Analysis of a parallel branch in the mitomycin biosynthetic pathway involving the mitN-encoded aziridine N-methyltransferase

Mitomycin C is a natural product with potent alkylating activity, and it is an important anticancer drug and antibiotic. mitN, one of three genes with high similarity to methyltransferases, is located within the mitomycin biosynthetic gene cluster. An inframe deletion in mitN of the mitomycin biosynthetic pathway was generated in Streptomyces lavendulae to produce the DHS5373 mutant strain. Investigation of DHS5373 revealed continued production of mitomycin A and mitomycin C in addition to the accumulation of a new mitomycin analog, 9-epi-mitomycin C. The mitN gene was overexpressed in Escherichia coli, and the histidine-tagged protein (MitN) was purified to homogeneity. Reaction of 9-epi-mitomycin C with MitN in the presence of S-adenosylmethionine yielded mitomycin E showing that the enzyme functions as an aziridine N-methyltransferase. Likewise, MitN was also shown to convert mitomycin A to mitomycin F under the same reaction conditions. We conclude that MitN plays an important role in a parallel biosynthetic pathway leading to the subclass of mitomycins with 9alpha-stereochemistry but is not involved directly in the biosynthesis of mitomycins A and C.     

Myocardial Infarction-induced N-terminal Fragment of Cardiac Myosin-binding Protein C (cMyBP-C) Impairs Myofilament Function in Human Myocardium

Myocardial infarction (MI) is associated with depressed cardiac contractile function and progression to heart failure. Cardiac myosin-binding protein C, a cardiac-specific myofilament protein, is proteolyzed post-MI in humans, which results in an N-terminal fragment, C0-C1f. The presence of C0-C1f in cultured cardiomyocytes results in decreased Ca²⁺ transients and cell shortening, abnormalities sufficient for the induction of heart failure in a mouse model. However, the underlying mechanisms remain unclear. Here, we investigate the association between C0-C1f and altered contractility in human cardiac myofilaments in vitro. To accomplish this, we generated recombinant human C0-C1f (hC0C1f) and incorporated it into permeabilized human left ventricular myocardium. Mechanical properties were studied at short (2 μm) and long (2.3 μm) sarcomere length (SL). Our data demonstrate that the presence of hC0C1f in the sarcomere had the greatest effect at short, but not long, SL, decreasing maximal force and myofilament Ca²⁺ sensitivity. Moreover, hC0C1f led to increased cooperative activation, cross-bridge cycling kinetics, and tension cost, with greater effects at short SL. We further established that the effects of hC0C1f occur through direct interaction with actin and α-tropomyosin. Our data demonstrate that the presence of hC0C1f in the sarcomere is sufficient to induce depressed myofilament function and Ca²⁺ sensitivity in otherwise healthy human donor myocardium. Decreased cardiac function post-MI may result, in part, from the ability of hC0C1f to bind actin and α-tropomyosin, suggesting that cleaved C0-C1f could act as a poison polypeptide and disrupt the interaction of native cardiac myosin-binding protein C with the thin filament.     

Effects of Escherichia coli ribosomal protein S12 mutations on cell-free protein synthesis.

We examined the effects of Escherichia coli ribosomal protein S12 mutations on the efficiency of cell-free protein synthesis. By screening 150 spontaneous streptomycin-resistant isolates from E. coli BL21, we successfully obtained seven mutants of the S12 protein, including two streptomycin-dependent mutants. The mutations occurred at Lys42, Lys87, Pro90 and Gly91 of the 30S ribosomal protein S12. We prepared S30 extracts from mutant cells harvested in the mid-log phase. Their protein synthesis activities were compared by measuring the yields of the active chloramphenicol acetyltransferase. Higher protein production (1.3-fold) than the wild-type was observed with the mutant that replaced Lys42 with Thr (K42T). The K42R, K42N, and K42I strains showed lower activities, while the other mutant strains with Lys87, Pro90 and Pro91 did not show any significant difference from the wild-type. We also assessed the frequency of Leu misincorporation in poly(U)-dependent poly(Phe) synthesis. In this assay system, almost all mutants showed higher accuracy and lower activity than the wild-type. However, K42T offered higher activity, in addition to high accuracy. Furthermore, when 14 mouse cDNA sequences were used as test templates, the protein yields of nine templates in the K42T system were 1.2-2 times higher than that of the wild-type.     

Mapping airborne pollen of papaya (Carica papaya L.) and its distribution related to land use using GIS and remote sensing

Papaya is an economically important plant in Thailand for domestic consumption and export. However, papaya is extremely susceptible to disease caused by the papaya ring spot virus. Although transgenic papaya has been developed, commercial cultivation of transgenic plants in Thailand is still illegal. One concern is cross-pollination to conventional varieties. In this study, windborne-pollen dispersion of papaya (Carica papaya L.) was investigated using geographic information systems (GIS) and remotely sensed data. Pollen traps were placed around a papaya plot in eight geographic directions, with radiuses varying from 5 to 900 m from the plot. Pollen counts were made for 12 different dates, and data were input into a GIS database. The distribution of pollen and its relation to land use were analyzed using land use data obtained from Quickbird imagery acquired during 2007. Comparative analyses of pollen dispersal, wind direction, and speed were made using data collected from a micro-climatic station set up at a papaya plot. The furthest distance from the plot that pollen was found was at 0.9 km, a distance at which only 1 pollen grain was found. The number of pollen grains carried by wind decreased as distance increased. The direction of dispersal was not in accordance with wind direction data. Most pollen grains were found in agricultural areas and bare land. The total number of pollen grains found in exposed areas was considerably higher than the total found in areas sheltered by dense tree lines.     

Enzymatic conversion of β-carotene to astaxanthin by cell-extracts of a green alga Haematococcus pluvialis

Astaxanthin was synthesized from β-carotene by the membrane-bound enzyme fraction prepared from a unicellular green alga Haematococcus pluvialis. Zeaxanthin was identified as one of the possible intermediates. NADPH addition and O₂ supply were essential for the conversion. A monooxygenase inhibitor, tetcyclacis blocked the astaxanthin formation. This revised version was published online in November 2006 with corrections to the Cover Date.