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To reach their proper targets, axons rely upon the actions of highly conserved families of attractive and repulsive guidance molecules, including the netrins, Slits, semaphorins and ephrins. These guidance systems are used to generate an astonishingly varied set of neuronal circuits. Here we consider the mechanisms by which a few guidance systems can be used to generate diverse outcomes. Recent studies have revealed extensive transcriptional and post-transcriptional regulation of guidance cues and their receptors, as well as combinatorial mechanisms that integrate information from different families of guidance cues.

Robo receptors interact with ligands of the Slit family. The nematode C. elegans has one Robo receptor (SAX-3) and one Slit protein (SLT-1), which direct ventral axon guidance and guidance at the midline. In larvae, slt-1 expression in dorsal muscles repels axons to promote ventral guidance. SLT-1 acts through the SAX-3 receptor, in parallel with the ventral attractant UNC-6 (Netrin). Removing both UNC-6 and SLT-1 eliminates all ventral guidance information for some axons, revealing an underlying longitudinal guidance pathway. In the embryo, slt-1 is expressed at high levels in anterior epidermis. Embryonic expression of SLT-1 provides anterior-posterior guidance information to migrating CAN neurons. Surprisingly, slt-1 mutants do not exhibit the nerve ring and epithelial defects of sax-3 mutants, suggesting that SAX-3 has both Slit-dependent and Slit-independent functions in development.

Rabbit muscle creatine kinase (CK) was modified by 5,5'-dithio-bis(2-nitrobenzoic acid) accompanied by 3 M guanidine hydrochloride denaturation to produce a partially folded state with modified thiol groups. The partially folded CK was in a monomeric state detected by size exclusion chromatography, native-polyacrylamide gel electrophoresis, circular dichroism, and intrinsic fluorescence studies. After dithiothreitol (DTT) treatment, about 70% CK activity was regained with a two-phase kinetic course. Rate constants calculated for regaining of activity and refolding were compared with those for CK modified with various treatments to show that refolding and recovery of activity were synchronized. To further characterize the partially folded CK state and its folding pathway, the molecular chaperone GroEL was used to evaluate whether it can bind with partly folded CK during refolding, and 1-anilinonaphthalene-8-sulfonate was used to detect the hydrophobic surface of the monomeric state of CK. The monomeric state of CK did not bind with GroEL, although it had a larger area of hydrophobic surface relative to the native state. These results may provide different evidence for the structural requirement of GroEL recognition to the substrate protein compared with previously reported results that GroEL bound with substrate proteins mainly through hydrophobic surface. The present study provides data for a monomeric intermediate trapped by the modification of the SH groups during the refolding of CK. Schemes are given for explaining both the partial folding CK pathway and the refolding pathway.

The role of two thioesterase genes in the premature release of polyketide synthase intermediates during rifamycin biosynthesis in the Amycolatopsis mediterranei S699 strain was investigated. Creation of an in-frame deletion in the rifR gene led to a 30 approximately 60% decrease in the production of both rifamycin B by the S699 strain or a series of tetra- to decaketide shunt products of polyketide chain assembly by the rifF strain. Since a similar percentage decrease was seen in both genetic backgrounds, we conclude that the RifR thioesterase 2 is not involved in premature release of the carbon chain assembly intermediates. Similarly, fusion of the Saccharopolyspora erythraea DEBS3 thioesterase I domain to the C-terminus of the RifE PKS subunit did not result in a noticeable increase in the amount of the undecaketide intermediate formed nor in the amounts of the tetra- to decaketide shunt products. Hence, premature release of the carbon chain assembly intermediates is an unusual property of the Rif PKS itself.

Epidermal nevus syndrome is seldom encountered, and its association with hypermelanosis and the chronic syndrome of inappropriate antidiuretic hormone secretion (SIADH) has never been reported. A male neonate who developed intractable seizures and hyponatremia soon after birth is reported. He had alopecic patches on the scalp at birth. Large areas of skin hyperpigmentation, and epidermal nevi developed gradually. The clinical picture of hypotonic hyponatremia, high urine osmolality, elevated urine sodium, and euvolemia was compatible with SIADH. The seizures did not correlate with the hyponatremia, and no other cause for the seizures could be identified. The hyponatremia became chronic and was treated with a direct supply of sodium chloride. The development of the patient was markedly delayed at the last visit when he was 1 year of age. It is suggested that hypermelanosis and chronic SIADH may also be a variant presentation of epidermal nevus syndrome.

The application of a simple lag-time assay for antioxidant capacity using myoglobin and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) or ABTS has been studied for its general application conditions. In the presence of an antioxidant, the ABTS(*+) radical-cation-forming chromogenic reaction, catalyzed by myoglobin and initiated by hydrogen peroxide (H(2)O(2)), has a lag period, and its duration is linearly correlated to the concentration of that antioxidant. The high linearity between the lag time and the antioxidant concentration remained unchanged regardless of the assay conditions. It was also found that the linearity was better for antioxidants at lower concentrations. The change of assay condition could significantly affect the relative antioxidant value of a chemical to the standard (ascorbic acid), although not to a large extent. Most of antioxidants investigated were found suitable to be assayed using this method. Some antioxidants, e.g., genistein, however, were not, probably due to their low reactivity toward ferrylmyoglobin or ABTS(*+). In conclusion, the lag-time assay is a reliable method for measuring the antioxidant capacity, provided caution is taken for antioxidants that mainly act through lowering the rate of the chromogenic reaction., (Copyright 1999 Academic Press.)

There is currently intense interest in unravelling the modus operandi of type I modular polyketide synthases in order to lay the ground work for their use in the combinatorial biosynthesis of new bioactive molecules. Much of our knowledge is derived from studies on 6-deoxyerythronolide B (DEBS), the enzyme assembling the polyketide backbone of erythromycin. Work on the rifamycin polyketide synthase has revealed a number of features that differ from those seen with DEBS.

The biosynthesis of ansamycin antibiotics, including rifamycin B, involves the synthesis of an aromatic precursor, 3-amino-5-hydroxybenzoic acid (AHBA), which serves as starter for the assembly of the antibiotics' polyketide backbone. The terminal enzyme of AHBA formation, AHBA synthase, is a dimeric, pyridoxal 5'-phosphate (PLP) dependent enzyme with pronounced sequence homology to a number of PLP enzymes involved in the biosynthesis of antibiotic sugar moieties. The structure of AHBA synthase from Amycolatopsis mediterranei has been determined to 2.0 A resolution, with bound cofactor, PLP, and in a complex with PLP and an inhibitor (gabaculine). The overall fold of AHBA synthase is similar to that of the aspartate aminotransferase family of PLP-dependent enzymes, with a large domain containing a seven-stranded beta-sheet surrounded by alpha-helices and a smaller domain consisting of a four-stranded antiparallel beta-sheet and four alpha-helices. The uninhibited form of the enzyme shows the cofactor covalently linked to Lys188 in an internal aldimine linkage. On binding the inhibitor, gabaculine, the internal aldimine linkage is broken, and a covalent bond is observed between the cofactor and inhibitor. The active site is composed of residues from two subunits of AHBA synthase, indicating that AHBA synthase is active as a dimer.

The assembly of the polyketide backbone of rifamycin B on the type I rifamycin polyketide synthase (PKS), encoded by the rifA-rifE genes, is terminated by the product of the rifF gene, an amide synthase that releases the completed undecaketide as its macrocyclic lactam. Inactivation of rifF gives a rifamycin B nonproducing mutant that still accumulates a series of linear polyketides ranging from the tetra- to a decaketide, also detected in the wild type, demonstrating that the PKS operates in a processive manner. Disruptions of the rifD module 8 and rifE module 9 and module 10 genes also result in accumulation of such linear polyketides as a consequence of premature termination of polyketide assembly. Whereas the tetraketide carries an unmodified aromatic chromophore, the penta- through decaketides have undergone oxidative cyclization to the naphthoquinone, suggesting that this modification occurs during, not after, PKS assembly. The structure of one of the accumulated compounds together with (18)O experiments suggests that this oxidative cyclization produces an 8-hydroxy-7, 8-dihydronaphthoquinone structure that, after the stage of proansamycin X, is dehydrogenated to an 8-hydroxynaphthoquinone.

Creatine kinase with its thiol groups modified by 5, 5'-dithio-bis(2-nitrobenzoic acid) has been shown to be partially folded in a monomeric state using fluorescence, circular dichroism, proteolysis, and size exclusion chromatography studies. In the presence of DTT, the partially folded modified creatine kinase can be reactivated and refolded following a biphasic course, suggesting the existence of a monomeric intermediate during the refolding of CK. The results provide evidence for our previously suggested model of the refolding pathway of urea-denatured creatine kinase., (Copyright 1999 Academic Press.)

The single recombinant expressing the Streptomyces coelicolor minimal whiE (spore pigment) polyketide synthase (PKS) is uniquely capable of generating a large array of well more than 30 polyketides, many of which, so far, are novel to this recombinant. The characterized polyketides represent a diverse set of molecules that differ in size (chain length) and shape (cyclization pattern). This combinatorial biosynthetic library is, by far, the largest and most complex of its kind described to date and indicates that the minimal whiE PKS does not independently control polyketide chain length nor dictate the first cyclization event. Rather, the minimal PKS enzyme complex must rely on the stabilizing effects of additional subunits (i.e., the cyclase whiE-ORFVI) to ensure that the chain reaches the full 24 carbons and cyclizes correctly. This dramatic loss of control implies that the growing polyketide chain does not remain enzyme bound, resulting in the spontaneous cyclization of the methyl terminus. Among the six characterized dodecaketides, four different first-ring cyclization regiochemistries are represented, including C7/C12, C8/C13, C10/C15, and C13/C15. The dodecaketide TW93h possesses a unique 2,4-dioxaadamantane ring system and represents a new structural class of polyketides with no related structures isolated from natural or engineered organisms, thus supporting the claim that engineered biosynthesis is capable of producing novel chemotypes.

In previous studies, N-(N'-acetyl-L-propyl)-N-nitrosoglycine (APNG) has been shown to be a potent mutagen in a variety of genotoxicity assays and a carcinogen in a limited cancer study. APNG decomposes to a carboxymethyldiazonium ion, which can also be generated from potassium diazoacetate (KDA). KDA is particularly interesting because it is a stable nitrosated derivative of glycine, one of the most common dietary amino acids. KDA has been shown to produce more O6 carboxymethyl- and O6 methyl-adducts than APNG, so it was anticipated that it might also be a potent genotoxic agent. Thus in the present study KDA has been investigated in the single cell gel electrophoresis (Comet) assay, which primarily measures DNA strand breakage. Since KDA has been shown to be formed in the gut, the genotoxic effects of KDA were investigated in vitro in human adenocarcinoma colon Caco-2 cells, and in rat primary colon cells and compared to responses from human peripheral lymphocytes. KDA induced DNA damage in the three cell types, confirming that KDA is genotoxic in a range of mammalian cells.

Over 200 agents/factors have been examined in the single cell gel electrophoresis assay, more commonly known as the Comet assay, performed either in vitro or in vivo in a variety of species. Unequivocal carcinogenicity data are available for 119 of them, amongst which unequivocal Comet assay data exist for 95 agents. Of these 95 agents the prevalence of carcinogens was 88% (84/95). The carcinogens that were Comet positive (sensitivity) formed 88% (74/84), the non-carcinogens that were Comet negative (specificity) formed 64% (7/11). This simple analysis of the Comet assay has not taken account of the difference between in vitro and in vivo responses, species differences or organ and tissue differences. Also, limitations as to the conduct of the assay have not been examined in any depth. Thus, at the present time the Comet assay has high sensitivity for carcinogens, but its specificity is uncertain because few non-carcinogens have been tested.