Post-translational heterogeneity of the human vitamin D-binding protein (group-specific component)

The vitamin D-binding protein in human serum (the group-specific component) is an alpha 2-globulin which is genetically polymorphic in all populations studied. Previous work (J. Svasti and B. H. Bowman (1978) J. Biol. Chem. 253, 5188-5194, and J. Svasti, A. Kurosky, A. Bennett, and B. H. Bowman (1979) Biochemistry 18, 1611-1617) has shown that the electrophoretic variations of the proteins controlled by two allelic genes, Gc1 and Gc2, are due to at least three amino acid substitutions between Gc1 and Gc2 (Svasti et al. (1979] and to heterogeneity in the Gc1 phenotype arising from carbohydrate dissimilarities. Gc1 migrates electrophoretically as two protein bands, while Gc2 migrates cathodally as a single band. This study demonstrates a post-translational glycosylation difference occurring in a single area of the Gc1 sequence which accounts for the heterogeneity observed previously. The glycosylation site, a threonine residue, appears to be in a sequence which differs between Gc1 and Gc2. The O-glycosidic bond, which is typical of mucins, is rare in plasma proteins. The cyanogen bromide fragment containing the galactosamine-containing carbohydrate in Gc1 was partially sequenced through 20 residues from the amino terminus. No detectable galactosamine could be found in the homologous cyanogen bromide fragment in Gc2. A new purification procedure for the vitamin D-binding protein in human plasma has been developed. Three chromatographic steps provide purified protein.     

The new biology of ageing

Human life expectancy in developed countries has increased steadily for over 150 years, through improvements in public health and lifestyle. More people are hence living long enough to suffer age-related loss of function and disease, and there is a need to improve the health of older people. Ageing is a complex process of damage accumulation, and has been viewed as experimentally and medically intractable. This view has been reinforced by the realization that ageing is a disadvantageous trait that evolves as a side effect of mutation accumulation or a benefit to the young, because of the decline in the force of natural selection at later ages. However, important recent discoveries are that mutations in single genes can extend lifespan of laboratory model organisms and that the mechanisms involved are conserved across large evolutionary distances, including to mammals. These mutations keep the animals functional and pathology-free to later ages, and they can protect against specific ageing-related diseases, including neurodegenerative disease and cancer. Preliminary indications suggest that these new findings from the laboratory may well also apply to humans. Translating these discoveries into medical treatments poses new challenges, including changing clinical thinking towards broad-spectrum, preventative medicine and finding novel routes to drug development.     

Spectrophotometric assay of phenolpthalein in biological fluids.

An assay for phenolphthalein in biological fluids has been developed utilizing methods previously applied to the assay of bromosulphalein and to the deconjugation of steroidal compounds in urine. Intestinal perfusate, serum, and urine samples containing phenolphthalein are deproteinized with acidified acetone, the samples dried, and the phenolphthalein redissolved in ethanol. Color is developed with 0.5 m glycine buffer, pH 12, and the samples read at 550 nm after blanking the spectrophotometer with one of the replicates to which acidic glycine buffer is added. To measure conjugated phenolphthalein in urine, the sample is incubated overnight with β-glucuronidase/arylsulphatase prior to phenolphthalein determination as noted above. This method gives an accurate assay of phenolphthalein to 10^?5m concentrations with coefficients of variation between 2 and 8% and with no resulting interference from hemoglobin or bilirubin.     

Residue Glu83 plays a major role in negatively regulating α-synuclein amyloid formation

α-Synuclein (α-syn) amyloid filaments are the major ultrastructural component of pathological inclusions that define several neurodegenerative disorders, including Parkinson disease and other disorders that are collectively termed synucleinopathies. Since the aggregation of α-syn is associated with the etiology of these diseases, defining the molecular elements that influence this process may have important therapeutics implication. The deletions of major portions of the hydrophobic region of α-syn (Δ74-79 and Δ71-82) impair the ability to form amyloid. However, mutating residue E83 to an A restored the ability of these proteins to form amyloid. Additionally supporting an inhibitory role of residue E83 on amyloid formation, mutating this residue to an A enhanced amyloid formation in the presence of small molecule inhibitors, such as dopamine and EGCG. Our data, therefore, suggest that the presence and placement of the highly charged E83 residue plays a significant inhibitory role in α-syn amyloid formation and these findings provide important insights in the planning of therapeutic agents that may be capable of preventing α-syn amyloid formation.     

Breeding-site productivity of the swamp forest mosquitoes Mansonia bonneae and Ma.dives in Sarawak, East Malaysia

Studies on larval population densities and adult emergence rates of the Brugian filariasis vectors Mansonia bonneae Edwards and Ma. dives Schiner were conducted in freshwater swamp forest bordering the Sadong River, Serian District, Sarawak, East Malaysia, during 1984-85. Three species of aquatic host-plants in the Family Araceae were identified as supporting immature stages of the Ma. bonneae/dives complex. Proportions of positive plants were 4.7%, 6.5% and 3.4% with 6.4 +/- 2.6, 7.3 +/- 2.8 and 10.1 +/- 1.1 larvae per positive plant, respectively, for the plant species Homalomena cordata Schott, H. rostrata Griffiths and Hydrostemma motleyi (Hook. f.) Mabberley. These data indicate no significant preferences between the three types of host-plant. Detailed monitoring of the host-plant H. cordata revealed no significant monthly fluctuations in larval density per plant nor the proportion of positive plants. 11.6% of larvae were Ma. dives and 88.4% were Ma. bonneae. Mean daily yields of Ma. bonneae/dives adults per square metre of H. cordata vegetated water surface were 0.45 males plus 0.57 females during the wet season (December-February) compared with 0.2 males plus 0.31 females during the dry season (June-August). Thus output of adults per plant was approximately halved, and suitable breeding areas were further reduced, during the dry season. By extrapolation from these rates, a crude mean estimate for productivity of Ma. bonneae/dives females is 1.6 million per hectare per annum in swamp forest habitats vegetated with any of the host-plants studied.     

Potential interspecific competition and host specificity: hispine beetles on Heliconia

Abstract. 1. Adults of rolled-leaf hispine beetles (Chrysomelidae, Coleoptera) spend their lives in the scrolls formed by immature leaves of Heliconia (Heli-coniaceae, Monocotyledonae) in Tropical America. As many as eight hispine species can intermingle in the host scrolls at a single site. Scrolls of single host species are invariably occupied by adults of more than one hispine species, and as many as five species can simultaneously occupy one scroll.
2. I made virgin scrolls, which had never contained any insects, by growing leaves under the cover of plastic bags.
3. Thirty-seven combinations of single hispine and Heliconia species were experimentally created in the virgin scrolls, at two sites and in two seasons, in Costa Rica. Combinations included all abundant host species at the sites.
4. All beetles left all leaves of the twenty-eight unnatural beetle—host combinations, within 24 h. On the other hand, none of the nine natural combinations was completely abandoned within the experimental period.
5. Thus, host specificity is not broader in the absence of similar hispine species that might be competitors, and interspecies competition does not affect this obvious aspect of resource utilization for these phytophagous insects.     

Toxicity of Dopamine to Striatal Neurons In Vitro and Potentiation of Cell Death by a Mitochondrial Inhibitor

Abstract: Intrastriatal injections of the mitochondrial toxins malonate and 3-nitropropionic acid produce selective cell death similar to that seen in transient ischemia and Huntington's disease. The extent of cell death can be attenuated by pharmacological or surgical blockade of cortical glutamatergic input. It is not known, however, if dopamine contributes to toxicity caused by inhibition of mitochondrial function. Exposure of primary striatal cultures to dopamine resulted in dose-dependent death of neurons. Addition of medium supplement containing free radical scavengers and antioxidants decreased neuronal loss. At high concentrations of the amine, cell death was predominantly apoptotic. Methyl malonate was used to inhibit activity of the mitochondrial respiratory chain. Neither methyl malonate (50 µ M ) nor dopamine (2.5 µ M ) caused significant toxicity when added individually to cultures, whereas simultaneous addition of both compounds killed 60% of neurons. Addition of antioxidants and free radical scavengers to the incubation medium prevented this cell death. Dopamine (up to 250 µ M ) did not alter the ATP/ADP ratio after a 6-h incubation. Methyl malonate, at 500 µ M , reduced the ATP/ADP ratio by ∼30% after 6 h; this decrease was not augmented by coincubation with 25 µ M dopamine. Our results suggest that dopamine causes primarily apoptotic death of striatal neurons in culture without damaging cells by an early adverse action on oxidative phosphorylation. However, when combined with minimal inhibition of mitochondrial function, dopamine neurotoxicity is markedly enhanced.     

Fixed interval and fixed time treadle pressing in the pigeon: A comparison with FI and FT keypecking

Experiment I used non-naive pigeons having previously performed on both keypecking and treadlepressing Fixed Interval schedules. In condition IT, treadlepressing was reinforced on successive Fixed Interval 60 seconds, Fixed Time 60 seconds and Fixed Interval 60 seconds schedules. Subsequently (condition IK), the same subjects pecked a key on an identical schedule sequence (FI60, FT60, FI60). In Experiment II, separate groups of naïve subjects were assigned either to treadlepressing (condition IIT) or keypecking (condition IIK) and to the same schedule sequence (FI60, FT60, FI60). Treadle pressing and keypecking decreased greatly in Fixed Time schedules. Curvature indices, pauses and running rates were less sensitive than response rates to the switching from one schedule to the other. Experiments I and II yielded similar results, experimental history accounting only for minor differences. The results were discussed in relation to interspecies differences in the temporal regulation of behavior and operant versus respondent control of the response and schedule-induced behaviour.     

Radioiodination and characterization of the plasma membrane of sea urchin sperm

Two methods were used to radioiodinate sea urchin sperm: lactoperoxidase-glucose oxidase and Iodo-Gen. Following iodination the sperm are viable, they undergo the acrosome reaction, and they fertilize eggs. Of the radioactivity associated with the labeled sperm, 28–50% is presumed to be free ¹²⁵I^?, 37–47% is incorporated in lipid, and 8–15% is in trypsin-digestible material believed to be protein. Digestion of the labeled, living sperm with trypsin removes 95.6–99.5% of the macromolecular label (the cells are alive after digestion) suggesting that almost all the protein label is on the external surface of the cell. Thin-layer chromatography of the lipid fraction shows that the major membrane phospholipids and cholesterol are labeled. SDS-PAGE analysis shows the protein-incorporated ¹²⁵I is distributed among four glycoproteins of >250K, 84K, 64K, and 52K dalton apparent molecular weight. Twenty-eight percent of the total protein (trypsin-digestible) label is in the 84K component and 46% in the 64K band. Although both molecules contain much of the label, they are relatively minor components of the TX-100 extract of sperm. The methods outlined will be useful in determining the role of sperm surface components in fertilization.     

Driving forces and current-voltage characteristics of amino acid transport in Riccia fluitans

The complete steady-state I–V relationship of α-aminoisobutyric acid transport across the plasmalemma of rhizoid cells from Riccia fluitans has been measured and analysed with special emphasis on α-aminoisobutyric acid equilibrium and saturation conditions. (A) The electrical data show that: (1) the amino acid-induced electrical current saturates after the addition of the amino acid, regardless of the concentration; (2) a steady state is reached 1–2 h after incubation in α-aminoisobutyric acid, but after less that 5 min in the presence of 1 mM CN⁻; (3) the steady-state I–V characteristic of α-aminoisobutyric acid transport is a sigmoid curve and fairly symmetric in current with respect to the voltage axis; and (4) the equilibrium potential is clearly a function of the amino acid accumulation ratio. It is suggested that the sigmoid curve represents the characteristic of carrier-mediated α-aminoisobutyric acid transport with a voltage-insensitive step, possibly the translocation of the unloaded carrier, rate-limiting. Since under normal conditions the voltage-sensitive rate constant k_oi is much greater than k_io, it is further suggested that the energy to drive this system is put into the transfer of positive charge from outside to the cytoplasm. (B) Accumulation ratios have been determined by inspection of current-voltage data, and additionally by compartmental analysis on green thalli from Riccia fluitans. Both methods give ratios far too low compared with the thermodynamically possible accumulation of about 10⁴. It is suggested that substantial leakages via different non-electrical pathways prevent equilibrium at steady state, and it is concluded that in such leaky systems the thermodynamic equilibrium condition is not suitable for estimating stoichiometries.