Alzheimer's disease is characterized by progressive loss of neurons accompanied by the formation of intraneural neurofibrillary tangles and extracellular amyloid plaques. Human neuronal growth inhibitory factor, classified as metallothionein-3 (MT-3), was found to be related to the neurotrophic activity promoting cortical neuron survival and dendrite outgrowth in the cell culture studies. We have determined the solution structure of the a-domain of human MT-3 (residues 32-68) by multinuclear and multidimensional NMR spectroscopy in combination with the molecular dynamic simulated annealing approach. The human MT-3 shows two metal-thiolate clusters, one in the Nterminus (b-domain) and one in the C-terminus (a-domain). The overall fold of the a-domain is similar to that of mouse MT-3. However, human MT-3 has a longer loop in the acidic hexapeptide insertion than that of mouse MT-3. Surprisingly, the backbone dynamics of the protein revealed that the b-domain exhibits similar internal motion to the a-domain, although the N-terminal residues are more flexible. Our results may provide useful information for understanding the structure-function relationship of human MT-3.

Two yellow pigments were isolated for the first time from the entomopathogenic bacterium Photorhabdus luminescens in liquid culture and were identified as the anthraquinone derivatives 3,8-dimethoxy-1-hydroxy-9,10-anthraquinone (minor) and 1,3-dimethoxy-8-hydroxy-9,10-anthraquinone (major). A known antibiotic, 3,5-dihydroxy-4-isopropylstilbene, was also detected and for the first time showed strong fungicidal activity against several fungi of medical and agricultural importance. Nematodes of the genus Heterorhabditis carry a luminous bacterial symbiont, Photorhabdus luminescens, in their gut (18). These parasitic nematodes penetrate insects and expel their bacterial symbionts into the insects' hemocoels (16, 17). Once released in an insect, the bacteria multiply rapidly, the insect dies within 24 to 48 h, and the nematodes feed on the multiplying bacteria. The insect cadaver becomes deep red but does not putrefy, apparently because of an antibiotic(s) produced by the bacteria (1, 9, 14, 15, 19). Recently, it was reported that antibiotics from P. luminescens and from Xenorhabdus spp., bacterial symbionts from Steinernema spp., have strong activities against fungal pathogens of plants (7, 13). These observations led to an investigation into the chemical natures of these substances, and we report here the identification of two pigments and one antibiotic from P. luminescens. MATERIALS AND METHODS Microorganisms. P. luminescens C9 and its nematode symbiont used in this study, Heterorhabditis megidis 90, were collected from soil in British Columbia, Canada, and maintained in culture in this laboratory (7). Last-instar larvae of the greater wax moth, Galleria mellonella, were infected with infective juvenile nematodes carrying P. luminescens at a rate of 25 infective juveniles per larva (7). After 24 to 48 h, the dead insect larvae were surface disinfected by being dipped into 95% ethyl alcohol and ignited. The cadavers were aseptically dissected, and hemolymph was streaked onto an NBTA medium (nutrient agar supplemented with 0.025 g of bromothymol blue and 0.04 g of 2,3,5-triphenyltetrazolium chloride per liter) and incubated in the dark at room temperature. Inocula of the primary form of the bacterium (7) were prepared by adding one loopful of the culture to 50 ml of tryptic soy broth in 100-ml Erlenmeyer flasks. Cultures were shaken at 120 rpm on a gyrorotary shaker for 24 h at 25ЊC. Bacterial fermentation was initiated by adding 100 ml of this bacterial culture to 900 ml of tryptic soy broth in a 2,000-ml flask, which was then incubated in the dark at 25ЊC on a gyrorotary shaker. After 96 h, the culture was centrifuged (12,000 ϫ g, 20 min, 4ЊC) to remove the bacterial cells. Isolation of chemical components. The cell-free culture broth (5 liters) was extracted with the same volume of ethyl acetate three times. The extracts were combined, dried over anhydrous sodium sulfate, and filtered through glass wool. The level of bioactivity was tested at this and each subsequent step in the isolation process, with Bacillus s...

RET protein functions as a receptor-type tyrosine kinase and has been found to be aberrantly expressed in a wide range of human diseases. A highly GC-rich region upstream of the promoter plays an important role in the transcriptional regulation of RET. Here, we report the NMR solution structure of the major intramolecular G-quadruplex formed on the G-rich strand of this region in K+ solution. The overall G-quadruplex is composed of three stacked G-tetrad and four syn guanines, which shows distinct features for all parallel-stranded folding topology. The core structure contains one G-tetrad with all syn guanines and two other with all anti-guanines. There are three double-chain reversal loops: the first and the third loops are made of 3 nt G-C-G segments, while the second one contains only 1 nt C10. These loops interact with the core G-tetrads in a specific way that defines and stabilizes the overall G-quadruplex structure and their conformations are in accord with the experimental mutations. The distinct RET promoter G-quadruplex structure suggests that it can be specifically involved in gene regulation and can be an attractive target for pathway-specific drug design.