Making the most of Papua New Guinea's biodiversity: Establishment of an integrated set of programs that link botanical survey with pharmacological assessment in "The Land of the Unexpected"

An integrated and coordinated set of programs has been established to meet ICBG goals in Papua New Guinea (PNG). Here we give an overview of the PNG ICBG and focus on the key elements and major steps taken to establish a program necessary for the pharmacological assessment of botanicals and traditional medicines in PNG and, by extrapolation, in other developing countries.

The identification of a gene family in the Saccharomyces cerevisiae ergosterol biosynthesis pathway.

The Saccharomyces cerevisiae ERG24 gene, encoding sterol delta 14 reductase (Erg24p), was cloned by selecting strains carrying sequences on a 2 mu-based vector for resistance to the morpholine fungicide, fenpropimorph (Fp). Four distinct plasmid inserts which conferred Fp resistance (FpR) were recovered (plasmids pML99, pML100, pML101 and pM103). Although Fp is reported to inhibit activity of Erg24p and sterol delta 8-delta 7 isomerase (Erg2p; encoded by ERG2), none of the inserts had restriction maps resembling ERG2. In addition, a 2 mu plasmid overexpression of the ERG2 sequence did not produce FpR. Characterization studies were focused on plasmid pML100, because it was the only plasmid to confer FpR consistently when tested in a number of different genetic backgrounds. Tests with a panel of fungicides indicated that pML100 conferred significant resistance only to compounds (Fp, tridemorph, fenpropidin and azasterol) which have a shared site of action, Erg24p. An insertional disruption of pML100 resulted in an obligate anaerobic phenotype, indicating a lesion in sterol biosynthesis. Sterol analysis of the disrupted mutant demonstrated the accumulation of ignosterol, indicating a loss of Erg24p activity. A SphI-XbaI fragment of pML100 was sequenced, revealing the presence of an ORF encoding a 438-amino-acid protein, which is highly similar to those encoded by two previously reported yeast drug sensitivity genes, sts1+ (Schizosaccharomyces pombe) and YGL022 (S. cerevisiae). Analyses of these genes demonstrated that strains carrying disruptions of sts1+ or YGL022 have ergosterol biosynthesis defects in the enzyme, sterol C-24(28) reductase (Erg4p; encoded by ERG4).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of voluntary wheel-running exercise on muscles of the mdx mouse.

The purpose of this study is to determine whether dystrophin-deficient mdx mice are more susceptible to muscle injury and functional impairment than normal C57 mice when allowed to exercise voluntarily on mouse wheels. The mdx mice were significantly impaired when compared to controls as shown by functional, contractile and morphometric responses. The distance young mdx mice ran was 67-78% of young C57 mice, while adult mdx mice ran 31-48% of adult controls. After exercise the slow, oxidative soleus of young and adult mdx mice exhibited hypertrophy with no changes in strength or fatiguability, while the young C57 mice increased strength and the adults became less fatiguable. In the adult mdx mice the fast EDL, which is primarily glycolytic, exhibits slight hypertrophy with a loss of strength, while the young exhibit no changes. These results indicate that the mdx mouse adapts differently than the C57 mouse to even moderate exercise.

Charcot-Marie-Tooth neuropathy: clinical phenotypes of four novel mutations in the MPZ and Cx 32 genes.

Charcot-Marie-Tooth Hereditary Neuropathy is a heterogeneous syndrome associated with mutations in several different genes including peripheral myelin protein 22, myelin P0, connexin 32, and early growth response 2. There is considerable variability in the phenotypic expression of this syndrome and the relationship of this variability to mutation genotypes requires extensive analysis. Here we describe the phenotypes and genotypes of four new mutations underlying the Charcot-Marie-Tooth syndrome and document segregation with disease. Four families with Charcot-Marie-Tooth were ascertained, examined, and evaluated electrophysiologically. Each family had peripheral blood DNA screened for mutations in myelin protein 22, myelin P0, and connexin 32. Two families were found with new mutations in the myelin P0 gene: S140T in the extracellular domain and K236del in the cytoplasmic domain. All families showed segregation of the mutations with the Charcot-Marie-Tooth phenotype as did a new family with the rare G163R mutation in the membrane domain. A 49-year-old man with the S140T mutation demonstrated conduction block on electrophysiological testing. A family with a novel S49P mutation in the connexin 32 gene had a neuropathy with very slow nerve conduction. These new mutations in the myelin P0 and connexin 32 genes help to clarify the pathophysiology of the clinical Charcot-Marie-Tooth syndrome. The S140T mutation in myelin P0 can be associated with conduction block and Charcot-Marie-Tooth should be part of the differential diagnosis of that phenomenon. Mutations in the cytoplasmic domain of myelin P0 can cause clinical neuropathy. The S49P mutation in the connexin 32 gene can produce aspects of a demyelinating type of X-linked hereditary neuropathy.

The use of fluorescent dextrans as a marker of sarcolemmal injury.

We investigated the use of intravenously injected fluorescent dextran molecules (FDx) as a histological marker of sarcolemmal injury. Using fluorescent microscopy, uptake of FDx (average MW 10 kD) was assessed in sections of quadriceps muscles from three models: 1) normal (C57BL/10SnJ) mice, 2) normal mice run downhill (0, 3, and 7 days post exercise), and 3) non-exercised mdx (dystrophin-deficient) mice. These were compared to serial sections stained with hematoxylin and eosin (H&E). In control muscles, strong fluorescence was seen between fibers (intercellular). Intracellular FDx was observed within cells of the quadriceps from normal mice run downhill at days 0 and 3 post exercise, but not at day 7. On H&E staining, muscle pathology was not observed until day 3, with regeneration by day 7. Intracellular FDx was also observed within mdx muscles, particularly in fibers that appeared pre-necrotic on H&E stained sections. FDx appears to be useful as a histological marker of changes in sarcolemmal integrity associated with muscle injury from eccentric exercise or muscle disease.

Relative and absolute configurations of ganefromycin alpha.

The full structure of ganefromycin alpha has been determined. The relative configurations were determined from 3JH,H coupling constants and NOE data, while the absolute configurations in moleties A and B were determined separately by difference CD of their acylate derivatives, which showed typical exciton couplets. The configurations of the stereogenic centers in ganefromycin alpha are 8S, 9S, 11R, 12S, 13S, 21S, 22R, 23R, 24R, and 26S.

Peak homogeneity determination for the validation of high-performance liquid chromatographic assay methods.

To validate high-performance liquid chromatographic assay procedures with regard to specificity, methods were developed to determine the homogeneity of the chromatographic peaks. These methods employed a rapid-scanning UV-visible spectrophotometer to monitor the chromatographic effluent. The absorption data were processed to nullify the signal due to the drug substances specifically, while allowing the detection of coincident impurities. Results from three model systems indicated the ability of these methods to detect as little as 0.1% of a coincident impurity.

New biotransformation products of nemadectins.

Selected nemadectins (formerly LL-F28249 series) have been fed to a panel of microorganisms with the aim of generating new derivatives. In addition to products resulting from the oxidation of the terminal methyl group (C-29), a unique phosphorylated nemadectin was isolated. The phosphate group was determined to be at C-23 by HMBC between phosphorus and H-23. Milbemycin or nemadectin derivatives with natural substituents involving the 23-hydroxyl group were hitherto unknown.

Base-catalyzed isomerism in elfamycin antibiotics. Structures of ganefromycins epsilon and epsilon 1.

The structures of ganefromycins epsilon (2a) and epsilon 1 (2b) have been determined by spectroscopic techniques. The compounds are isomeric deoxygenated precursors of the parent antibiotics ganefromycins alpha and beta. The nature of the isomerism was determined by chemical interconversion experiments and spectroscope analysis to be a change in configuration at C-21. Evidence is provided for other cases of this type of isomerism in the elfamycin class of antibiotics.

Hongoquercins A and B, new sesquiterpenoid antibiotics: isolation, structure elucidation, and antibacterial activity.

Two new sesquiterpenoid antibiotics, hongoquercins A and B, were isolated from the extracts of an unidentified fungus. The structures of both metabolites were determined by spectroscopic analysis. They are related to a class of compounds commonly found in brown algae and dictyoceratid sponges. Hongoquercin A exhibited moderate activity against gram-positive bacteria.

Cell wall active antifungal compounds produced by the marine fungus Hypoxylon oceanicum LL-15G256. II. Isolation and structure determination.

Fermentations of the marine fungus Hypoxylon oceanicum (LL-15G256) were found to have potent antifungal activity. Isolation and purification of the antifungal agents provided two classes of compounds, macrocyclic polylactones and the lipodepsipeptides 15G256 gamma (1), 15G256 delta (2) and 15G256 epsilon (3). The isolation and structure elucidation of the lipodepsipeptides, all containing D-glutamate, L-serine, and the rare amino acid beta-ketotryptohan, are described in this paper.

Biosynthesis of chrysomycins A and B. Origin of the chromophore.

The biosynthetic origin of the carbon atoms in the chromophores of chrysomycins A and B was investigated in feeding experiments using 13C labeled acetates and propionate. A biosynthetic scheme is proposed involving the condensation and rearrangement of a decaketide intermediate which contains either propionate (chrysomycin A) or acetate (chrysomycin B) as the chain initiator.

Citreamicins, novel antibiotics from Micromonospora citrea: isolation, characterization, and structure determination.

A new family of antibacterial antibiotics has been isolated from Micromonospora citrea. The compounds, designated citreamicins alpha, beta, gamma, zeta and eta are of the polycyclic xanthone structure type. Their isolation, characterization and structure determination are presented.

The effect of methylation inhibitors on citreamicin biosynthesis in Micromonospora citrea.

When the citreamicin-producing organism Micromonospora citrea NRRL 189351 was incubated in the presence of the methylation inhibitors sinefungin or aminopterin, biosynthesis of the zeta component was stimulated approximately 20 to 200-fold above the level normally produced. Inhibition of a second methylation reaction, which is superficially very similar to the first, was not detected. Other known methylation inhibitors failed to yield any change in the natural pattern of citreamicins produced. This approach is an excellent route for preparing citreamicin zeta, which can be used as a substrate for semi-synthesis or for further biosynthetic studies.

LL-F28249 antibiotic complex: a new family of antiparasitic macrocyclic lactones. Isolation, characterization and structures of LL-F28249 alpha, beta, gamma, lambda.

A new family of antiparasitic macrolides has been isolated from Streptomyces cyaneogriseus sp. noncyanogenus. The compounds, designated LL-F28249 alpha, beta, gamma and lambda, possess potent antiparasitic activity. The isolation, purification and structure determination by spectroscopic methods are presented.

Biosynthetic origin of the carbon skeleton and oxygen atoms of the LL-F28249 alpha, a potent antiparasitic macrolide.

The biosynthesis of LL-F28249 alpha in a culture of Streptomyces cyaneogriseus has been studied using 13C, 14C and 18O labeled precursors. A complete 13C NMR spectrum of F28249 alpha has been assigned. Incorporation studies using 13C labeled precursors indicate that the carbon skeleton of F28249 alpha is derived from seven acetate, six propionate and one 2-methylpropionate units. The origin of the oxygen atoms of F28249 alpha has been examined by feeding [1-13C,18O2]acetate, [1-13C,18O2]propionate, [2-13C]acetate/18O2 and 18O2 separately to the fermentation culture and analyzing the resulting labeled LL-F28249 alpha samples by 13C NMR, electron impact MS and chemical ionization MS. Out of a total of eight oxygen atoms in LL-F28249 alpha, four oxygen atoms are derived from acetate, three from propionate and one from molecular oxygen.

Isolation, characterization and structure of a new allenic polyine antibiotic produced by fungus LL-07F275.

Antibiotic 07F275 (1), produced by submerged fermentations of fungal culture LL-07F275, was isolated and characterized despite its inherent instability. Its UV spectrum was identical with that of nemotin, a member of the allenic polyacetylene family, but a molecular weight of 218 daltons indicated a new compound. Structure 1 was determined on the basis of spectroscopic evidence, particularly NMR. Since 1 is a thirteen carbon-containing allenic diyne, it is closely related to mycomycin.

Martinomycin, a new polyether antibiotic produced by Streptomyces salvialis. I. Taxonomy, fermentation and biological activity.

Actinomycete culture LL-D37187 has been found to produce the new polyether antibiotic martinomycin. Taxonomic studies, including morphological, physiological, and cell wall chemistry analyses, revealed that culture LL-D37187 is a novel streptomycete species, and the proposed name is Streptomyces salvialis. Martinomycin exhibits activity against the Southern Army Worm (Spodoptera eridania) and Gram-positive bacteria.

Advances in the management of neuropathic pain.

Much progress has been made in the assessment and management of neuropathic pain over the past 5 years. Assessment has improved with the Neuropathic Pain Scale, a new, easily administered, diagnostic tool. Mechanistically, recent studies indicate that peripheral neuropathic pain is generated through a focal inflammatory process rather than axonal destruction. This process also appears to involve mRNA regulation of fast sodium channels, which produce ectopic discharges and are presumably responsible for pain generation. In addition the entire neuraxis undergoes neuroplastic changes as a result of peripheral nerve injury. The available clinical trial data indicate that newer antiepileptic drugs (AEDs), most notably gabapentin, are better alternatives to older medications such as carbamazepine or phenytoin in the treatment of neuropathic pain. Gabapentin is at least as good with respect to actual pain relief as the antidepressants, including amitriptyline, but has a much better safety profile with minimal drug-drug interactions and side effects. Mexiletine is a reasonable alternative agent in patients who have not had a satisfactory response to, or cannot tolerate, the AEDs or antidepressants. Long-acting opioids should be considered in patients refractory to these adjunctive agents. With the advent of the topical lidocaine patch, the first drug with an FDA-approved indication for postherpetic neuralgia, a revolutionary new agent is now available for the treatment of neuropathic pain that does not have any systemic side effects.

Comprehensive management of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive motor neuron disease that poses a myriad of clinical problems. Patients with ALS are best treated in a multidisciplinary setting involving physicians, clinical nursing specialists, and physical, occupational, speech, and respiratory therapists, as well as psychologists and social workers. Palliative and rehabilitative strategies may ease suffering, while new treatments provide hope for effective treatment of this disease.