Human placental sialidase complex: characterization of the 60 kDa protein that cross-reacts with anti-saposin antibodies

Sialidase isolated from human placenta is associated with several proteins including acid beta-galactosidase, carboxypeptidase, N-acetyl-alpha-galactosaminidase, and others. These proteins are thought to form an aggregated complex during isolation of sialidase. One of the proteins of 60 kDa was recently identified by Potier et al. (Biochem. Biophys. Res. Comm. 173, 449-456, 1990) as a sialidase protein: this protein also cross-reacted with anti-prosaposin antibodies. We have isolated this protein and from the following evidence identified it as a heavy chain component of immunoglobulin G and not sialidase or a derivative of prosaposin. On gel filtration HPLC, sialidase activity and the 60 kDa protein were clearly separated from one another. The 60 kDa protein cross-reacted not only with antibodies raised against human saposins A, C, and D, but also with second antibody (goat anti-rabbit immunoglobulin G antibody) alone. This 60 kDa protein strongly cross-reacted with anti-human immunoglobulin G antibodies. The sequence of the initial 15 amino acids from the N-terminus of the 60 kDa protein was identical to the sequence of an immunoglobulin G heavy chain protein Tie (gamma 1).     

Fusogenic activity of SIV (simian immunodeficiency virus) peptides located in the GP32 NH2 terminal domain

Peptides of 12, 16 and 24 amino acids length corresponding to the NH2 terminal sequence of SIV gp32 were synthesized. Fluorescence energy transfer studies have shown that those peptides can induce lipid mixing of SUV (Small Unilamellar Vesicles) of various compositions at pH 7.4 and 37 degrees C. LUV (Large Unilamellar Vesicles) were shown to undergo fusion, provided they contained PE in their lipid composition. This work is an attempt to determine how the fusogenic activity depends on the structure of the peptide inserted into a lipidic environment. The peptides secondary structure and orientation in the lipid bilayer were determined using Fourier Transform infrared spectroscopy (FTIR). They adopt mainly a beta-sheet conformation in the absence of lipids. After interaction with DOPC SUV, the beta-sheet is partly converted into alpha-helix oriented obliquely with respect to the membrane interface. We bring here evidence that this oblique orientation is a prerequisite to the fusion process.     

Species-specific differences in tissue-specific expression of alcohol dehydrogenase are under the control of complexcis-acting loci: Evidence fromDrosophila hybrids

Differences in the expression of alcohol dehydrogenase in the hindgut and testis of adult Drosophila virilis, D. texana, D. novamexicana and D. borealis flies were observed. These heritable differences do not arise due to chromosomal rearrangements, since the polytene chromosome banding patterns did not reveal any such gross chromosomal rearrangements near the Adh locus in any of the tested species. Analysis of the interspecific hybrids revealed that these differences are controlled by complex cis-acting genetic loci. Further, the cis-acting locus controlling the expression of ADH in testis was found to be separable by crossing-over.     

Fluorescence and kinetic properties of Ru(III) (NH3)5 modified transferrin

Diferric transferrin was modified using aquopentaammine ruthenium(II), a reagent for surface-accessible uncoordinated histidines. Introduction of the cationic Ru(III) (NH3)3 + 5 group on the imidazole of only 5.5 of the 17 uncoordinated histidines enhances the rates of pyrophosphate-assisted iron removal from the N-terminal and C-terminal binding sites by 16- and 2-fold, respectively. This differential effect on the kinetics of the two sites may partially explain why in the native protein the N-terminal site is more labile than the C-terminal site in acidic solutions where histidine residues become positively charged through protonation. The distance between the metal site and nearby uncoordinated histidines was estimated from fluorescence energy transfer measurements using Tb (III) as the donor and pentaammine ruthenium(III)-labeled imidazole of histidine as the acceptor chromophore. A Tsou Chen-Lu statistical analysis of the fluorescence quenching data suggest that two residues in each lobe of the protein are involved in quenching the fluorescence. By using estimates for the index of refraction and the quantum yield and assuming the energy transfer follows parallel first-order kinetics, an upper limit for the donor-acceptor distance of about 1.4 nm was obtained, assuming two uncoordinated histidine residues equidistant from the metal. His-207 and His-242 in the N-terminal lobe of transferrin and His-535 and His-577 in the C-terminal lobe are within this distance, based on information from the lactoferrin crystal structure. It is postulated that His-207 in the N-terminal lobe and His-535 in the C-terminal lobe are the uncoordinated residues that, when protonated or modified with Ru(III) (NH3)3 + 5, lead to accelerated loss of iron from the two binding sites of the protein.     

Growth and thiophene accumulation by hairy root cultures of Tagetes patula in media of varying initial pH

Summary Hairy roots of Tagetes patula were grown for 24 days in modified Murashige and Skoog's liquid medium at different initial pH levels of 4.0, 5.0, 5.7, 6.0 and 7.0. Irrespective of the initial pH, after 12 days, the pH of the culture medium was approximately 4.5. However the final pH, after 24 days of growth, did depend weakly on the initial pH of the medium. The biomass yield was lowest at an initial pH of 4.0, possibly due to lower utilization of ammonium at this pH. Similar patterns of thiophene accumulation was observed at all pH levels tested. Maximum thiophene accumulation occurred in root cultures which were 12–16 days old.Abbreviations BBTOH 5-(4-hydroxy-1-butenyl)-2,2-bithienyl - BBTOAc 5-(4-acetoxy-1-butenyl)-2,2-bithienyl - BBT 5-(3-buten-1-ynyl)-2,2-bithienyl - MS Murashige and Skoog's nutrient medium - B5 Gamborg's B5 nutrient salts - HPLC High pressure liquid chromatography     

Detection of ginkgolide A in Ginkgo biloba cell cultures

Ginkgo biloba cells were cultured in two 500 mL shake flasks and in 2 L and 6 L immobilization bioreactors using MS medium supplemented with 1 mg.L^–1 NAA, 0.1 mg.L^–1 K and 30 g.L^–1 sucrose. Specific growth rates were 0.06 d^–1, 0.11 d^–1 and 0.07 d^–1 for the 2 L and 6 L bioreactors and shake flask cultures, respectively. Extracellular phosphate, nitrate, ammonium and carbohydrate uptake rates of the bio reactor cultures were approximately 17 to 39% slower than those of shake flask cultures. The specific oxygen uptake and carbon dioxide transfer rates of immobilized Ginkgo biloba cells ranged from 0.027 to 0.041 mmol O₂.g^–1.d.w.hr^–1 (maximum uptake at 14 days) and 0.020 to 0.057 mmol CO₂g. ^–1.d.w.hr^–1 (maximum production at 14 days). Extracts from the biomass of the two immobilized and shake flask suspension cultures were analysed for ginkgolide A by GC-MS. Yields of 7, 17, 19 and 7 ng.g. ^–1d.w. of ginkgolide A were determined for shake flask 1, shake flask 2 and the 2 L and 6 L immobilized cultures, respectively. Traces of ginkgolide B were detected with the signal to noise ratio, however, being too low for positive confirmation of this last product.Abbreviations CTR Carbon dioxide transfer rate - DO Dissolved oxygen - g.d.w. Gram dry weight - GA Ginkgolide A - GB Ginkgolide B - GC Gas chromatography - GC-MS Gas chromatography-mass spectrometry - HPLC High performance liquid chromatography - K Kinetin - MS Murashige and Skoog salt medium - N₁K₁MS Complete Murashige and Skoog medium supplemented with 1 mg.L^–1 NAA, 0.1 mg.L^–1 K and 30g.L^–1 sucrose - NAA Naphthaleneacetic acid - OTR Oxygen transfer rate - PAF Platelet Aggregating Factor - q_CO2 Specific carbon dioxide production rate - q_O2 Specific oxygen uptake rate - u Specific growth rate     

Activity feedback to the mammalian circadian pacemaker: influence on observed measures of rhythm period length.

In the mouse, activity is precisely timed by the circadian clock and is normally most intense in the early subjective night. Since vigorous activity (e.g., wheel running) is thought to induce phase shifts in rodents, the temporal placement of daily exercise/activity could be a determinant of observed circadian rhythm period. The relationship between spontaneous running-wheel activity and the circadian period of free-running rhythms was studied to assess this possibility. With ad libitum access to a running wheel, mice exhibited a free-running period (tau) of 23.43 +/- 0.08 hr (mean +/- SEM). When running wheels were locked, tau increased (23.88 +/- 0.04 hr, p less than 0.03), and restoration of ad libitum wheel running again produced a shorter period (tau = 23.56 +/- 0.06 hr, p less than 0.05). A survey of free-running activity patterns in a population of 100 mice revealed a significant correlation between the observed circadian period and the time of day in which spontaneous wheel running occurred (r = 0.7314, p less than 0.0001). Significantly shorter periods were observed when running was concentrated at the beginning of the subjective night (tau = 23.23 +/- 0.04), and longer periods were observed if mice ran late in the subjective night (tau = 23.89 +/- 0.04), F (1, 99) = 34.96, p less than 0.0001. It was previously believed that the period of the circadian clock was primarily responsive to externally imposed tonic or phasic events. Systematic influences of spontaneous exercise on tau demonstrate that physiological and/or behavioral determinants of circadian timekeeping exist as well.     

mRNA localization studies suggest that murine FGF-5 plays a role in gastrulation.

During gastrulation in the mouse, the pluripotent embryonic ectoderm cells form the three primary germ layers, ectoderm, mesoderm and endoderm. Little is known about the mechanisms responsible for these processes, but evidence from previous studies in amphibians, as well as expression studies in mammals, suggest that signalling molecules of the Fibroblast Growth Factor (FGF) family may play a role in gastrulation. To determine whether this might be the case for FGF-5 in the mouse embryo, we carried out RNA in situ hybridization studies to determine when and where in the early postimplantation embryo the Fgf-5 gene is expressed. We chose to study this particular member of the FGF gene family because we had previously observed that its pattern of expression in cultures of teratocarcinoma cell aggregates is consistent with the proposal that Fgf-5 plays a role in gastrulation in vivo. The results reported here show that Fgf-5 expression increases dramatically in the pluripotent embryonic ectoderm just prior to gastrulation, is restricted to the cells forming the three primary germ layers during gastrulation, and is not detectable in any cells in the embryo once formation of the primary germ layers is virtually complete. Based on this provocative expression pattern and in light of what is known about the functions in vitro of other members of the FGF family, we hypothesize that in the mouse embryo Fgf-5 functions in an autocrine manner to stimulate the mobility of the cells that contribute to the embryonic germ layers or to render them competent to respond to other inductive or positional signals.     

Electrocatalytic properties of polypyrrole in amperometric electrodes

The electrocatalytic oxidation of NADH, ascorbate, urate, xanthine and H₂O₂ at different polypyrrole electrodes has been investigated. The conducting polymer was grown on platinum, glassy carbon, or graphite electrodes and modified by means of enclosed redox-active anions or other redox-active compounds covalently bound to either the N- or the β-position of the pyrrole. Copolymers of pyrrole and N-substituted pyrrole derivatives of chloranil or 2,3-dicholoro-1,4-naphthoquinone showed outstanding electrocatalytic properties for the oxidation of NADH. The application of these electrodes in amperometric steady-state measurements or flow-injection systems in combination with dehydrogenase reactions has been possible.     

Scatter hoarding by kangaroo rats (Dipodomys merriami) and pilferage from their caches

We observed radio-implanted Merriam's kangaroo rats disposingof 10-g bonanzas of rolled oats in 48 trials in the field. Theprincipal determinant of the initial disposition of discoveredfood was apparently its distance from the day burrow: food foundwithin about 10m was mainly larder hoarded, whereas food encounteredfarther afield was usually dispersed immediately in shallowcaches. Cache sites were newly dug for the purpose and not reused;most caches were nearer the current day burrow than was thefood source, but a few were placed far from both the cacher'sday burrow and its habitual nocturnal range. An experiment withartificial caches indicated that security from discovery increaseswith spacing and with proximity to perennial shrubs. Nine kangaroorats cached dyed food, and fecal dye traces revealed extensivepilferage from five of them, by both conspecifics and otherrodent species. Limited evidence indicates that food encounterednearer home and initially larder hoarded was more secure frompilferage than food initially scattered, and yet kangaroo ratswere observed to scatter caches soon after initial larder hoarding.A kangaroo rat whose dyed stores escaped pilferage fed fromthem at intervals for at least 12 days. Even cachers who incurredpilferage made as much, or more, use of their caches as anythief, suggesting that scattering caches may be a defense againstcatastrophic losses.