Synthesis of active Olisthodiscus luteus ribulose-1,5-bisphosphate carboxylase in Escherichia coli

The ribulose-1,5-bisphosphate carboxylase (Rubisco) large- and small-subunit genes are encoded on the chloroplast genome of the eukaryotic chromophytic alga Olisthodiscus luteus. Northern blot experiments indicate that both genes are co-transcribed into a single (>6 kb) mRNA molecule. Clones from the O. luteus rbc gene region were constructed with deleted 5 non-coding regions and placed under control of the lac promoter, resulting in the expression of high levels of O. luteus Rubisco large and small subunits in Escherichia coli. Sucrose gradient centrifugation of soluble extracts fractionated a minute amount of carboxylase activity that cosedimented with native hexadecameric O. luteus Rubisco. Most of the large subunit synthesized in E. coli appeared insoluble or formed an aggregate with the small subunit possessing an altered charge: mass ratio compared to the native holoenzyme. The presence in O. luteus of a polypeptide that has an identical molecular mass and cross reacts with antiserum generated against pea large-subunit binding protein may indicate that a protein of similar function is required for Rubisco assembly in O. luteus.     

The respiratory burst response to Histoplasma capsulatum by human neutrophils. Evidence for intracellular trapping of superoxide anion

Human neutrophils (PMN) have received little attention as to the role they play in host defense against Histoplasma capsulatum (Hc). We have characterized the binding and phagocytosis of Hc yeasts by human PMN and quantified the PMN respiratory burst in response to this organism. mAb specific for CD11a, CD11b, and CD11c all partially blocked the attachment of unopsonized yeasts to PMN; a mAb to CD18 inhibited attachment by greater than 90%. Thus, human PMN recognize and bind Hc yeasts via CD18 adhesion receptors as has been found for human cultured macrophages and alveolar macrophages. Unopsonized yeasts were phagocytosed by PMN, but phagocytosis was increased markedly by heat-labile and heat-stable serum opsonins. These opsonins promoted enhanced phagocytosis of yeasts by increasing the attachment of Hc yeasts to the PMN membrane. Phagocytosis of viable or heat-killed Hc yeasts by PMN did not induce the secretion of superoxide anion (O2-) as quantified by the reduction of cytochrome c. O2- was not detected when yeasts were opsonized in normal serum or immune serum, or at a ratio of yeasts to PMN of up to a 100:1. However, phagocytosis of opsonized yeasts by PMN did not prevent them from subsequently releasing O2- after further incubation with opsonized zymosan or PMA. Opsonized Hc yeasts clearly stimulated the PMN respiratory burst as quantified by intracellular reduction of nitroblue tetrazolium, reduction of cytochrome c in the presence of cytochalasin D, oxygen consumption, luminol-enhanced and nonenhanced chemiluminescence, and H2O2 production. These data suggest that phagocytosis of Hc yeasts by PMN is associated with intracellular entrapment of O2- that is not detectable by reduction of extracellular cytochrome c.     

A single mutation affects L-serine deaminase, L-leucyl-, L-phenylalanyl-tRNA protein transferase, and proline oxidase activity in Escherichia coli K-12.

A mutation at a single locus, wyb, results in several phenotypic changes in Escherichia coli K-12. The Wyb- phenotype includes: (i) an increase in L-serine deaminase activity, together with a loss of inducibility by L-leucine; (ii) an absence of L-leucyl-, L-phenylalanyl-tRNA protein transferase activity; (iii) inducibility of proline oxidase by proline; and (iv) a loss of ability to use maltose as a carbon and energy source.     

Lactose-proton symport by purified lac carrier protein

The lac carrier protein of Escherichia coli was purified by an improved procedure and its activity assayed by a rapid filter method. Following reconstitution of the carrier by octyl glucoside dilution, proteoliposomes were concentrated by filtration on a microporous filter. Lactose accumulation by adsorbed or entrapped proteoliposomes is driven by an artificially imposed pH gradient (interior alkaline), by a membrane potential (interior negative), or by a combination of both forces. Activity is almost completely abolished by the protonophore carbonyl cyanide m-chlorophenylhydrazone or by the competitive inhibitor thiodigalactoside. Addition of lactose to proteoliposomes under appropriate conditions results in alkalinization of the external medium. This effect is not observed with liposomes devoid of lac carrier or in the presence of proton conducting agents. The results provide a strong indication that the lac gamma gene product is the only protein in the cytoplasmic membrane of Escherichia coli required for lactose-proton symport.     

Structural studies on lamellated osmiophilic bodies isolated from pig lung. 31P NMR results and water content

1. Lamellated osmiophilic bodies are intracellular organelles in which pulmonary surfactant is stored prior to secretion. They contain about 85% phospholipid (per dry weight) and dipalmitoyl phosphatidylcholine is a major constituent, and although their ultrastructure is uncertain it is generally supposed that they resemble liposomes. However, liposomes are stable because layers of water are interposed between the lipid bilayers whereas an essential aspect of the function of lamellated bodies is that, subsequent to their secretion, they are rapidly disrupted to form a surface-active film which covers the respiratory epithelium of the lung. 2. A new method for isolating lamellated bodies from pig lung is described which has the advantage of speed and simplicity and which results in increased yields. The homogeneity of the preparation is similar to that obtained by other methods. 3. 31P NMR spectra of lamellated bodies showed that at 40 degrees C about 95% of the phospholipid was present as extended bilayers and that about 5% was present in a phase exhibiting isotropic head group mobility (tau R less than 10(-5) s). It is suggested that this phase may be due to apolar proteins which are present both in lamellated bodies and in liposomes prepared from lipids extracted from them. 4. The internal water content of lamellated bodies has been measured gravimetrically and the hydration of the phospholipid head groups has been examined by 31P NMR. The two methods gave results in good agreement and show that there are about seven molecules of water/molecule of phospholipid. It is concluded that although the phospholipid head groups in lamellated bodies are fully hydrated, there is no zone of free water. 5. Lamellated bodies are osmotically insensitive to NaCl whereas liposomes prepared from lipids extracted from them behave like perfect osmometers. It is suggested that the osmotic insensitivity and restricted water content of lamellated bodies are important to their function and dependent upon polar proteins in the outer limiting membrane.     

Comparison of propranolol,metoprolol, and acebutolol on insulin-induced hypoglycaemia.

Metoprolol and acebutolol, two supposedly cardio-selective beta-adrenergic recptor blocking agents, were tested in healthy volunteers against propranolol, a non-selective drug, for their effect on blood glucose levels during insulin-induced hypoglycaemia. There was not significant difference between propranolol and metoprolol, which both potentiated the initial hypoglycaemic action of the insulin and delayed the return to normoglycaemia. Acebutolol, even though potentiating the initial hypoglycaemia, did not possess a significant delaying effect. A similar trial should be undertaken in diabetics to determine with certainty the safety of such drugs in diabetes mellitus.