoms, who gave a positive skin prick test (SPT) for Betulaceae and/or Corylaceae. Twenty-five patients (29.4%; 19 M; 6 F; mean age, 38.8 years) all with oculorhinitis, were SPT positive to an allergenic extract ofCryptomeria. RAST confirmed this positivity in 44% of the cases. No patients showed monosensitization forCryptomeria to either SPT or RAST. Two subjects gave a positive result on specific nasal provocation. RAST inhibition showed no cross-reaction betweenCryptomeria and birch pollen. During the pollen season each patient made a list, scoring symptoms and specifying any drugs used, so we could correlate these elements with aerobiological observations. The pollen concentration probably exceeded the allergizing threshold forCryptomeria on 8 days during 3 months of recording. The intensity and duration of symptoms seemed to be mainly influenced by sensitization to Betulaceae and Corylaceae. It is thus possible that a combination of minor pollinosis may produce seasonal symptoms in allergic patients.     

Heme geometry in the 10 K photoproduct from sperm whale carbonmonoxymyoglobin

We have measured the Soret band of the photoproduct obtained by complete photolysis of sperm whale carbonmonoxymyoglobin at 10 K. The experimental spectrum has been modeled with an analytical expression that takes into account the homogeneous bandwidth, the coupling of the electronic transition with both high and low frequency vibrational modes, and the effects of static conformational heterogeneity. The comparison with deoxymyoglobin at low temperature reveals three main differences. In the photoproduct, the Soret band is shifted to red. The band is less asymmetric, and an enhanced coupling to the heme vibrational mode at 674 cm⁻¹ is observed. These differences reflect incomplete relaxation of the active site after ligand dissociation. The smaller band asymmetry of the photoproduct can be explained by a smaller displacement of the iron atom from the mean porphyrin plane, in quantitative agreement with the X-ray structure analysis. The enhanced vibrational coupling is attributed to a subtle heme distortion from the planar geometry that is barely detectable in the X-ray structure.     

The modulation of calcium currents by the activation of mGluRs

Glutamatergic transmission in the central nervous system (CNS) is mediated by ionotropic, ligand-gated receptors (iGluRs), and metabotropic receptors (mGluRs). mGluRs are coupled to GTP-binding regulatory proteins (G-proteins) and modulate different second messenger pathways. Multiple effects have been described following their activation; among others, regulation of fast synaptic transmission, changes in synaptic plasticity, and modification of the threshold for seizure generation. Some of the major roles played by the activation of mGluRs might depend on the modulation of high-voltage-activated (HVA) calcium (Ca²⁺) currents. Some HVA Ca²⁺ channels (N-, P-, and Q-type channels) are signaling components at most presynaptic active zones. Their mGluR-mediated inhibition reduces synaptic transmission. The interference, by agonists at mGluRs, on L-type channels might affect the repetitive neuronal firing behavior and the integration of complex events at the somatic level. In addition, the mGluR-mediated effects on voltagegated Ca²⁺ signals have been suggested to strongly influence neurotoxicity. Rather different coupling mechanisms underlie the relation between mGluRs and Ca²⁺ currents: Together with a fast, membrane-delimited mechanism of action, much slower responses, involving intracellular second messengers, have also been postulated. In the recent past, the relative paucity of selective agonists and antagonists for the different subclasses of mGluRs had hampered the clear definition of the roles of mGluRs in brain function. However, the recent availability of new pharmacological tools is promising to provide a better understanding of the neuronal functions related to different mGluR subtypes. The analysis of the mGluR-mediated modulation of Ca²⁺ conductances will probably offer new insights into the characterization of synaptic transmission and the development of neuroprotective agents.     

Enzymatic Preparation of Mono- and Di-Stearin by Glycerolysis of Ethyl Stearate and Direct Esterification of Glycerol in the Presence of a Lipase from Candida Antarctica (Novozym 435)

Mixtures of 1(3)-monostearin and distearin were prepared by direct esterification of glycerol with stearic acid or transesterification using ethyl stearate as acyl donor in the presence of Candida antarctica lipase (Novozym 435) using a variety of solvents of differing polarity. In all cases, the transesterification resulted in higher product yields. In n-heptane as reaction medium the addition of water (3%) was essential for high product yields, with mono- and distearin being produced in almost equal amounts. Using more polar solvents as reaction media, such as acetonitrile or acetone, again the highest yields were obtained in the transesterification mode; employing these solvents the reactions were much more selective towards the formation of monostearin.     

The proliferating cell marker monoclonal antibody Ki-67 recognizes specific antigens associated with the nuclear envelope of the early Drosophila embryo

Summary— Immunofluorescence and immunoelectron microscopy indicated that the antibody raised against the nuclear antigen Ki-67 of mammalian cells recognized antigenic determinants of early Drosophila embryos, localized on the outside of the nuclear envelope. Hence, the nuclear envelope of Drosophila appears to share a similar epitope with the chromosome scaffold of mitotic mammalian cells. With the progression of mitosis the antigen persisted around the mitotic spindle region and was also found in the pole regions at metaphase and anaphase. The antibody also stained the equatorial regions of the spindles from anaphase to late telophase. The antibody may therefore be used as a biochemical marker of the nuclear envelope for studying nuclear membrane biogenesis and behavior during the mitotic divisions of the Drosophila embryo.