Physicochemical characteristics of human sex hormone binding globulin: evidence for two identical subunits

We have developed a rapid protocol for the purification of human sex hormone binding globulin (SHBG) which allows the protein to be purified from pregnancy serum within 48 h. This minimizes any possible degradation of the protein by serum proteases, and has enabled us to re-examine some important and controversial aspects of its structural composition. Our physicochemical data are consistent with the hypothesis that SHBG is a dimeric glycoprotein composed of 2 protomers that exhibit size heterogeneity (approximately 50 and approximately 52 K daltons). The dimeric SHBG molecule appears to contain only approximately 8% carbohydrate, and sequence information indicates that an N-linked oligosaccharide chain may be attached to residue 7 (asparagine) from the NH2-terminal amino acid (leucine). When compared with earlier reports, differences in the relative amounts of heavy (approximately 52 K) and light (approximately 50 K) protomers, and the microheterogeneity of NH2-terminal amino acids, have led us to conclude that they may be caused by proteolytic degradation in vivo as well as during the storage of blood samples prior to protein purification. However, the NH2-terminal amino acid sequence data indicate that the primary structures of the heavy protomers, which evidently interact to form the majority of SHBG dimer in serum, are similar and may even be identical. Evidence to support this is provided by the observation that a monoclonal antibody, which recognises a configurational epitope, interacts with two epitopes per native dimeric form of human SHBG.     

Multilocus genotyping and molecular phylogenetics resolve a novel head blight pathogen within the Fusarium graminearum species complex from Ethiopia

A survey of Fusarium head blight (FHB)-contaminated wheat in Ethiopia recovered 31 isolates resembling members of the Fusarium graminearum species complex. Results of a multilocus genotyping (MLGT) assay for FHB species and trichothecene chemotype determination suggested that 22 of these isolates might represent a new species within the Fg complex. Phylogenetic analyses of multilocus DNA sequence data resolved the 22 Ethiopian isolates as a novel, phylogenetically distinct species. The new species also appears to be novel in that MLGT probe data and sequence analysis of both ends of the TRI-cluster identified 15ADON and NIV recombination blocks, documenting inter-chemotype recombination involving the chemotype-determining genes near the ends of the TRI-cluster. Results of pathogenicity experiments and analyses of trichothecene mycotoxins demonstrated that this novel Fg complex species could induce FHB on wheat and elaborate 15ADON in planta. Herein the FHB pathogen from Ethiopia is formally described as a novel species.     

Function of Head-Cocking in Garnett's Greater Bush Baby (Otolemur garnettii)

Head-cocking is rotation of the head about the rostrocaudal axis with a fixed direction of orientation. The behavior is a response to either visual or auditory stimuli according to species. Although head-cocking is prevalent in small primates, its functional significance is unclear. We studied head-cocking in response to a variety of novel visual and acoustic stimuli in Garnett's greater bush babies (Otolemur garnettii). We systematically varied stimulus type (animate vs. inanimate image) and mode of presentation (NON-VIDEO vs. VIDEO) to assess their effects on the head-cocking response. A higher incidence of head-cocking occurred with novel animal images and for NON-VIDEO presentations. Acoustic stimuli suppressed rather than facilitated head-cocking. Juveniles head-cocked much more than adults did. Clearly head-cocking in Otolemur garnettii is primarily involved in visual rather than auditory function. It does not serve simple sensory/perceptual functions such as depth perception or acuity. Instead, in consideration of the importance of novelty to the elicitation of the behavior, the higher incidence in younger animals, and the structure of the visual system, we propose that head-cocking is a motor strategy to encode the parameters of novel images in the process of form learning.     

Sulfate reduction and S-oxidation in a moorland pool sediment

In an oligotrophic moorland pool in The Netherlands, S cycling near the sediment/water boundary was investigated by measuring (1) SO₄ ^2– reduction rates in the sediment, (2) depletion of SO₄ ^2– in the overlying water column and (3) release of³⁵S from the sediment into the water column. Two locations differing in sediment type (highly organic and sandy) were compared, with respect to reduction rates and depletion of SO₄ ^2– in the overlying water.Sulfate reduction rates in sediments of an oligotrophic moorland pool were estimated by diagenetic modelling and whole core³⁵SO₄ ^2– injection. Rates of SO₄ ^2– consumption in the overlying water were estimated by changes in SO₄ ^2– concentration over time in in situ enclosures. Reduction rates ranged from 0.27–11.2 mmol m^–2 d^–1. Rates of SO₄ ^2– uptake from the enclosed water column varied from –0.5, –0.3 mmol m^–2 d^–1 (November) to 0.43–1.81 mmol m^–2 d^–1 (July, August and April). Maximum rates of oxidation to SO₄ ^2– in July 1990 estimated by combination of SO₄ ^2– reduction rates and rates of in situ SO₄ ^2– uptake in the enclosed water column were 10.3 and 10.5 mmol m^–2 d^–1 at an organic rich and at a sandy site respectively.Experiments with³⁵S^2– and³⁵SO₄ ^2– tracer suggested (1) a rapid formation of organically bound S from dissimilatory reduced SO₄ ^2– and (2) the presence of mainly non SO₄ ^2–-S derived from reduced S transported from the sediment into the overlying water. A³⁵S^2– tracer experiment showed that about 7% of³⁵S^2– injected at 1 cm depth in a sediment core was recovered in the overlying water column.Sulfate reduction rates in sediments with higher volumetric mass fraction of organic matter did not significantly differ from those in sediments with a lower mass fraction of organic matter.Corresponding author     

Plasma-mediated release of morphine from synthesized prodrugs

Two morphine prodrugs (‘PDA’ and ‘PDB’) were synthesized and the kinetics of esterase-mediated morphine release from these prodrugs were determined when incubated with plasma from different animal species. Morphine was rapidly released from PDA by all species plasma with the maximum reached within 5–10 min; the released morphine was biologically active as determined by an in vitro cAMP assay. The morphine was released from PDB at a slower and species-dependent rate (mouse > rat > guinea pig > human). Morphine’s release from PDB appeared to be mediated by carboxyl esterases as the release was inhibited by the carboxyl esterase inhibitor benzil. PDA nor PDB induce cytotoxicity in the neuronal cell lines SK-NSH and SH-SY5Y. The carboxyl and amino functional moieties present on the linker portions of PDA and PDB, respectively, may facilitate their conjugation to nanoparticles to tailor morphine pharmacokinetics and specific targeting. These studies suggest the potential clinical utility of these prodrugs for morphine release at desired rates by administration of their mixture at selected ratios.     

Studies in conservation genetics of tarpon (Megalops atlanticus) — V. Isolation and characterization of microsatellite loci

The tarpon, Megalops atlanticus, supports important recreational fisheries in the Atlantic Ocean, Caribbean Sea and Gulf of Mexico. Declines in segments of these fisheries have prompted questions concerning genetic stock structure in this species. Preparatory to a survey of genetic variation in tarpon, 15 microsatellite markers were isolated. The number of alleles per locus ranged from two to 10 and observed heterozygosity ranged from 0.091 to 0.765, providing potentially useful markers for the detection of within‐ and among‐population genetic variability.     

Effect of Docosahexaenoic Acid on the Synthesis of Phosphatidylserine in Rat Brain Microsomes and C6 Glioma Cells

Abstract: Docosahexaenoic acid (22:6n-3) is the major polyunsaturated fatty acid (PUFA) in the CNS and accumulates particularly in phosphatidylserine (PS). We have investigated the effect of the 22:6n-3 compositional status on the synthesis of PS. The fatty acid composition of brain microsomes from offspring of rats artificially reared on an n-3-deficient diet showed a dramatic reduction of 22:6n-3 content (1.7 ± 0.1%) when compared with control animals (15.0 ± 0.2%). The decrease was accompanied by an increase in docosapentaenoic acid (22:5n-6) content, which replaced the 22:6n-3 phospholipids with 22:5n-6 molecular species, as demonstrated using HPLC/electrospray mass spectrometry. The n-3 deficiency did not affect the total amount of polyunsaturated phospholipids in brain microsomes; however, it was associated with a decrease in the total polyunsaturated PS content and with increased levels of 1-stearoyl-2-docosapentanoyl (18:0/22:5n-6) species, particularly in phosphatidylcholine. Incorporation of [³H]serine into PS in rat brain microsomes from n-3-deficient animals was slightly but significantly less than that of the control animals. Similarly, C6 glioma cells cultured for 24 h in 22:6n-3-supplemented media (10–40 µ M ) showed a significant increase in the synthesis of [³H]PS when compared with unsupplemented cells. Our data show that neuronal and glial PS synthesis is sensitive to changes in the docosahexaenoate levels of phospholipids and suggest that 22:6n-3 may be a modulator of PS synthesis.     

Protein Brownian Rotation at the Glass Transition Temperature of a Freeze-Concentrated Buffer Probed by Superparamagnetic Nanoparticles

For applications from food science to the freeze-thawing of proteins it is important to understand the often complex freezing behavior of solutions of biomolecules. Here we use a magnetic method to monitor the Brownian rotation of a quasi-spherical cage-shaped protein, apoferritin, approaching the glass transition T_g in a freeze-concentrated buffer (Tris-HCl). The protein incorporates a synthetic magnetic nanoparticle (Co-doped Fe₃O₄ (magnetite)). We use the magnetic signal from the nanoparticles to monitor the protein orientation. As T decreases toward T_g of the buffer solution the protein’s rotational relaxation time increases exponentially, taking values in the range from a few seconds up to thousands of seconds, i.e., orders of magnitude greater than usually accessed, e.g., by NMR. The longest relaxation times measured correspond to estimated viscosities >2 MPa s. As well as being a means to study low-temperature, high-viscosity environments, our method provides evidence that, for the cooling protocol used, the following applies: 1), the concentration of the freeze-concentrated buffer at T_g is independent of its initial concentration; 2), little protein adsorption takes place at the interface between ice and buffer; and 3), the protein is free to rotate even at temperatures as low as 207 K.