Glycerol production in relation to the ATP pool and heat production rate of the yeasts Debaryomyces hansenii and Saccharomyces cerevisiae during salt stress

Changes in glycerol production and two parameters related to energy metabolism i. e. the heat production rate and the ATP pool, were assayed during growth of Saccharomyces cerevisiae and Debaryomyces hansenii in 4 mM and 1.35 M NaCl media. For both of the yeasts, the specific ATP pool changed during the growth cycle and reached maximum values around 10 nmol per mg dry weight in both types of media. The levels of glycerol were markedly enhanced by high salinity. In the presence of 1.35 M NaCl, D. hansenii retained most of its glycerol produced intracellularly, while S. cerevisiae extruded most of the glycerol to the environment. The intracellular glycerol level of S. cerevisiae equalled or exceeded that of D. hansenii, however, with values never lower than 3 mol per mg dry weight at all phases of growth. When D. hansenii was grown at this high salinity the intracellular level of glycerol was found to correlate with the specific heat production rate. No such correlation was found for S. cerevisiae. We concluded that during salt stress, D. hansenii possesses the capacity to regulate the metabolism of glycerol to optimize growth, while S. cerevisiae may not be able to regulate when exposed to different demands on the glycerol metabolism.     

Base pair opening dynamics of a 2-aminopurine substituted Eco RI restriction sequence and its unsubstituted counterpart in oligonucleotides.

Studies of 1H NMR selective saturation recovery were performed to determine the imino proton exchange with solvent water of the base pairs in the Eco RI endonuclease recognition sequence GAATTC, placed at the center of self-complementary decamer and dodecamer oligonucleotides. In one oligonucleotide the innermost adenine was replaced by the fluorescent base analogue 2-aminopurine (2AP). From the measurements at different concentrations of TRIS buffer acting as proton exchange catalyst, base pair lifetimes were evaluated. The results at 25 degrees show that the AT base pairs have lifetimes of the order of a few ms, whereas the surrounding GC base pairs in a dodecamer have lifetimes of about 100 ms. The (2AP)T base pair has a shorter lifetime than the corresponding AT base pair. The temperature dependent optical absorption, and for the 2AP containing oligonucleotide fluorescence, were used to study the single strand-duplex equilibrium of the decamers. The results indicate that NMR and the optical techniques, although applied at very different concentrations, monitor the same conformational transition of the oligonucleotide.     

Interaction of monoclonal antiparathyroid antibody with Ca2+ agonistic actions of Mn2+ in normal human parathyroid cells

Effects of the monoclonal antiparathyroid antibodies G11 and E11 on Mn2+ interaction with individual normal human parathyroid cells were studied. At 0.5mM Ca2+, 3mM Mn2+ induced a rapid transient increase in cytoplasmic Ca2+ [Ca2+i] followed by quenching of the fluorescence from the Ca2+ indicator fura-2 as Mn2+ entered into the cells. Whereas the antibody E11 had no effects, treatment with G11 abolished the Ca2+i transient and considerably delayed the entry of Mn2+. The results support the presence of a cation-sensitive receptor mechanism on parathyroid cells and indicate that the antibody G11 not only blocks the interaction between Ca2+ and this receptor mechanism but also that of Mn2+.     

Kinetic characterization of GABA-transaminase from cultured neurons and astrocytes

The enzymatic mechanism and the kinetic parameters of GABA-transaminase extracted from cultured mouse cerebral cortex neurons and astrocytes were studied. Neuronal as well as astrocytic GABA-transaminase obeyed a bi bi ping-pong reaction mechanism. The estimated K_m-values for -ketoglutarate and GABA were significantly lower for astroglial GABA-transaminase compared to the neuronal enzyme suggesting a possible existence of cell specific isozymes of GABA-transaminase. The observed enzymatic mechanism and the magnitude of the estimated kinetic parameters imply that GABA-transaminase synthesized in the two types of cultured neural cells is mechanistically and kinetically equivalent to the enzyme synthesized in the brainin vivo.     

Characterization of MSH/ACTH-like immunoreactivity in sciatic nerves of Xenopus laevis by immunocytochemistry,Western blotting and radioimmunoassay

Summary Previous studies have indicated that rat neurofilament protein may contain an endogenous MSH-like epitope with neuroregenerative properties. The presence of such an epitope has now been studied in nerve tissue from Xenopus laevis. Western blot analyses of sciatic nerve tissue using an assortment of sequence-specific MSH/ACTH antisera revealed the presence of two major immunoreactive protein bands of 52 and 50 kDa, which contained a mid-region MSH-like epitope. Weaker staining occurred in another protein band at 135 kDa. Immunocytochemistry revealed the immunoreactivity to reside in the axis cylinders of the nerve fibers. Other antisera, recognizing other regions of MSH/ACTH produced strong staining of Xenopus intermediate lobes, but failed to stain sciatic nerves. Thus, the proteins detected have no clear relation to either Xenopus neurofilament proteins or proopiomelanocortin.     

Neonatal screening for congenital adrenal hyperplasia using 17-hydroxyprogesterone assay in filter paper blood spots

Screening of infants for congenital adrenal hyperplasia (CAH) using filter paper blood samples collected on the 5th day of life was performed with a radioimmunoassay for 17-hydroxyprogesterone without extraction with organic solvents. A total of 153,000 newborns were screened and 12 cases of CAH were detected (1:12,800). With recall levels related to gestational age, the recall rate could be lowered to 0.05%.     

Detection of proopiomelanocortin mRNA by in situ hybridization, using a biotinylated oligodeoxynucleotide probe and avidin-alkaline phosphatase histochemistry

A synthetic 24-mer oligodeoxynucleotide complementary to the region of proopiomelanocortin (POMC) mRNA that codes for the MSH core sequence (alpha MSH/ACTH[4-11]), was synthesized and labelled in the 3'-end by use of terminal transferase. Probes tailed with either [3H]- or biotin-labelled nucleotides could be used for in situ hybridization studies. Biotinylated probes, hybridized to mouse and rat pituitary sections, were detected by avidin-alkaline phosphatase or streptavidin-alkaline phosphatase procedures and development in 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-nitroblue tetrazolium (NBT). Proteinase K pretreatment of sections produced a drastic enhancement of the signal obtained, particularly in strongly fixed, paraffin-embedded material. The non-radioactive in situ hybridization technique compared favourably to radioactive in situ hybridization in terms of rapidity and precision of the localization. Controls involved deletion of the probe to prove that other components of the reaction sequence did not yield stain, digestion with RNase to prove that tissue RNA was necessary to bind the probe, prehybridization (blocking) with unlabelled probe to prove that the biotinylated probe reacted with its anti-sense region and not its tail and Northern blotting to show that the probe reacted with only one species of pituitary RNA, having the size of mouse pituitary POMC mRNA. In addition, adrenalectomy, known to increase anterior lobe POMC levels, resulted in both increased numbers and increased intensity of positive corticotroph-like cells. Synthetic oligodeoxynucleotides labelled with biotin appear to constitute attractive reagents for in situ hybridization studies when supported by appropriate control procedures.     

Identification of a calmodulin-stimulated (Ca2++ Mg2+)-ATPase in a plasma membrane fraction isolated from maize (Zea mays) leaves

Plasma membranes were isolated from light-grown, 14-day-old maize leaves ( Zea mays L . cv. Golden Cross Bantam) using aqueous two-phase partitioning. The plasma membrane (PM) fraction contained < 0.3% of the total chlorophyll, < 0.2% of the mitochondrial marker enzyme activity, minimal contamination by endomembranes and 34% of the total PM.
A calmodulin-stimulated (Ca2++ Mg2+)-ATPase was identified in the PM-enriched fraction. The Ca2++ calmodulin stimulation was dependent on Mg2+, saturated at ca 25 μM total Ca2+, had a pH maximum at 7.2 and was maximally stimulated by 600 n M bovine brain calmodulin. The stimulation was not greatly affected by the anion present and showed a divalent cation specificity of Ca2+ > Sr+2 ± Mn+2 > Co2+± Cu2+ > Ba2+. The napthalenesulfonamide W7, an antagonist of calmodulin action, completely inhibited the calmodulin stimulation at 175 μM , while the less active analogue W5 was ineffective at this concentration. La3+, an inhibitor of PM Ca2+ transport, showed a 50% inhibition of calmodulin-stimulated ATPase activity at ca 200 μM . Taken as a whole, these data demonstrate the presence of a calmodulinstimulated, (Ca2++ Mg2+)-ATPase on the cytoplasmic surface of the plasma membrane of maize leaf cells.