The origin and age of haplochromine fishes in Lake Victoria, east Africa

According to a widely held view, the more than 300 species of haplochromine cichlid fishes in Lake Victoria (LV), East Africa, originated from a single founder species in less than 12,000 years. This view, however, does not follow from the published geological and molecular evidence. The former does indeed suggest that the LV basin dried out less than 15,000 years ago, but it does not provide any information about the species that re-colonized the new lake or that remained in the rivers draining the area. The molecular evidence is inconclusive with respect to the origin of the LV haplochromines because cichlids from critical regions around LV were not adequately sampled; and as far as the age of the LV haplochromines is concerned, it in fact led to an estimate of 250,000-750,000 years old. In the present study, mitochondrial DNA (control region) variation was determined by heteroduplex and sequencing analyses of more than 670 specimens collected at widely distributed East African riverine and lacustrine localities. The analyses revealed the existence of seven haplogroups (I-VII) distinguishable by characteristic substitutions. All endemic LV samples tested fell into one of these haplogroups (V) which, however, was also found to be present at various other localities, both riverine and lacustrine, outside LV. Within this haplogroup, four subgroups (VA through VD) could be distinguished, two of which (VB and VC) were represented in LV and at other localities. The great majority of the LV haplochromine species could be classified as belonging to the VC subgroup, which was found only in LV and in the rivers draining into it. Hence, while the endemic haplochromine species of LV could not have originated from a single founding population, the lake does harbour a large species flock which probably arose in situ.     

Phosphatidylinositol 4,5-bisphosphate regulates two steps of homotypic vacuole fusion

Yeast vacuoles undergo cycles of fragmentation and fusion as part of their transmission to the daughter cell and in response to changes of nutrients and the environment. Vacuole fusion can be reconstituted in a cell free system. We now show that the vacuoles synthesize phosphoinositides during in vitro fusion. Of these phosphoinositides, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) are important for fusion. Monoclonal antibodies to PI(4,5)P(2), neomycin (a phosphoinositide ligand), and phosphatidylinositol-specific phospholipase C interfere with the reaction. Readdition of PI(4, 5)P(2) restores fusion in each case. Phosphatidylinositol 3-phosphate and PI(3,5)P(2) synthesis are not required. PI(4,5)P(2) is necessary for priming, i.e., for the Sec18p (NSF)-driven release of Sec17p (alpha-SNAP), which activates the vacuoles for subsequent tethering and docking. Therefore, it represents the kinetically earliest requirement identified for vacuole fusion so far. Furthermore, PI(4,5)P(2) is required at a step that can only occur after docking but before the BAPTA sensitive step in the latest stage of the reaction. We hence propose that PI(4,5)P(2) controls two steps of vacuole fusion.     

Multistep mechanism of substrate binding determines chaperone activity of Hsp70

The 70 kDa heat shock proteins (the Hsp70 family) assist refolding of their substrates through ATP-controlled binding. We have analyzed mutants of DnaK, an Hsp70 homolog, altered in key residues of its substrate binding domain. Substrate binding occurs by a dynamic mechanism involving: a hydrophobic pocket for a single residue that is crucial for affinity, a two-layered closing device involving independent action of an alpha-helical lid and an arch, and a superimposed allosteric mechanism of ATP-controlled opening of the substrate binding cavity that operates largely through a beta-structured subdomain. Correlative evidence from mutational analysis suggests that the ADP and ATP states of DnaK differ in the frequency of the conformational changes in the alpha-helical lid and beta-domain that cause opening of the substrate binding cavity. The affinity for substrates, as defined by this mechanism, determines the efficiency of DnaJ-mediated and ATP hydrolysis mediated locking-in of substrates and chaperone activity of DnaK.     

Corticotropin-releasing factor receptor 1 mediates acute and delayed stress-induced visceral hyperalgesia in maternally separated Long-Evans rats

In rodents, maternal pup interactions play an important role in programming the stress responsiveness of the adult organism. The aims of this study were 1) to determine the effect of different neonatal rearing conditions on acute and delayed stress-induced visceral sensitivity as well as on other measures of stress sensitivity of the adult animal; and 2) to determine the role of corticotropin-releasing factor receptor (CRF-R) subtype 1 (CRF(1)R) in mediating visceral hypersensitivity. Three groups of male Long-Evans rat pups were used: separation from their dam for 180 min daily from postnatal days 2-14 (MS180), daily separation (handling) for 15 min (H), or no handling. The visceromotor responses (VMR) to colorectal distension, stress-induced colonic motility, and anxiety-like behavior were assessed in the adult rats. The VMR was assessed at baseline, immediately after a 1-h water avoidance (WA) stress, and 24 h poststress. Astressin B, a nonselective CRF-R antagonist, or CP-154,526, a selective CRF(1)R antagonist, was administered before the stressor and/or before the 24-h measurement. MS rats developed acute and delayed stress-induced visceral hyperalgesia. In contrast, H rats showed hypoalgesia immediately after WA and no change in VMR on day 2. MS rats with visceral hyperalgesia also exhibited enhanced stress-induced colonic motility and increased anxiety-like behavior. In MS rats, both CRF-R antagonists abolished acute and delayed increases in VMR. Rearing conditions have a significant effect on adult stress responsiveness including immediate and delayed visceral pain responses to an acute stressor. Both acute and delayed stress-induced visceral hypersensitivity in MS rats are mediated by the CRF/CRF(1)R system.     

Phase diagram of the system sodium alginate/water: a model for biofilms

Sodium alginate is a polyelectrolyte consisting of the monomer units β-d-mannuronate and -l-guluronate. Mainly based on the theory of Khokhlow et al., the state diagram of the binary system alginate/water has been calculated using different sets of parameters like degree of ionization, degree of polymerization and interaction function. The calculations comprise miscibility gaps, liquidus curves, eutectic points and the behaviour at temperatures below the melting point of water. Also gel and swelling curves have been treated, where gels are physically crosslinked. The DSC diagram of a 0.5 by wt.% polymer sol shows a double melting peak, which is explained by a heterogeneity above 0 °C. The crystallization of water seems to concentrate the gelled system irreversibly.     

Backbone solution structures of proteins using residual dipolar couplings: Application to a novel structural genomics target

Structural genomics (or proteomics) activities are critically dependent on the availability of high-throughput structure determination methodology. Development of such methodology has been a particular challenge for NMR based structure determination because of the demands for isotopic labeling of proteins and the requirements for very long data acquisition times. We present here a methodology that gains efficiency from a focus on determination of backbone structures of proteins as opposed to full structures with all sidechains in place. This focus is appropriate given the presumption that many protein structures in the future will be built using computational methods that start from representative fold family structures and replace as many as 70% of the sidechains in the course of structure determination. The methodology we present is based primarily on residual dipolar couplings (RDCs), readily accessible NMR observables that constrain the orientation of backbone fragments irrespective of separation in space. A new software tool is described for the assembly of backbone fragments under RDC constraints and an application to a structural genomics target is presented. The target is an 8.7 kDa protein from Pyrococcus furiosus, PF1061, that was previously not well annotated, and had a nearest structurally characterized neighbor with only 33% sequence identity. The structure produced shows structural similarity to this sequence homologue, but also shows similarity to other proteins, which suggests a functional role in sulfur transfer. Given the backbone structure and a possible functional link this should be an ideal target for development of modeling methods. This revised version was published online in March 2005 with corrections to the references.