A mechanobiological model to study upstream cell migration guided by tensotaxis

Biomechanics and Modeling in Mechanobiology - Cell migration is a process of crucial importance for the human body. It is responsible for important processes such as wound healing and tumor...     

Reassessment of the vocal repertoire of a nest-building gladiator frog,Boana pardalis (Anura,Hylidae, Cophomantinae): implications for its diagnosis within the B. faber species group

In this study, we re-describe the advertisement and territorial calls of Boana pardalis, carry out an acoustic comparison between the studied species and the other congeners of the B. faber group, and report for the first time the tympanic amplexus for the studied species. The advertisement call of B. pardalis can be used to supplement its diagnosis in the B. faber group based on temporal call traits, e.g. emission rate and emission pattern, as well as the call envelope.     

A new human growth hormone production process using a recombinant Bacillus subtilis strain.

We constructed a series of hybrid plasmids which directed the synthesis of different human growth hormone (hGH) precursor sequences in Bacillus subtilis. In addition to the 191 amino acids of the hormone, the precursors had in common an amino-terminal extension characterized by the presence of a methionine at position 1 and of the tetrapeptide Ile-Glu-Gly-Arg preceding the first residue (Phe) of hGH. The sequence between the methionine and the tetrapeptide was specific for each precursor and, because of the presence of charged residues, conferred particular properties to the molecules. Long homopolymeric tail-containing precursors such as MRRRRRRIILM-IEGR appeared insoluble whereas shorter sequences of the type MRR-IEGR and MEELM-IEGR augmented the solubility of the precursors with respect to Met-hGH. The soluble precursors could be easily purified from the bulk proteins taking advantage of the charged residues present on the N-terminal tail. After purification, the natural hGH was obtained by treating the precursors with the protease Factor Xa which cleaves after the arginine residue of the tetrapeptide IEGR. A protocol for the production and purification of authentic hGH from a strain expressing one of these soluble precursors is reported.     

Effect of dissolved oxygen concentration on repeated production of gluconic acid by immobilised mycelia of Aspergillus niger

Summary Gluconic acid production from corn starch hydrolysates by immobilised mycelia of Aspergillus niger was studied in a laboratory-scale stirred fermentor at different concentrations of glucose (S ₀) and dissolved oxygen (DO) in the culture broth. Its evolution was simulated quite well by applying the same unstructured model set up in previous experiments using stirred and airlift fermentors. In particular, increasing S ₀ in the range 70–160 g/l, although uninfluential upon the yield coefficient, resulted in an exponential decrease in the gluconic acid formation rate constant. Nevertheless, the greater the oxygen transfer rate used in the fermentor, the smaller the inhibitor effect of the higher concentrations of glucose on gluconate productivity became. This was achieved by enriching the inlet air with pure oxygen so as to maintain the DO level above 75% saturation throughout the fermentation. Offprint requests to: M. Moresi     

Prochymosin expression in Bacillus subtilis

Prochymosin (PC) sequence was cloned in Bacillus subtilis using two kinds of plasmid constructions. In plasmid pSM316 the cDNA was inserted to obtain the intracellular expression of the enzyme. The enzyme turned out to be expressed in an insoluble form which could be converted to native enzyme under proper denaturing and refolding conditions. The levels of intracellular expression of PC were further enhanced by modifying the 5' region of the gene in a way that a two-cistron expression system was created. For the PC secretion, the cDNA was fused to the subtilisin leader sequence and expressed under the control of the B. subtilis neutral protease promoter. A properly folded PC was secreted by the cells, although to low levels.     

Advantages and limitations of 1-palmitoyl-2-[[2-[4- (6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]carbonyl]-3- sn-phosphatidylcholine as a fluorescent membrane probe

We have investigated the behavior of 1-palmitoyl-2-[[2-[4- (6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]carbonyl]-3-sn -phosphatidylcholine (DPHpPC) in synthetic, multilamellar phosphatidylcholine vesicles. This fluorescent phospholipid has photophysical properties similar to its parent fluorophore, diphenylhexatriene (DPH). DPHpPC preferentially partitioned into fluid phase lipid (Kf/s = 3.3) and reported a lower phase transition temperature as detected by fluorescence anisotropy than that observed by differential scanning calorimetry. Calorimetric measurements of the bilayer phase transition in samples having different phospholipid to probe ratios demonstrated very slight changes in membrane phase transition temperature (0.1-0.2 degree C) and showed no measurable change in transition width. Nonetheless, measurements of probe fluorescence properties suggested that DPHpPC disrupts its local environment in the membrane and may even induce perturbed probe-rich local domains below the phospholipid phase transition. Temperature profiles of steady-state fluorescence anisotropy, limiting anisotropy, differential tangent, and rotational rate were similar to those of DPH below the main lipid phase transition but indicated more restricted rotational motion above the lipid phase transition temperature. As for DPH, the fluorescence decay of DPHpPC could be described by either a single or double exponential both above and below the DPPC phase transition. The choice seemed dependent on the treatment of the sample. The intensity-weighted average lifetime of DPHpPC was roughly 1.5 ns shorter than that of DPH. In summary, the measured properties of DPHpPC and its lipid-like structure make it a powerful probe of membrane structure and dynamics.     

Morphology and phase behavior of two types of unilamellar vesicles prepared from synthetic phosphatidylcholines studied by freeze-fracture electron microscopy and calorimetry

Differential scanning calorimetry and freeze-fracture electron microscopy have been used to characterize the phase behavior and morphology of two types of unilamellar vesicles composed of synthetic phosphatidylcholines. The first type displayed an average diameter of roughly 100 nm and was formed by slow dilution and dialysis of octylglucoside-solubilized lipid. These large, unilamellar vesicles were termed dialyzed, octylglucoside vesicles and could be obtained as a fairly well defined and uniform population of vesicles. The second vesicle type was prepared by a unique procedure involving dialysis of deoxycholate-solubilized lipid at its pre-transition temperature. This procedure produced a much more heterogeneous distribution of vesicle sizes (500 to 4000 nm in diameter) and left some dilamellar and oligolamellar species which could not be conveniently separated from the giant, unilamellar vesicles constituting the major portion of the sample. Both populations of vesicles displayed phase behavior similar, but not identical to that of large, multilamellar vesicles (LMV). Fracture-face morphology of the gel phase was also observed to differ between the two unilamellar and the multilamellar species. LMV have previously been shown to have clear undulated or banded fracture-faces in the P beta phase, while octylglucoside vesicles are shown here to have facetted fracture-faces. Giant, unilamellar vesicles displayed a faint banded morphology similar to but less distinct than that of the LMV P beta phase. These results have demonstrated that bilayer apposition is not required to support the banded fracture-face morphology characteristic of the P beta phase but that a limiting curvature is necessary.     

Molecular basis of superreactivity of cysteine residues 31 and 32 of seminal ribonuclease

The molecular basis of the high reactivity toward reducing agents of intersubunit disulfides at positions 31 and 32 of dimeric bovine seminal ribonuclease was investigated by studying in the monomeric enzyme the fast reaction kinetics with disulfides of the adjacent cysteine-31 and -32, exposed by selective reduction of the intersubunit disulfides. Negatively charged and neutral disulfide reagents were used for measuring the thiol reaction rates at neutral pH. The kinetics studied as a function of pH permitted us to define pK values for the thiols of interest and indicated the possibility of determining pK values of SH groups in proteins indirectly by measuring the kinetics of reactivity of the SH groups with a disulfide reagent. The results were compared with those obtained under identical conditions with synthetic thiol peptides and model compounds. The data indicate that the superreactivity of intersubunit disulfides of seminal ribonuclease is matched by the high reactivity at neutral pH of adjacent cysteine residues 31 and 32, as compared to all small thiol compounds tested. The synthetic hexapeptide segment of seminal ribonuclease Ac-Met-Cys-Cys-Arg-Lys-Met-OH, which includes the two cysteine residues of interest, was even more reactive. These data, and the other results reported in this paper, led to the conclusion that the superreactivity at neutral pH of cysteine residues at positions 31 and 32 of bovine seminal ribonuclease is primarily dependent on the nearby presence of positively charged groups, particularly the epsilon-NH2 of lysine-34, and is influenced by the adjacency of the two thiols and by the protein tertiary structure.     

Low species turnover of upland Amazonian birds in the absence of physical barriers

Aim

One of the oldest and most powerful ways for ecologists to explain distinct biological communities is to invoke underlying environmental differences. But in hyper-diverse systems, which often display high species richness and low species abundance, these sorts of community comparisons are especially challenging. The classic view for Amazonian birds posits that riverine barriers and habitat specialization determine local and regional community composition. We test the tacit, complementary assumption that similar bird communities should therefore permeate uniform habitat between major rivers, regardless of distance.

Location

Upland (terra firme) rainforests of central Amazonia.

Methods

We conducted intensive whole-community surveys of birds in three pairs of 100-ha plots, separated by 40–60 km. We then used dissimilarity indices, cluster analysis, and ordination to characterize differences among the six avian communities.

Results

In all, we detected 244 forest-dependent birds, with an average of 190 species (78%) per plot. Species turnover was negligible, no unique indicator species were found among plot pairs, and all documented species were already known from a complete inventory at one of the three sites.

Main Conclusions

Our study corroborates the classic biogeographical pattern and suggests that turnover contributes little to regional avian diversity within upland forests. Using a grain size of 100 ha, this implies that upland birds perceive the environment as uniform, at least over distances of ~60 km. Therefore, to maximize both local species richness and population persistence, our findings support the conservation of very large tracts of upland rainforest. Our analyses also revealed that the avifauna at Reserva Ducke, encroached by urban sprawl from the city of Manaus, shows the hallmarks of a disturbed community, with fewer vulnerable insectivores. This defaunation signals that even an enormous preserve (10 × 10 km) in lowland Amazonia is not insulated from anthropogenic degradation within the surrounding landscape.