Purification of Pregnancy Zone Protein and Its Receptor Binding Domain from Human Plasma

A new significantly improved method for purification of pregnancy zone protein (PZP), α₂-macroglobulin (α₂M), and the C-terminal PZP receptor binding domain is presented. Several steps in an earlier procedure have been deleted, and modifications in the gradients in the DEAE step leave most of the contaminants bound to a DEAE-Sephacel gel. This procedure makes possible the rapid, simultaneous purification of both of these closely related unstable proteins in native form from human plasma, with no thiolester cleavage or formation of tetrameric PZP. The final preparations of both α₂M and PZP are pure as determined by nonreducing and reducing polyacrylamide gel electrophoresis following silver staining and no cross-contamination can be observed. The yield has been significantly improved and typically more than 500 mg PZP can be obtained from 1 liter pregnancy plasma. Furthermore, the stability of PZP at different temperatures on storage was studied. In liquid nitrogen PZP can be maintained in native dimeric form with intact thiolester for many years. The storage of native PZP with intact functional properties during and after purification is an obligatory prerequisite to elucidate the biological role of PZP. The receptor binding domain of PZP can be cleaved from the PZP–methylamine complex by papain and isolated from the other peptides by S-200 gel filtration. The cleavage site was determined and the C-terminal fragment was identified with several site-specific monoclonal antibodies against PZP.     

Improving sodium dodecyl sulfate polyacrylamide gel electrophoresis detection of low-abundance protein samples by rapid freeze centrifugation

This work presents a rapid and simple freeze centrifugation method to concentrate dilute protein solutions for detection by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) Coomassie blue staining. Moreover, a simple way to assemble a cryoconcentration device is presented, and its use is discussed. Commercial purified protein standard and an enzyme with high fructosyltransferase (FTase) activity, coming from target fractions obtained by chromatographic separation, were used as an example. FTase, coming directly from the chromatographic fractions, was difficult to view through SDS–PAGE analysis; however, it was easily visualized, and its activity was enhanced, after the application of the freeze centrifugation protocol presented here.     

Ecological aspects of thermoregulation at high altitudes: the case of andean Liolaemus lizards in northern Chile

Summary We document activity field temperatures, daily activity patterns, and extent of thermoregulation in four species of Liolaemus lizards inhabiting at high altitude (above 3500 m) in the Andes of northern Chile. These four species have similar activity field temperature (Tb near 29°C) despite their being distributed at different altitudinal belts. However, conspicuous differences exist between higher-altitude (L. alticolor and L. jamesi) and lower-altitude (L. islugensis and L. ornatus) lizards regarding extent of thermoregulation and activity period. Some differences in morphology, behavior, and patterns of microhabitat occupancy are also apparent among these four species and are seemingly related to the thermal environment to which they are subjected. In comparison to eight low-altitude Liolaemus species in central Chile (Tb near 35°C) the four high-altitude species in northern Chile have lower activity field temperature. The latter is apparently due to the constraints imposed by the harsh Andean thermal environment, a hypothesis supported by the fact that high-altitude Liolaemus lizards under laboratory conditions demonstrate body temperatures that exceed by 5°C or more, those recorded in the field.     

Morphology of neurons and axon terminals associated with descending and ascending pathways of the lateral forebrain bundle in Rana esculenta

Summary The cells of origin of afferent and efferent pathways of the lateral forebrain bundle were studied with the aid of the cobalt-filling technique. Ascending afferents originated from the lateral thalamic nucleus, central thalamic nucleus, posterior tuberculum and the cerebellar nucleus. They terminated in the anterior entopeduncular nucleus, amygdala and the striatum. Telencephalic projection neurons, which are related to the lateral forebrain bundle, were located mainly in the ventral striatum and the anterior entopeduncular nucleus, but were not so numerous in the dorsal striatum. Irrespective of their location, most of the neurons projecting axons into the lateral forebrain bundle had piriform or pyramidal perikarya. Long apical dendrites usually arborized in a narrow space, whereas widely arborizing secondary dendrites originated from short dendritic trunks. The other neurons that contributed to the lateral forebrain bundle were fusiform or multipolar cells. Striatal efferents terminated in the pretectal area and in the anterodorsal, anteroventral and posteroventral tegmental nuclei.     

Prevention of anaphylactic death by macrophage blockade.

Data in the literature concerning the role of macrophages in anaphylaxis are contradictory. In the present study the effect of macrophage blockade induced by gadolinium chloride (GdCl3) on anaphylactic shock was investigated. Our observations show that GdCl3 prevents the lethal anaphylactic shock of mice sensitized to ovalbumin. GdCl3 given i.v. in a dose of 1 mg/100 g body weight 24 or 48 h before the elicitation of anaphylactic shock resulted in 90% survival, compared to the 43% survival in the control group. The same dose of this rare earth metal salt also greatly reduced the mortality in mice sensitized with ovalbumin containing Bordetella pertussis vaccine, and the symptoms of anaphylaxis including the accumulation of 5-hydroxytryptamine in the liver. Our results suggest that macrophages play an important role in anaphylaxis.     

Effect of prolactin and glucocorticoids on P-enolpyruvate carboxykinase activity in liver and mammary gland from diabetic and lactating rats

Summary The administration of 2 bromo--ergocryptine, to reduce serum prolactin decreased the activity of cytosolic P-enolpyruvatc carboxykinase (GTP) (EC4.1.1.32) about 50% in both liver and mammary gland of lactating animals. Adrenalectomy had similar effects to those of bromo-a-ergocryptine. In contrast, there was a 50% increase in enzyme activity in the mammary gland of diabetic, lactating rats and a 10-fold increase in liver as compared with normal rats. P-enolpyruvate carboxykinase activity in mammary gland as liver is coordinately regulated by prolactin, glucocorticoids and insulin.     

Urinary excretion of mandelic and phenylglyoxylic acids after human exposure to styrene vapour

The kinetics of the urinary excretion of mandelic and phenylglyoxylic acids were studied in volunteers exposed to the known concentrations of styrene vapour. The level and the time of exposure were suitably changed to simulate situations in the industrial environment. The aim was to find out the reasons for the contradictory reports in the literature and to verify parameters characterizing the course of excretion of both metabolites. It was found that the course of mandelic acid excretion might be influenced by the length of styrene exposure. If exposure was longer than 4 hours the maximum of excretion was at the end of the exposure time; after short-term exposures (4 h or less) it was somewhat delayed. Maximum excretion of phenylglyoxylic acid was delayed both after short-term and 8-hour exposures. Excretion of the metabolites was diphasic (biexponential). The effective half-lives were found to be independent of the level of exposure. The apparent half-lives (determined in the post-exposure time of 0-16 hours) tended to become prolonged at daily repeated exposures. The ratio of mandelic to phenylglyoxylic acid changed considerably with the level of exposure. In biological monitoring it is advisable to determine both metabolites.