Molecular cloning and analysis of CDC28 and cyclin homologues from the human fungal pathogen Candida albicans

In the budding yeast Saccharomyces cerevisiae, progress of the cell cycle beyond the major control point in G1 phase, termed START, requires activation of the evolutionarily conserved Cdc28 protein kinase by direct association with GI cyclins. We have used a conditional lethal mutation in CDC28 of S. cerevisiae to clone a functional homologue from the human fungal pathogen Candida albicans. The protein sequence, deduced from the nucleotide sequence, is 79% identical to that of S. cerevisiae Cdc28 and as such is the most closely related protein yet identified. We have also isolated from C. albicans two genes encoding putative G1 cyclins, by their ability to rescue a conditional GI cyclin defect in S. cerevisiae; one of these genes encodes a protein of 697 amino acids and is identical to the product of the previously described CCN1 gene. The second gene codes for a protein of 465 residues, which has significant homology to S. cerevisiae Cln3. These data suggest that the events and regulatory mechanisms operating at START are highly conserved between these two organisms.     

Effects of elevated cytosolic calcium on ACh-induced swine tracheal smooth muscle contraction

Increased intracellular calcium concentration ([Ca²⁺]_i) is required for smooth muscle contraction. In tracheal and other tonic smooth muscles, contraction and elevated [Ca²⁺]_i are maintained as long as an agonist is present. To evaluate the physiological role of steady-state increases in Ca²⁺ on tension maintenance, [Ca²⁺]_i was elevated using ionomycin, a Ca²⁺ ionophore or charybdotoxin, a large-conductance calcium-activated potassium channel (K_Ca) blocker prior to or during exposure of tracheal smooth muscle strips to Ach (10^–9 to 10^–4 M). Ionomycin (5 µM) in resting muscles induced increases in [Ca²⁺]_i to 500±230 nM and small increases in force of 2.6±2.3 N/cm². This tension is only 10% of the maximal tension induced by ACh. Charybdotoxin had no effect on [Ca²⁺]_i or tension in resting muscle. After pretreatment of muscle with ionomycin, the concentration-response relationship for ACh-induced changes in tension shifted to the left (EC₅₀=0.07±0.05 µM ionomycin; 0.17±0.07 µM, control, p<0.05). When applied to the muscles during steady-state responses to submaximal concentrations of ACh, both ionomycin and charybdotoxin induced further increases in tension. The same magnitude increase in tension occurs after ionomycin and charybdotoxin treatment, even though the increase in [Ca²⁺]_i induced by charybdotoxin is much smaller than that induced by ionomycin. We conclude that the resting muscle is much less sensitive to elevation of [Ca²⁺]_i when compared to muscles stimulated with ACh. Steady-state [Ca²⁺]_i limits tension development induced by submaximal concentrations of ACh. The activity of K_Ca moderates the response of the muscle to ACh at concentrations less than 1 µM.     

Multiple residues specify external tetraethylammonium blockade in voltage-gated potassium channels.

We have mapped residues in the carboxyl half of the P region of a voltage-gated K+ channel that influence external tetraethylammonium (TEA) block. Fifteen amino acids were substituted with cysteine and expressed in oocytes from monomeric or heterodimeric cRNAs. From a total of six mutant channels with altered TEA sensitivity, three were susceptible to modification by extracellularly applied charged methanethiosulfonates (MTSX). Another residue did not affect TEA block but was protected from MTSX by TEA. MTSX modification of position Y380C, thought to form the TEA binding site, affected TEA affinity only moderately, and this effect could be reversed by additional charge transfer from an oppositely charged MTSX analog. The results show that TEA block is modulated from multiple sites, including residues located deep in the pore and that several side chains besides that of Y380 are exposed at the TEA receptor.     

Stereochemical control of yeast reductions. 2. Quantitative treatment of the kinetics of competing enzyme systems for a single substrate

Quantitative expressions have been developed for systems such as yeast reductions where competing enzymes act on one substrate to yield two enantiomeric products. These expressions relate the observed stereochemical variables, the extent of conversion (C), the optical purity expressed as enantiomeric excess (ee), and the initial substrate concentration (A₀) to the kinetic parameters K_R and K_S (apparent Michaelis constants) and y ($\text{V}{}_{\mathrm{R}}\text{V}{}_{\mathrm{S}}$, the ratio of maximal velocities) of such competing enzymes. The expressions have been experimentally verified using a purified competing enzyme system of l- and d-lactic dehydrogenases. Furthermore, the enantioselective reduction of β-keto esters by intact yeast cells has been examined by means of this kinetic analysis.     

Properties of S-adenosyl-L-methionine-magnesium-protoporphyrin IX methyltransferase from barley

S-Adenosyl-L-methionine-magnesium-protoporphyrin IX methyltransferase(EC 2.1.1.11 [EC] ) is present in greening barley seedlings associatedwith the particulate fraction. This enzyme was purified 20 foldusing protamine and ammonium sulfate precipitation. The enzymewas active over a wide pH range with highest activity at pH7.5. The Km values for Mg-protoporphyrin IX and S-adenosylmethioninewere 48 and 39 µM, respectively; S-adenosylethionine andS-adenosyihomocysteine were competitive inhibitors with respectto S-adenosylmethionine; hemin inhibition was non-competitivewith respect to Mg-protoporphyrin IX; thiol compounds exhibiteda stimulatory effect on enzyme activity. The properties of theenzyme are discussed and compared with the enzyme from otherorganisms. ¹ This research was supported in part by the Utah State AgriculturalExperiment Station. ² Present address: Department of Chemistry, Boston University,Boston, Massachusetts, U. S. A. ³ Present address: Department of Biochemistry and Microbiology,Faculty of Pharmacy, Comenius University, Bratislava, Czechoslovakia. (Received February 20, 1978; )     

Photosynthetic Characteristics of Portulaca grandiflora, a Succulent C(4) Dicot : CELLULAR COMPARTMENTATION OF ENZYMES AND ACID METABOLISM

The succulent, cylindrical leaves of the C₄ dicot Portulaca grandiflora possess three distinct green cell types: bundle sheath cells (BSC) in radial arrangement around the vascular bundles; mesophyll cells (MC) in an outer layer adjacent to the BSC; and water storage cells (WSC) in the leaf center. Unlike typical Kranz leaf anatomy, the MC do not surround the bundle sheath tissue but occur only in the area between the bundle sheath and the epidermis. Intercellular localization of photosynthetic enzymes was characterized using protoplasts isolated enzymatically from all three green cell types.     

Interfacial preference of anesthetic action upon the phase transition of phospholipid bilayers and partition equilibrium of inhalation anesthetics between membrane and deuterium oxide

The half-height linewidth (v 1/2) of the 1H-NMR spectra of dipalmitoylphosphatidylcholine vesicles changes abruptly at the phase transition temperature. In the absence of inhalation anesthetics, proton signals from the choline head group (hydrophilic interface) and acyl-chain tails (lipid core) change at the same temperature of 39.6 degrees C. The present study compared the effect of four inhalation anesthetics, i.e., methoxyflurane, chloroform, halothane and enflurane, upon the ligand-induced phase transition of phosphatidylcholine vesicle membranes at 37 degrees C. The anesthetics showed differential action upon the phase transition of the phospholipid vesicle membranes between the lipid core and the hydrophilic interface. The concentrations of anesthetics which induced the phase transition of the lipid core were about 2-fold greater than those required for the phase transition of the interfacial choline head groups. From the area under the proton signals of inhalation anesthetics in the NMR spectra, the maximum solubilities of methoxyflurane, chloroform and halothane in 2H2O at 37 degrees C were determined to be 0.671 . 10(-4), 2.637 . 10(-4) and 1.398 . 10(-4) (expressed as mole fractions), or 3.35, 13.17 and 6.98 mmol/1000 g 2H2O, respectively. The solubilities of the anesthetic vapor in 2H2O expressed as mole fractions according to Henry's law ere 9.586 . 10(-4), 6.432 . 10(-4) and 2.311 10(-4)/atm (1.013 . 10(5) Pa) partial pressure, respectively. The presence of phospholipid vesicles in 2H2O increased the solubility of the inhalation anesthetics. From difference between solubility in 2H2O and a dipalmitoylphosphatidylcholine vesicle suspension, the partition coefficients of methoxyflurane, chloroform and halothane between the phospholipid vesicle membranes and 2H2O were estimated. These values, calculated from the mole fractions, were 3364, 1660 and 3850, respectively at 37 degrees C.     

Blood changes in brook trout induced by infection with Aeromonas salmonicida.

Furunculosis was induced in brook trout, Salvelinus fontinalis, by experimental inoculation with Aeromonas salmonicida. Total protein, hemoglobin, sialic acid, fatty acids, triglycerides, cholesterol, inorganic-phosphorus, acid-soluble phosphorus, and lipid-phosphorus decreased in the blood of the infected fish while amino acids, urea, total creatinine, ammonia, and glucose increased. Pyruvic acid, lactic acid, and ascorbic acid values showed no significant change.