Concentrations of Amino Acids in Extracellular Fluid After Opening of the Blood-Brain Barrier by Intracarotid Infusion of Protamine Sulfate

Abstract: This article evaluates the influence of an opening of the blood-brain barrier (BBB) on compounds in brain extracellular fluid. The concentrations of amino acids and some other primary amines were determined in dialysates sampled from the right parietal cortex of rats before and after an intracarotid infusion of protamine sulfate. Extravasated plasma proteins were visualized by Evans blue/albumin and immunohistochemistry. CSF albumin— an indicator of blood-CSF barrier opening—was quantified with immunoelectrophoresis. The brains were macroscopically edematous after 10 mg but not after 5 mg of protamine sulfate. The higher dose led to a 50% death rate. The concentrations of amino acids did not change 10 min after the BBB opening. No significant alterations in the amino acid concentrations were observed after the lower dose. The concentrations of glutamate, aspartate, GABA, glycine, taurine, and phosphoethanolamine increased significantly within 50–80 min after the infusion of 10 mg of protamine sulfate. CSF albumin levels were significantly increased 1 h after infusion. We conclude that a dysfunction of the BBB, of a degree known to induce brain edema (10 mg of protamine sulfate), significantly increases the extracellular concentration of excitatory amino acids, GABA, taurine, and phosphoethanolamine in the extracellular space.     

A chimeric gene encoding the methionine-rich 2S albumin of the Brazil nut (Bertlrolletia excelsa H.B.K.) is stably expressed and inherited in transgenic grain legumes

The coding region of the 2S albumin gene of Brazil nut (Bertholletia excelsa H.B.K.) was completely synthesized, placed under control of the cauliflower mosaic virus (CaMV) 35S promoter and inserted into the binary vector plasmid pGSGLUC1, thus giving rise to pGSGLUC1-2S. This was used for transformation of tobacco (Nicotiana tabacum L. cv. Petit Havanna) and of the grain legume Vicia narbonensis L., mediated by the supervirulent Agrobacterium tumefaciens strain EHA 101. Putative transformants were selected by screening for neomycin phosphotransferase (NPT II) and -glucuronidase (GUS) activities. Transgenic plants were grown until flowering and fruiting occurred. The presence of the foreign gene was confirmed by Southern analysis. GUS activity was found in all organs of the regenerated transgenic tobacco and legume plants, including the seeds. In the legume, the highest expression levels of the CaMV 35S promoter-controlled 2S albumin gene were observed in leaves and roots. 2S albumin was localized in the vacuoles of leaf mesophyll cells of transgenic tobacco. The Brazil nut protein was present in the 2S fraction after gel filtration chromatography of the legume seed proteins and could be clearly identified by immunoblotting. Analysis of seeds from the R₂ progenies of the legume and of transgenic tobacco plants revealed Mendelian inheritance of the foreign gene. Agrobacterium rhizogenes strain RifR 15834 harbouring the binary vector pGSGLUCl2S was also used to transform Pisum sativum L. and Vicia faba L. Hairy roots expressed the 2S albumin-specific gene. Several shoots were raised but they never completely rooted and no fertile plants were obtained from these transformants.     

D-Amino acids in chronic renal failure and the effects of dialysis and urinary losses

Summary Total D-amino acids were measured in plasma for 20 non-dialysed patients (creatinine clearance < 12 ml/minute), 20 on CAPD, 20 on haemodialysis and 20 normals. Plasma D-tyrosine and D-phenylalanine were measured in 8 of each group by HPLC. Total D-amino acids, D-tyrosine and D-phenylalanine were significantly greater for patients than normals. D-amino acids and D-tyrosine correlated with creatinine and were decreased during HD. During dialysis, the mean losses for D-tyrosine and D-phenylalanine were similar, about 0.2 mg/CAPD exchange and 3 mg/4 hour haemodialysis (i.e. 2% of the total amino acid, as in plasma). Clearance was unaffected by stereochemical configuration. Urinary losses/24 hour in the non-dialysed patients were 0.35 mg D-tyrosine and 0.25 mg D-phenylalanine. Clearance for D-phenylalanine was greater than for the L-enantiomer. Increases in D-amino acids in renal failure are probably due to depletion of D-amino acid oxidase, but may be enhanced by a D-amino acid rich diet, peptide antibiotics and D-amino acid oxidase inhibiting drugs and metabolites. Possible toxic effects need further investigation.     

Ectopic expression of β-lactoglobulin/human serum albumin fusion genes in transgenic mice: hormonal regulation andin situ localization

We produced transgenic mice carrying the native sheep -lactoglobulin (BLG) or fusion genes composed of the BLG promoter and human serum albumin (HSA) minigenes. BLG was expressed exclusively in the mammary glands of the virgin and lactating transgenic mice evaluated. In contrast, transgenic females carrying the BLG/HSA fusion constructs also expressed the HSA RNA ectopically in skeletal muscle, kidney, brain, spleen, salivary gland and skin. Ectopic expression of HSA RNA was detected only in strains that express the transgene in the mammary gland. There was no obvious correlation between the level of the HSA RNA expressed in the mammary gland and that found ectopically. In three transgenic strains analysed, the expression of HSA RNA in kidney and skeletal muscle increased during pregnancy and lactation, whereas in the brain HSA expression decreased during lactation in one of the strains. HSA protein was synthesized in skeletal muscle and skin of strain #23 and its level was higher in lactating mice compared with virgin mice. Expression of HSA was also analysed in males and was found to be more stringently controlled than in females of the same strains.In situ hybridization analyses localized the expressed transgene in the skin, kidney, brain and salivary glands of various transgenic strains. Distinct strain-specific and cell-type specific HSA expression patterns were observed in the skin. This is in contrast to the exclusive expression of the HSA transgene in epithelial cells surrounding the alveoli of the mammary gland. Taken together, these results suggest that the absence of sufficient mammary-specific regulatory elements in the BLG promoter sequences and/or the juxtaposition of the BLG promoter with the HSA coding sequences leads to novel tissue- and cell-specific expression in ectopic tissues of transgenic mice.     

Zinc content of normal human serum and its correlation with some hematic parameters

Numerous studies have demonstrated that several diseases and stress conditions are associated with changes in the levels of zinc in the blood plasma and cellular elements. In this research the association between serum zinc concentrations and other hematic parameters of diagnostic interest has been evaluated. Quantitative determinations of zinc, total plasmatic proteins, albumin, hemoglobin and calculation of mean corpuscular volume were performed on blood samples from 58 males aged 20–61 years. Concentrations measured in our sample are comparable with reference values. Statistically significant correlation coefficients were found between age and albumin (r = - 0.562, P < 0.001), serum zinc and albumin (r = 0.328, P < 0.05), serum zinc and hemoglobin (r = 0.291, P < 0.05), and total plasmatic proteins and albumin (r = 0.463, P < 0.001). These correlation coefficients were significant even after adjustment for age effect. The determination of serum zinc concentration may be useful in the assessment of clinical scenarios. Particularly, it may provide additional information for the diagnosis of specific pathologies, such as hepatic malfunctions. It could also be useful in the identification of different stages of anemia.     

Molecular heterogeneity of Cowpea (Vigna unguiculata Fabaceae) seed storage proteins

81 wild forms and 110 cultivated cowpea,Vigna unguiculata, accessions from 21 countries of Africa were screened for variability in seed storage proteins. Total seed proteins, albumin and globulin fractions were investigated by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) of nonreduced and/or reduced samples in one- and two-dimensional procedures. The globulin fraction is heterogeneous in molecular weight and contains both legumin-like components and three to six nondisulfide-linked subunits. Three globulin subunits, with molecular weights 110, 76, and 41 kD were found to be composed of disulfide-linked polypeptides. In the nondisulfide-linked fraction, both cultivated and wild forms exhibited patterns of four types (A–D). This fraction contains polypeptide subunits of molecular weights 62, 56, and 52 kD for A type, 62, 56, 54, and 52 kD for B type, 62, 56, 52, and 50 kD for C type, and at least 62, 56, 54, 52, 50, and 49 kD for D type. These subunits present similar multiple charge forms but C and D types possess more basic specific 50 and 49 kD nondisulfide linked components. Major albumin fraction contains subunits of 94, 86, 32, and 24kD. No infraspecific variation was observed in albumin or legumin-like fractions. The discussion is focussed on the relations between genetic variability assessed by storage protein coding genes and phenotypic variability.     

Rat epididymis-specific sperm maturation antigens. I. Evidence that the 26 kD 4E9 antigen found on rat caudal epididymal sperm tail is derived from a protein secreted by the epididymis

Monoclonal antibody 4E9, which was raised against a partially purified detergent extract of rat caudal epididymal sperm, recognizes the tail of sperm from the cauda, but not from caput epididymidis, as well as epithelial cells in a restricted region of the distal caput/corpus epididymidis and proteins in epididymal fluid from corpus and cauda epididymidis. The antigen is apparently a glycoprotein, since it is retained on a Ricinus communis agglutinin l lectin column. Epididymal fluid antigens have apparent M_rS of 38–26 kD, whereas the memrane-associated form of the molecule has an M_r of 26 kD. Immunocytochemical data and Western immunoblot data suggest that the membrane antigen is derived from the fluid antigen, which, in turn, is secrteted by the epididymal epithelium. Characterization of the membrane antigen indicates that it is tightly associated with the sperm surface, behaving as though it is an integral membrane protein. The antigen persists on ejaculated sperm. © 1994 Wiley-Liss, Inc.     

Basis for Phospholipid Incorporation into Peripheral Nerve Myelin

Abstract: To characterize the mechanism(s) for targeting of phospholipids to peripheral nerve myelin, we examined the kinetics of incorporation of tritiated choline-, glycerol-, and ethanolamine-labeled phospholipids into four subfractions: microsomes, mitochondria, myelin-like material, and purified myelin at 1, 6, and 24 h after precursors were injected into sciatic nerves of 23–24-day-old rats. As validation of the fractionation scheme, a lag (> 1 h) in the accumulation of labeled phospholipids in the myelin-containing subfractions was found. This lag signifies the time between synthesis on organelles in Schwann cell cytoplasm and transport to myelin. In the present study, we find that sphingomyelin (choline-labeled) accumulated in myelin-rich subfractions only at 6 and 24 h, whereas phosphatidylserine (glycerol-labeled) and plasmalogen (ethanolamine-labeled) accumulated in the myelin-rich fractions by 1 h. The later phospholipids accumulate preferentially in the myelin-like fraction. These results are consistent with the notion that the targeting of sphingomyelin, a lipid present in the outer myelin leaflet, is different from the targeting of phosphatidylserine and ethanolamine plasmalogen, lipids in the inner leaflet. These findings are discussed in light of the possibility that sphingomyelin targeting is Golgi apparatus based, whereas phosphatidylserine and ethanolamine plasmalogen use a more direct transport system. Furthermore, the routes of phospholipid targeting mimic routes taken by myelin proteins P₀ (Golgi) and myelin basic proteins (more direct).     

Adsorption of BSA on QAE-dextran: equilibria

Equilibrium isotherms for adsorption of bovine serum albumin (BSA) on a strong-base (QAE) dextran-type ion exchanger have been determined experimentally. They were not affected by the initial concentration of BSA but were affected by pH considerably. They were correlated by the Langmuir equation when pH >/= 5.05 and by the Freundlich equation of pH 4.8, which is close to pl approximately 4.8 of BSA. The contribution of ion exchange to adsorption of BSA on the ion exchanger was determined experimentally. The maximum amounts of inorganic anion exchanged for BSA were 1% and 0.4% of the exchange capacity of the ion exchanger at pH 6.9, respectively. Since the effect of the ion exchange on the adsorption appeared small, BSA may be adsorbed mainly by electrostatic attraction when pH >/= 5.05 and by hydrophobic interaction or hydrogen bonding at pH 4.8. When NaCl coexisted in the solution, the shape of the isotherm was similar to the Langmuir isotherm, but it is shifted to the right. When the concentration of NaCl was 0.2 mol/dm(3), BsA was not adsorbed on the resin. When BSA was dissolved in pure water, the saturation capacity of BSA on HPO(4) (2-),-orm resin was about 2 times larger than that for adsorption from the solution with buffer (pH 6.9 and 8.79). The saturation capacity for adsorption of BSA in pure water on HPO(4) (2-) + H(2)O(4) (-)-from resin was much smaller than that from the solution with buffer. The isotherms for univalent Cl(-)-and H(2)PO(4) (-)-form resin was peculiar; that is, the amount of BSA adsorbed decreased with increasing the liquid-phase equilibrium concentration of BSA. (c) 1993 John Wiley & Sons, Inc.