The protoplasts of Acremonium chrysogenum. Biochemical and morphological studies

A procedure for preparing stable A. chrysogenum protoplasts capable of 60 per cent regeneration was developed. Two morphogenetic types of the regeneration were detected. The variants isolated after the protoplast regeneration were characterized by wide ranges of morphological variation. Capacity for the antibiotic production varied from 60 to 160 per cent of the activity of the starting strain. A procedure for isolating functionally active mitochondria from protoplasts of A. chrysogenum was also developed. Their main bioenergetic parameters were studied. In the respiratory chain of the A. chrysogenum mitochondria there were detected three conjugation sites of oxidative phosphorylation.     

Composition and structure of the antibiotic polypeptide 308-I from Actinomadura recticatena

Antibiotic 308-I isolated from Actinomadura recticatena and the products of its degradation were studied with the methods of electron impact mass spectrometry, chemical ionization with ammonia, field desorption and ion extraction from solution under atmospheric pressure. It was shown that by its composition and structure antibiotic 308-I was identical to antibiotic BBM-928 A.     

Hybridization of A-factor deficient mutants of Streptomyces griseus by means of fusion of protoplasts

Characteristics of 6 A-factor deficient mutants of S. griseus are presented. The common feature of the mutants was impairment of sporulation, formation of aerial mycelium and streptomycin synthesis. Pair-by-pair hybridization of the mutants was performed with protoplast fusion followed by regeneration. 9 pair couplings of the mutants were performed. In 3 of them sporulating recombinants were detected. The antibiotic production level in 70 hybrids was different and ranged from 0 to 1700 micrograms/ml. The morphological features of the colonies and the number of the spores formed were also different. The common feature of all the 70 sporulating hybrid strains was recovery of synthesis of A-factor, an endogenic regulator of S. griseus development. Therefore, in the A-factor deficient mutants impairment of A-factor synthesis was induced not by the plasmid elimination, as was suggested, but by mutation of separate genes.     

Comparison of the action of pristan and Freund's incomplete adjuvant on the development of ascites in mice--the recipients of hybridoma cells

Production of immunodiagnostic preparations based on monoclonal antibodies requires large amounts of such antibodies. Most frequently preparative quantities of monoclonal antibodies are provided by ascitic fluid from hybridoma-carrying mice. Prior to intraperitoneal administration of hybridoma cells mice are subjected to stimulation with pristan, a mineral oil component. The authors showed that the Freund's incomplete adjuvant (FIA) may be as well used for this purpose. The effect of pristan and the FIA on ascites development in mice with hybridomas producing monoclonal antibodies to the Ia-like antigens of man was studied. The stimulants were administered in amounts of 0.5 ml per a mouse. It was shown that irrespective of the stimulant injection time and the number of the administered hybridoma cells the amount of the ascitic fluid formed in female mice BALB/c stimulated with the FIA was 1.5-3 times higher as that in the animals stimulated with Pristane, the antibody titer in the ascitic fluid being unchanged.     

Use of a protoplasting method for altering the qualitative and quantitative composition of the anthracycline complex in Streptomyces peucetius var. caesius

Conditions for efficient regeneration in mutant strains of the doxorubicin-producing organism Str. peucetius var. caesius were developed. The effect of the protoplast regeneration on changes in the proportion of the components of the anthracycline complex produced by these strains was shown. Variants with doxorubicin productivity 2 times higher than that of the parent strain were isolated.     

The structure of Staphylococcus aureus alpha-toxin-induced ionic channel

Polyethylene glycols (PEG) with molecular weight less than or equal to 3000 were shown to effectively protect human erythrocytes from osmotic lysis induced by alpha-staphylotoxin (ST). PEG with MW less than 3000 do not change the conductivity of ion channels induced by ST in bilayer lipid membranes (BLM). Changing the bilayer from a pure phosphatidylcholine (PC) to a negatively charged phosphatidylserine (PS) film results in an asymmetry of the current-voltage characteristics. This is evidenced by the asymmetrical position of the ST-channel pore in bilayer membranes. The results obtained allow to conclude that the ST-channel is an interprotein pore filled with water (with an inner diameter of 2.5-3 nm and a length of approximately 10 nm). It is composed of six molecules of alpha-toxin from Staphylococcus aureus. The ST-channel incorporates into a membrane with only one mouth in contact with the polar lipid heads and the other one protruding 4.5-5 nm from the bilayer plane in water solution.     

A single-step purification of an extracellular fungal laccase

Summary A single-step purification procedure forNeurospora crassa laccase is reported. It used Celite chromatography which permits the concentration of the extracellular enzyme from large culture volumes. This method is useful to isolate any fungal or plant laccase as well as other coppercontaining proteins. This work also takes advantage of the 100-fold induction of the enzyme by low concentrations of cycloheximide.

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Resumen Aquí se reporta un procedimiento de purificación en un solo paso para le enzima lacasa deNeurospora crassa. Consiste en el empleo de la cromatografía en Celite que permite la concentración de la enzima exocelular de grandes volúmenes de cultivo. Este método es útil para aislar cualquier lacasa de hongos o plantas así como otras proteínas que contienen sobre. En este trabajo, hemos aprovechado también la inducción de la enzima en más de 100 veces por bajas concentraciones de cicloheximida.

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Résumé Nous rapportons ici une procédure de purification en une étape de la laccase deNeurospora crassa. Elle consiste à employer la chromatographie sur Celite qui permet la concentration de l'enzyme exo-cellulaire à partir de grands volumes de milieu de culture. Cette méthode permet d'isoler n'importe quelle laccase fungique ou de plante ainsi que d'autres protéines à cuivre. Dans le présent travail, nous avons aussi tiré parti de l'induction au centuple de l'enzyme par de faibles concentrations en cycloheximide.

     

Brain Metabolism and Intracellular pH During Ischaemia: Effects of Systemic Glucose and Bicarbonate Administration Studied by 31P and 1H Nuclear Magnetic Resonance Spectroscopy In Vivo in the Lamb

Brain metabolism and intracellular pH were studied during and after episodes of incomplete cerebral ischaemia in lambs under sodium pentobarbitone anaesthesia. 31P and 1H magnetic resonance spectroscopy was used to monitor brain pHi and brain concentrations of inorganic phosphate (Pi), phosphocreatine (PCr), beta-nucleoside triphosphate (beta NTP), and lactate. Simultaneous measurements were made of arterio-cerebral venous concentration differences (AVDs) for oxygen, glucose, and lactate. Cerebral ischaemia was induced by a combination of bilateral carotid clamping and hypotension, and the acute effects of systemic administration of glucose and sodium bicarbonate were examined. The molar ratio of glucose to oxygen uptake by the brain (6G/O2) increased above unity during cerebral ischaemia. Statistically significant AVDs for lactate were not observed. Cerebral ischaemia was associated with a reduction in brain pHi PCr/Pi ratio, and an increase in brain lactate. No effect of arterial plasma glucose on brain lactate concentration or brain pHi was evident during cerebral ischaemia or in the postischaemic period. Administration of sodium bicarbonate systemically in the postischaemic period was associated with a rise in arterial and brain tissue PCO2. A fall in brain pHi occurred which was attributable in part to coincidental brain lactate accumulation. The increase in brain lactate measured by 1H nuclear magnetic resonance in vivo during ischaemia was insufficient to account for the change in buffer base calculated to have occurred from previous estimates of brain buffering capacity.     

N-terminal sequence analysis of human placental protein 14, purified in high yield from decidual cytosol

Human placental protein 14 (PP14) has been purified in high yield from first trimester decidual cytosol. High-performance liquid chromatography on anion exchange, gel filtration and reverse-phase chromatography were used. The protein obtained is approximately 97% pure with an overall recovery of about 50% from the original tissue extract. The first 24 amino acids of the N-terminal were found to be Met-Asp-Ile-Pro-Gln-Thr-Lys-Gln-Asp-Leu-Glu-Leu-Pro-Lys-Leu-Ala-Gly-Thr-Glu-His - Glu-Met-Ala-Met. PP14 has been characterized in this study to be a dimeric glycoprotein of Mr 60,000, with homologous subunits having an Mr of 28,000.     

Structural Features of Human Monoamine Oxidase A Elucidated from cDNA and Peptide Sequences

Monoamine oxidase (MAO), an important enzyme for the degradation of amine neurotransmitters, has been implicated in neuropsychiatric illness. The amino acid sequence for one form of the enzyme, MAO-A, has been deduced from human cDNA clones and verified against proteolytic peptides. The covalent binding site for the flavin adenine dinucleotide (FAD) cofactor is near the C-terminal region. The presence of features characteristic of the ADP-binding fold suggests that the N-terminal region is also involved in the binding of FAD. These cDNAs should facilitate the study of the structure, function, and intracellular targeting of MAO, as well as the analysis of its expression in normal and pathological states.