Evidence for the formation of covalent bonds between macromolecules in the domain of the wall of Candida albicans mycelial cells

An O-glycosylated mannoprotein, after its incorporation into the wall, showed an increase in its molecular weight, due at least to its association with N-glycosidic sugar chain(s). This was shown by rendering the material soluble after partial degradation of the wall structure. At present it is unknown whether this phenomenon is due to an additional transglycosylation process or whether the partial degradation of the wall solubilizes a supramolecular structure formed between the original O-glycosylated protein which becomes linked either directly or indirectly through a protein to the N-sugar chain(s).     

Dicarboxylic acid anhydrides as dissociating agents of protein-containing structures

Dissociation of protein-containing structures by modification of protein amino groups with dicarboxylic acid anhydrides is a mild procedure which, in some cases, offers advantages over treatment with alternative dissociating agents, such as urea, guanidine hydrochloride, detergents, high ionic strength, and extremes of pH: In addition to dissociating multimeric proteins and protein aggregates, dicarboxylic acid anhydrides are effective dissociating agents for membrane-bound proteins and nucleoprotein particles. With most dicarboxylic acid anhydrides reviewed, the introduced reagent residues can be eliminated under moderate acid conditions, which allows the purification of unmodified individual components, and the use of disassembly-reconstitution systems valuable for investigating the structural and functional roles played by the individual components of complex particles:Each reagent can be suitable for a particular purpose, depending on the required specificity of the modification and stability of the modified groups: The stability of the acylated amino groups ranges from the very stable succinylated amino groups to the very labile acylation obtained with dimethylmaleic anhydride: Between these extremes, the stability of the modified amino groups decreases stepwise in the following order: maleic, exo-cis-3,6-endoxo-⁴-tetrahydrophthalic, citraconic, and 3,4,5,6-tetrahydrophthalic anhydride. With respect to the selectivity of the produced modification, little or no modification of hydroxyamino acid and cysteine residues has been observed with dimethylmaleic, exo-cis-3,6-endoxo-⁴-tetrahydrophthalic, and 3,4,5,6-tetrahydrophthalic anhydrides: With the other reagents, the extent of modification of hydroxyamino acid residues increases in the order citraconic, maleic and succinic anhydride: Citraconic and maleic anhydrides can produce irreversible modification of cysteine residues, the reactivity of sulfhydryl groups being higher with maleic anhydride:     

Influence of the solvent on the conformational-dependent properties of random coil polypeptides. II. Mean square optical anisotropies and Kerr constants

Mean square optical anisotropies and molar Kerr constants were calculated for homopolypeptides of the 20 natural amino acids and of several enzymes and proteins in the random-coil state. The effect of hydration was taken into account in constructing the molecular potential that gives the conformational energies as a function of the rotational angles phi and psi of the backbone and chi(1) of the side chain. The Rotational Isomeric State model was used in calculated energies, the Valence Optical Scheme and the matrix calculus technique of Flory being employed in the evaluation of the optical properties. The results are compared with calculations for the same substances that were performed without taking into account the solvent, as well as with other similar studies. The Kerr constant is confirmed as being one of the most sensitive properties of a given polypeptide to the residue class and to the sequence of those residues.     

The caudal spinal cord of coho salmon (Oncorhynchus kisutch): Immunocytochemical evidence of a “caudal serotoninergic system”

Summary The caudal spinal cord of the coho salmon was investigated by means of immunocytochemistry using antisera against serotonin, urotensin I, urotensin II, somatostatin and a urea-extract of bovine Reissner's fiber (AFRU). Populations of serotonin-immunoreactive (IR) neurons were found rostral and dorsal to the urophysis in close spatial association with caudal secretory neurons. Thick, smooth serotonin-IR processes extended toward the external surface of the spinal cord where they displayed conspicuous terminal dilatations. Thin, beaded serotonin-IR fibers appeared to innervate populations of caudal secretory and somatostatin-IR cerebrospinal fluid-contacting neurons. Most caudal neurosecretory cells displayed both urotensin I and urotensin II immunoreactivities; only a minority reacted exclusively with either urotensin I or urotensin II antisera. Urotensin II-IR and somatostatin-IR cerebrospinal fluid (CSF)-contacting neurons were found as an integral component of the central canal wall in the caudal spinal cord and filum terminale; their dendritic processes appeared to contact Reissner's fiber, which displayed a weak AFRU-immunoreactivity while inside the central canal, but became strongly reactive in the interior of the terminal ventricle as it formed the massa caudalis. The distribution of serotoninergic processes points to a regulatory role in the function of caudal secretory and CSF-contacting neurons and to a putative serotonin release into the subarachnoid space and/or meningeal vasculature. It is also suggested that the CSF-contacting neurons of the central canal may participate in a feedback mechanism controlling the secretory activity of the subcommissural organ.Supported by Grant A/1095-1 from the International Foundation for Science, Sweden, to C.Y.; Grant I/63-476 from Volkswagen-Stiftung to E.R.; and Grant S-85-39 from the Dirección de Investigaciones, Universidad Austral de Chile     

RNA helicase: a novel activity associated with a protein encoded by a positive strand RNA virus.

Most positive strand RNA viruses infecting plants and animals encode proteins containing the so-called nucleotide binding motif (NTBM) (1) in their amino acid sequences (2). As suggested from the high level of sequence similarity of these viral proteins with the recently described superfamilies of helicase-like proteins (3-5), the NTBM-containing cylindrical inclusion (CI) protein from plum pox virus (PPV), which belongs to the potyvirus group of positive strand RNA viruses, is shown to be able to unwind RNA duplexes. This activity was found to be dependent on the hydrolysis of NTP to NDP and Pi, and thus it can be considered as an RNA helicase activity. In the in vitro assay used, the PPV CI protein was only able to unwind double strand RNA substrates with 3' single strand overhangs. This result indicates that the helicase activity of the PPV CI protein functions in the 3' to 5' direction (6). To our knowledge, this is the first report on a helicase activity associated with a protein encoded by an RNA virus.     

Transposon-generated Tn10 insertion mutations at thearo genes ofEscherichia coli K-12

We have obtained a set ofEscherichia coli K-12 derivatives with transposon-generated Tn10 insertion mutations at thearo genes of their aromatic biosynthetic pathway. Bacteriophage NK561 (Tn10) has been used for transposon mutagenesis ofE. coli, strain BW545. Tetracycline (Tc)-resistant derivatives were screened by their Aro^– phenotype by growth on a minimal medium with adequate requirements. Sixaro mutant types were mapped; two strains werearoA, twoaroD, onearoB oraroE, and onearoC. A selective medium and ad-cycloserine enrichment in the presence of tetracycline were used to select for Aro^–, Tc-sensitive derivatives. The reversion index to aromatic-independent colonies of some derivatives was less than 2 × 10^–11 per bacterium per generation. P1 transduction experiments transferred an aroA::Tn10 insertion fromE. coli BW545 to an enterotoxigenicE. coli strain from porcine origin. Derivatives of this strain beingaro, Tc-sensitive and not reverting toaro ⁺ at a detectable frequency, and many others transduced at will, may prove their usefulness as live vaccines.     

Evidence of morphology-specific isozymes inCandida albicans

S-Adenosylmethionine (SAM) synthetase of yeast and hyphal-phase cells of the dimorphic fungusCandida albicans was characterized by kinetic analysis and response to inhibitors. The enzyme from yeast-phase cells has a Km of 0.17 mM for methionine, 0.14 mM for ATP, and is inhibited (in vitro) by dimethyl-sulfoxide, methionine sulfone, and methionine sulfoxide. The hyphal-phase SAM synthetase has a Km of 0.06 mM for methionine, 0.02 mM for ATP, and its activity (in vitro) is enhanced by the substances that inhibit the yeast-phase enzyme. These data strongly suggest that isozymes of SAM synthetase are present inC. albicans and that they are possibly morphology specific. In vivo studies revealed that synthesis of the enzyme is repressed by the addition of methionine to the growth medium and that specific activity of the enzyme increases when intracellular SAM levels are lowered. In addition, it was shown that the increase in specific activity seen during yeast hypha morphogenesis and in yeast cells grown in a methionine-free medium involves de novo protein synthesis.     

The daily variations of airborne fungal spores in Mexico City

The daily and seasonal distribution of airborne fungal particles was recorded in a high altitude tropical zone. Sampling was carried out in the southern part of Mexico City. An Andersen air sampler was used over a period of six months. Ten minutes sampling for each set of plates was done at fixed schedule: 07:30, 14:00 and 19:00 hours. The sampler was placed 10 m above the ground. Daily variation was found to be associated with the season, weather and atmospheric stability. The highest value of mold counts (3195 CFU m⁻³) was recorded in the evening on October, a transitional month between the rainy and the dry seasons, the lowest (45 CFU m⁻³) at noon during the rainy season. Mold counts were significantly correlated with temperature, having negative signs both in the morning and at noon, and being positive in the evening. The abundance of only three genera was recorded.Cladosporium, was isolated more frequently, and its abundance at 14:00 h was of 38%;Alternaria represented 4.0%, at 14:00 h, andAspergillus 3.0% at 7:30 h. Fifteen species belonging to the latter genera were identified and most of them are considered as opportunistic molds of clinical significance.     

A protocol for the purification of bovine serum albumin free of deoxyribonuclease activity

Bovine serum albumin, free of deoxyribonuclease activity, was obtained in our laboratory using ion-exchange chromatography followed by acetylation. Chromatography on four different resins (DEAE-52, P-11, hydroxylapatite and Q Sepharose fast-flow) was examined. Fractions from Q Sepharose chromatography, eluted with a linear gradient 0-1.0 M NaCl and subsequently acetylated, proved to be the most effective method for obtaining deoxyribonuclease-free bovine serum albumin.