Substrate requirements and subcellular distribution of calcium transport activities in brain membranes

Ca²⁺ transport activity in synaptosomal membranes has been identified as having two major components: Ca²⁺-stimulated ATP hydrolysis and ATP-dependent CA²⁺ uptake. Both processes exhibit similar affinities for Ca²⁺ and operate maximally under identical buffer conditions. Subcellular fractionation studies revealed the Ca²⁺/Mg²⁺ ATPase and ATP-dependent CA²⁺ uptake activities to be highest in synaptic plasma membrane fractions 1 and 2, with lesser activity in synaptic vesicles and mitochondria. Progressive treatment with Triton X-100 activated, then decreased Ca²⁺/Mg²⁺ ATPase, Mg²⁺ ATPase and Ca²⁺ ATPase. ATP-dependent Ca²⁺ uptake was progressively decreased by similar treatment with Triton X-100. These studies illustrate that Ca²⁺ ATPase and ATP-dependent Ca²⁺ uptake may provide two important mechanisms for buffering of cytosolic Ca²⁺ at the nerve terminal. These systems may function to rapidly sequester cytosolic Ca²⁺ following a rise during depolarization and then extrude Ca²⁺ from the terminal against a concentration gradient. This regulation of cytosolic Ca²⁺, represented by two processes of the type seen in other plasma membranes, may play critical roles in calcium homeostasis in nerve cells.Footnote: Portions of this research were submitted by K. M. Garrett in partial fulfillment of requirements for the Doctor of Philosophy Degree in Pharmacology at the University of Texas Health Science Center.     

Identification of canine pulmonary surfactant-associated glycoprotein A precursors

Surfactant-associated glycoproteins A were identified by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude surfactant from canine alveolar lavage: an unglycosylated form (protein A1), 27,000-28,000 daltons; glycoprotein A2, 32,000-34,000 daltons; and glycoprotein A3, 37,000-38,000 daltons; pH at isoelectric point (pI) 4.5-5.0. Glycoproteins A2 and A3 were electroeluted and used to prepare a monospecific antiserum that identified proteins A1, A2, and A3 in immunoblots of crude surfactant obtained from dog lung lavage. This antiserum precipitated several proteins from in vitro translated canine lung poly(A)+ mRNA; proteins of 27,000 daltons, pI 5.0, and 28,000 daltons, pI 4.8-5.0, which precisely comigrated with proteins A1 from canine surfactant. Cotranslational processing of the primary translation products by canine pancreatic microsomal membranes resulted in larger proteins of 31,000-34,000 daltons, pI 4.8-5.0. Treatment of these processed forms of glycoprotein A with endoglycosidase F, to remove N-linked carbohydrate, resulted in proteins of 27,000-28,000 daltons which precisely comigrated with surfactant protein A1. These observations demonstrate that the polypeptide precursors to the glycoproteins A complex are extensively modified by addition of asparagine N-linked complex carbohydrate and are subsequently secreted as glycoproteins A2 and A3.     

Antihypertensive treatment in pregnancy: analysis of different responses to oxprenolol and methyldopa.

One hundred and eighty three hypertensive pregnant women were randomly assigned to antihypertensive treatment with oxprenolol (96 women) or methyldopa (87 women). Control of hypertension was equivalent in both treatment groups, and in 64 (35%) cases hydralazine had to be added to the treatment to achieve the therapeutic goal (diastolic blood pressure below 85 mm Hg). Five perinatal deaths occurred, one in the oxprenolol group and four in the methyldopa group. Detailed analysis confirmed a previous report of greater fetal growth in the group treated with oxprenolol; this trend was present regardless of severity of hypertension and parity. With increasing duration of treatment the differences between the two groups diminished, and there was no difference after 10 weeks of treatment, a finding that may explain some of the reported discrepancies among therapeutic studies. As hypertension in pregnancy may pursue an accelerated course, necessitating urgent delivery, and there is no satisfactory method of predicting the duration of treatment in individual patients fetal benefit is most likely to be achieved by treatment with oxprenolol, provided that there is no maternal contraindication to treatment with beta blockers.     

Selection with gene-cytoplasm interactions. I. Maintenance of cytoplasm polymorphisms

General conditions for the protectedness of gene-cytoplasm polymorphisms are considered for a biallelic model with two cytoplasm types and under the assumption that nuclear polymorphisms cannot be maintained in the presence of only one cytoplasm type. Analytical results involving male fertilities, female fertilities, viabilities and selfing rates are obtained, and numerical results show spiral and cyclic behavior of population trajectories. It is shown that a maternally inherited cytoplasmic polymorphism cannot be maintained in the absence of a nuclear polymorphism, and that a gene-cytoplasm polymorphism can only be maintained if the population shows sexual asymmetry, i.e. , if the ratio of male to female fertility varies among genotypes. Thus, the classical viability selection model does not allow gene-cytoplasm polymorphisms.     

Expression in Bacillus subtilis of the gene for human urogastrone using synthetic ribosome binding sites

Summary A chemically synthesised gene coding for human urogastrone which was earlier cloned in E. coli (Smith et al. 1982) has now been cloned into expression vectors for Bacillus subtilis Two types of constructs have been made, one giving production of methionylurogastrone and the other giving rise to a methionyl-urogastrone- galactosidase fusion polypeptide facilitating quantification of expression levels.The ribosome binding sites used in the expression plasmids are synthetically made oligonucleotides residing on short restriction fragments to allow easy replacement by other ribosome binding sites.Using shuttle vectors and constitutive promoters from Bacillus phages 105 and SPP1, we were able to detect levels of expression amounting to a few thousand molecules per cell during logarithmic growth in both E. coli and B. subtilis.     

Characterization of Cholecystokinin from the Human Brain

Human forms of cholecystokinin have not previously been characterized chemically. In this study, we have extracted and purified the predominant molecular form of cholecystokinin present in human cerebral cortex. The peptide was characterized by amino acid analysis, automated peptide sequencing, and fast atom bombardment mass spectrometry. It appears to be identical to porcine cholecystokinin-octapeptide, with the sequence of Asp-Tyr(SO3)-Met-Gly-Trp-Met-Asp-Phe(NH2). This structural identity is consistent with the observations that the peptide in human brain and porcine cholecystokinin-octapeptide are recognized similarly by a battery of antisera to porcine cholecystokinin; that they coelute from several chromatographic systems, including gel filtration, ion exchange, and reversed-phase; and that they possess similar biological activities.     

The regulation of Mn2+ and Cu2+ uptake in cells of the yeast Candida utilis grown in continuous culture

Abstract Transport of Mn²⁺ was repressed in Candida utilis cells grown in continuous culture in high-Mn²⁺ (100 μM Mn²⁺) medium as compared to cells grown in basic (0.45 μM Mn²⁺) and low-Mn²⁺ (< 0.05 μM Mn²⁺) media. In contrast, no repression of Cu²⁺ uptake occurred in high-Cu²⁺-grown (25 μM Cu²⁺) cells as compared to cells grown in basic medium (0.54 μM Cu²⁺). Cu²⁺-limited cells did not hyperaccumulate Cu²⁺ and there was not significant difference in initial uptake rates for all 3 Cu²⁺ conditions. Mn²⁺ uptake appears to be regulated by a mechanism sensitive to the external Mn²⁺ concentration, whereas Cu²⁺ transport is not governed in this way by the external Cu²⁺.