Subunit function in eukaryote cytochrome c oxidase. A mutation in the nuclear-coded subunit IV allows assembly but alters the function and stability of yeast cytochrome c oxidase.

Strains of the yeast Saccharomyces cerevisiae disrupted in YCOX4, the nuclear gene encoding cytochrome c oxidase subunit IV, do not assemble a functional or spectrally visible oxidase. We report the characterization of a yeast strain, RM1, expressing a mutated YCOX4 gene which is temperature sensitive for respiration at 37 degrees C, but incorporates cytochrome aa3 over all growth temperatures. The mutant enzyme is less stable than the wild type, with subunit IV readily proteolyzed without gross denaturation of the complex but with a concomitant loss of oxidase activity. When grown fermentatively at 37 degrees C, cytochrome c oxidase from the mutant strain had a turnover number of less than 3% of the normal complex, while Km values and subunit levels were comparable to normal. Thus alterations in subunit IV can perturb the enzyme structure and alter its catalytic rate, implying a role for this subunit in cytochrome c oxidase function as distinct from assembly.     

Crystallization and preliminary X-ray analysis of gamma-aminobutyric acid transaminase

gamma-Aminobutyric acid transaminase from pig liver, an alpha 2 dimeric enzyme of Mr 110,100, has been crystallized by the vapour diffusion method with polyethylene glycol as precipitant. The crystals are monoclinic, space group P2(1), unit cell dimensions a = 82.1 A, b = 230.0 A, c = 70.3 A, beta = 123.9 degrees and diffract to 2.5 A resolution. There are two dimers per asymmetric unit.     

The Macrophage Inhibitor CNI-1493 Blocks Metastasis in a Mouse Model of Ewing Sarcoma through Inhibition of Extravasation

Metastatic Ewing Sarcoma carries a poor prognosis, and novel therapeutics to prevent and treat metastatic disease are greatly needed. Recent evidence demonstrates that tumor-associated macrophages in Ewing Sarcoma are associated with more advanced disease. While some macrophage phenotypes (M1) exhibit anti-tumor activity, distinct phenotypes (M2) may contribute to malignant progression and metastasis. In this study, we show that M2 macrophages promote Ewing Sarcoma invasion and extravasation, pointing to a potential target of anti-metastatic therapy. CNI-1493 is a selective inhibitor of macrophage function and has shown to be safe in clinical trials as an anti-inflammatory agent. In a xenograft mouse model of metastatic Ewing Sarcoma, CNI-1493 treatment dramatically reduces metastatic tumor burden. Furthermore, metastases in treated animals have a less invasive morphology. We show in vitro that CNI-1493 decreases M2-stimulated Ewing Sarcoma tumor cell invasion and extravasation, offering a functional mechanism through which CNI-1493 attenuates metastasis. These data indicate that CNI-1493 may be a safe and effective adjuvant agent for the prevention and treatment of metastatic Ewing Sarcoma.     

Determination of products of acetohydroxy acid synthase by the colorimetric method, revisited

The enzyme acetohydroxy acid synthase (AHAS, EC 4.1.3.18) catalyzes two competing reactions of physiological importance: condensation of two molecules of pyruvate to form acetolactate (AL) or condensation of pyruvate and 2-ketobutyrate to form acetohydroxybutyrate (AHB). The activity of AHAS is most frequently analyzed using the Westerfeld method, in which the acetoin formed upon decarboxylation of AL is determined by colorimetric reaction with creatine and alpha-naphthol. However, there has been confusion as to the interpretation of the results of this assay in the presence of both substrates, conditions which lead to formation of both AL and AHB. By applying this assay to enzymatically prepared samples of AL and AHB which have also been analyzed by two other independent methods, we show here that the color yield for AHB in the commonly used assay is 35-40% that for equivalent amounts of acetoin or AL. The relative color yield is not significantly affected by varying the time or temperature of various steps in the color-forming reaction. This information could in principle be used, together with an independent specific assay for AHB, to determine the composition of an AHAS product mixture; it would, however, be less accurate than a simultaneous chromatographic method.     

Modulation by polyelectrolytes of canine cardiac microsomal calcium uptake and the possible relationship to phospholamban

Calcium uptake and (Ca2+ + Mg2+)-ATPase activity in canine cardiac microsomes were found to be stimulated by heparin and various other polyanions. Prior treatment of the microsomes with the ionophores alamethicin or A23187 produced no change in the extent of stimulation of the ATPase activity by heparin yet eliminated net calcium uptake. This finding and a lack of change in the stoichiometric ratio of mol of calcium transported/mol of ATP hydrolyzed (calcium:ATP) suggest that the effect of heparin is on the calcium pump rather than on a parallel calcium efflux pathway. Certain polycationic compounds including poly-L-arginine and histone inhibited both cardiac and fast skeletal muscle microsomal calcium uptake and also produced no change in the stoichiometric ratio of calcium to ATP. Several lines of evidence indicate that the polyanionic compounds tested stimulate calcium uptake by interacting with phospholamban, the putative phosphorylatable regulator of the cardiac sarcoplasmic reticulum calcium pump, whereas polycationic compounds appear to interact with the pump. (i) Heparin stimulated calcium uptake to the same extent as protein kinase A or trypsin, whereas prior phosphorylation or tryptic cleavage of phospholamban from the membrane abolished the stimulatory effect of heparin. (ii) Calcium uptake and (Ca2+ + Mg2+)-ATPase activity in fast skeletal muscle microsomes, which lack phospholamban, were unaffected by heparin. (iii) Purified cardiac (Ca2+ + Mg2+)-ATPase activity was no longer stimulated by heparin yet was still inhibited by polycationic compounds. The heparin-induced stimulation of calcium uptake was dependent on the pH and ionic strength of the heparin-containing preincubation medium, hence electrostatic interactions appear to play a significant role in heparin's stimulatory action. The data are consistent with an inhibitory role of the positively charged cytoplasmic domain of phospholamban with respect to calcium pump activity and the relief of the inhibition upon reduction in phospholamban's positive charge by phosphorylation or binding of polyanions.     

Studies of the proliferation and differentiation of immature myeloid cells in vitro: 4: Preculture proto-oncogene expression and the behaviour of myeloid leukemia cells in vitro

Studies were conducted to determine the relationship between the pretherapy characteristics of leukemia cells and their behaviour during culture in vitro. Leukemia cells which proliferated well in vitro also proliferated well in vivo. Cells which manifested myeloid or monocytic differentiation in vivo tended to manifest differentiation along these lines in vitro. Cells which manifested high levels of expression of c-fms, c-fes, or triose phosphate isomerase prior to culture were likely to differentiate in vitro, with high levels of c-fes expression being related to myeloid maturation. These observations suggest that differentiation at the molecular level prior to culture is a requisite for leukemia cell differentiation in vitro. The same may be true for differentiation in vivo under the influence of exogenously administered agents such as cytotoxic chemotherapy or recombinant growth factors.     

Transport of zinc and manganese to developing wheat grains

An understanding of the transport pathway used by Zn and Mn to enter developing grains may allow measures to increase the Zn and Mn content of wheat grain grown on Zn/Mn deficient soils. For this reason, transport of Zn and Mn into developing grains of wheat ( Triticum aestivum L. cv. Aroona) was investigated. Detached ears (18–22 days post-anthesis) were cultured for 48 h in a solution containing 185 kBq of ⁶⁵Zn and 185 kBq of ⁵⁴Mn. Transport of ⁶⁵Zn to the grain was unaffected by removal of glumes but was slightly reduced after the lemma was removed. Heat girdling the peduncle slightly reduced the amount of ⁶⁵Zn transported to the grain, whilst heat girdling the rachilla reduced transport of ⁶⁵Zn to the grain to a greater degree, suggesting phloem transport to the rachilla. The transport inhibitor CCCP (carbonyl cyanide m -chlorophenyl hydrazone) blocked ⁶⁵Zn transport to grain but not to lemma and glumes. Removing glumes and lemma and heat girdling the peduncle did not affect transport of ⁵⁴Mn, but transport was slightly affected by heat girdling the rachilla, indicating xylem transport. CCCP blocked transport of ⁵⁴Mn into the grain but not to lemma and glumes. It was concluded that xylem-to-phloem transfer of Zn occurs in the rachis and to a lesser extent in peduncle and lemma. The results suggest that the lemma may be an important site for phloem loading when the concentration of Zn within the xylem is high. The data also suggest that Mn was predominantly translocated to the spikelets in the xylem, but that transport to the grain was dependent upon membrane transport before entering the grain. Phloem loading of Mn into the grain vascular system may have occurred at the site of xylem discontinuity in the floral axis.