Protein folding as a diffusional process.

A protein chain must move relative to the solvent molecules and explore many conformations when it folds from the extended unfolded state to the compact native state. Experimental and theoretical approaches suggest that diffusional processes in fact contribute to the kinetics of protein folding. We describe here how variations of the solvent viscosity can be employed to uncover the diffusional contributions to a folding reaction and assess the use of transition state theory and Kramers' rate theory for the analysis of protein folding reactions.     

Wnt-7a in feather morphogenesis: involvement of anterior-posterior asymmetry and proximal-distal elongation demonstrated with an in vitro reconstitution model.

How do vertebrate epithelial appendages form from the flat epithelia? Following the formation of feather placodes, the previously radially symmetrical primordia become anterior-posterior (A-P) asymmetrical and develop a proximo-distal (P-D) axis. Analysis of the molecular heterogeneity revealed a surprising parallel of molecular profiles in the A-P feather buds and the ventral-dorsal (V-D) Drosophila appendage imaginal discs. The functional significance was tested with an in vitro feather reconstitution model. Wnt-7a expression initiated all over the feather tract epithelium, intensifying as it became restricted first to the primordia domain, then to an accentuated ring pattern within the primordia border, and finally to the posterior bud. In contrast, sonic hedgehog expression was induced later as a dot within the primordia. RCAS was used to overexpress Wnt-7a in reconstituted feather explants derived from stage 29 dorsal skin to further test its function in feather formation. Control skin formed normal elongated, slender buds with A-P orientation, but Wnt-7a overexpression led to plateau-like skin appendages lacking an A-P axis. Feathers in the Wnt-7a overexpressing skin also had inhibited elongation of the P-D axes. This was not due to a lack of cell proliferation, which actually was increased although randomly distributed. While morphogenesis was perturbed, differentiation proceeded as indicated by the formation of barb ridges. Wnt-7a buds have reduced expression of anterior (Tenascin) bud markers. Middle (Notch-1) and posterior bud markers including Delta-1 and Serrate-1 were diffusely expressed. The results showed that ectopic Wnt-7a expression enhanced properties characteristic of the middle and posterior feather buds and suggest that P-D elongation of vertebrate skin appendages requires balanced interactions between the anterior and posterior buds.     

Isolation and characterization of lipopeptide antibiotics produced by Bacillus subtilis

Aims:  Antibiotics from Bacillus subtilis JA show strong pathogen inhibition ability, which has potential market application; yet, the composition of these antibiotics has not been elucidated. The aim of this paper is to isolate and identify these antibiotics.
Methods and Results:  The antagonistic activity of JA was tested in vitro ; it exhibited strong inhibition against some important phytopathogens and postharvest pathogens. Crude antibiotic production was extracted with methanol from the precipitate by adding 6 mol l⁻¹ HCl to the bacillus-free culture broth. The crude extract was run on Diamonsil C₁₈ column (5  μ m, 250 × 4·6 mm) in HPLC system to separate the antibiotics. Major antibiotics were classified into three lipopeptide families according to electrospray ionization–mass spectrometry analysis. Subsequently, the classification of antibiotics was confirmed with typical collision-induced dissociation fragments.
Conclusions:  Three kinds of antibiotics were isolated from B. subtilis JA and were identified to the lipopeptide families, surfactin, iturin and fengycin. These compounds could function as biocontrol agents against a large spectrum of pathogens.
Significance and Impact of the Study:  This study provided a reliable and rapid method for isolation and structural characterization of lipopeptide antibiotics from B. subtilis .     

An iso-random Bi Bi mechanism for adenylate kinase.

An iso-random Bi Bi mechanism has been proposed for adenylate kinase. In this mechanism, one of the enzyme forms can bind the substrates MgATP and AMP, whereas the other form can bind the products MgADP and ADP. In a catalytic cycle, the conformational changes of the free enzyme and the ternary complexes are the rate-limiting steps. The AP(5)A inhibition equations derived from this mechanism show theoretically that AP(5)A acts as a competitive inhibitor for the forward reaction and a mixed noncompetitive inhibitor for the backward reaction.     

Role of tumor-derived cytokines on the immune system of mice bearing a mammary adenocarcinoma. II. Down-regulation of macrophage-mediated cytotoxicity by tumor-derived granulocyte-macrophage colony-stimulating factor

Peritoneal elicited macrophages (PEM) from mammary tumor-bearing mice have a decreased capacity to become cytotoxic against syngeneic, allogeneic, and xenogeneic target cells upon in vitro stimulation with LPS, as compared with PEM of normal mice. A regulatory mechanism other than PG release is suggested because the addition of both indomethacin and LPS to macrophage cultures from tumor-bearing mice caused no changes in their cytotoxic capability. Because tumor products have been implicated in the down-regulation of immune responses, we investigated whether pretreatment with supernatants from the tumor cell line DA-3, derived from the in vivo mammary adenocarcinoma D1-DMBA-3, affects the cytolytic capacity of macrophages. This treatment inhibits, in a dose-dependent fashion, the ability of stimulated normal PEM to kill target cells. Partial purification of DA-3 cell line supernatant showed that most of the inhibitory activity was exerted by factors with a molecular mass greater than 10 kDa and less than 30 kDa. However, slight inhibition could also be observed with fractions containing molecules less than 10 kDa. The data suggest that more than one factor released by the mammary tumor cells may be involved in the down-regulation of macrophage-mediated cytotoxicity. Because the DA-3 cells constitutively produce granulocyte-macrophage CSF (GM-CSF), which has a molecular mass of 27 kDa, we pretreated PEM from normal mice in vitro with rGM-CSF for 24 h. This resulted in a dose-dependent decrease in their capacity to kill tumor target cells upon LPS stimulation. Furthermore, PEM from normal mice injected with rGM-CSF for 25 days displayed a profound decrease in their cytolytic ability against DA-3 targets upon in vitro stimulation with increasing amounts of LPS. The pretreatment of PEM from normal mice with a combination of DA-3 cell supernatants and specific anti-GM-CSF partially neutralized the inhibitory effect of the DA-3 supernatant on macrophage tumoricidal capability. These results indicate that tumor-derived GM-CSF is an important factor involved in the decreased macrophage cytotoxicity during mammary adenocarcinoma progression.     

Bone marrow depletion by 89Sr complements a preleukemic defect in a long terminal repeat variant of Moloney murine leukemia virus.

We previously described a preleukemic state induced by Moloney murine leukemia virus (Mo-MuLV) characterized by hematopoietic hyperplasia in the spleen. Further experiments suggested that splenic hyperplasia results from inhibitory effects in the bone marrow, leading to compensatory extramedullary hematopoiesis. An enhancer variant of Mo-MuLV, Mo + PyF101 Mo-MuLV, fails to induce preleukemic hyperplasia and has greatly reduced leukemogenicity, indicating the importance of this state to efficient leukemogenesis. An alternative method for induction of preleukemic hyperplasia was sought. Treatment of mice with 89Sr causes specific ablation of bone marrow hematopoiesis and compensatory extramedullary hematopoiesis in spleen and nodes. NIH Swiss mice were inoculated neonatally with Mo + PyF101 Mo-MuLV and treated with 89Sr at 6 weeks of age. Approximately 85% developed lymphoid leukemia with a time course resembling that caused by wild-type Mo-MuLV. In contrast, very few animals treated with Mo + PyF101 Mo-MuLV or 89Sr alone developed disease. In approximately one-third of cases, the Mo + PyF101 Mo-MuLV proviruses were found at common sites for wild-type Mo-MuLV-induced tumors (c-myc, pvt-1, and pim-1), indicating that this virus is capable of performing insertional activation in T-lymphoid cells. These results support the proposal that splenic hyperplasia results from inhibitory effects in the bone marrow. They also indicate that Mo + PyF101 Mo-MuLV is blocked in early and not late events in leukemogenesis.     

Predictive modeling for allopatric Strix (Strigiformes: Strigidae) owls in South America: determinants of their distributions and ecological niche‐based processes

Strix (Strigidae) is a worldwide genus of 17 owl species typical of forested habitats, including Rusty‐barred Owls (S. hylophila), Chaco Owls (S. chacoensis), and Rufous‐legged Owls (S. rufipes) in South America. These species are distributed allopatrically, but the ecological traits that determine their distributions remain largely unknown and their phylogenetic relationships are unclear. We used species distribution models (SDMs) to identify variables explaining their distribution patterns and test hypotheses about ecological divergence and conservatism based on niche overlap analysis. For Rusty‐barred Owls and Chaco Owls, climatic factors related to temperature played a major role, whereas a rainfall variable was more important for Rufous‐legged Owls. When niche overlaps were compared, accounting for regional similarities in the habitat available to each species, an ecological niche divergence process was supported for Chaco Owl‐Rusty‐barred Owl and Chaco Owl‐Rufous‐legged Owl, whereas a niche conservatism process was supported for Rusty‐barred Owl‐Rufous‐legged Owl. Different ecological requirements support current species delimitation, but they are in disagreement with the two main hypotheses currently envisaged about their phylogenetic relationships (Chaco Owls as the sister taxa of either Rufous‐legged Owls or Rusty‐barred Owls) and support a new phylogenetic hypothesis (Rufous‐legged Owls as sister taxa of Rusty‐barred Owls). Our findings suggest that speciation of Rusty‐barred Owls and Rufous‐legged Owls was a vicariant event resulting from Atlantic marine transgressions in southern South America in the Miocene, but their niche was conserved because habitat changed little in their respective ranges. In contrast, Chaco Owls diverged ecologically from the other two species as a result of their adaptations to the habitat they currently occupy. Ecological and historical approaches in biogeography can be embedded to explain distribution patterns, and results provided by SDMs can be used to infer historical and ecological processes in an integrative way.