Automated image analysis to improve bead ingestion toxicity test counts in the protozoan Tetrahymena pyriformis

AIMS: To improve bead ingestion counts in Tetrahymena pyriformis by automated image analysis as an alternative to direct-counts. METHODS AND RESULTS: Fluorescent latex beads were added to T. pyriformis cultures for ingestion tests. The number of beads ingested by 25 cells was counted directly by epifluorescence microscopy and compared with similar data from image analysis. anova indicated that counts were not significantly different (P < 0.05). The image analysis particularly provided advantages in terms of speed. CONCLUSIONS: The image analysis is superior to direct beads counting in T. pyriformis particularly in terms of speed of analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: The image analysis method is very rapid and will allow many more toxicological analyses to be undertaken with less operator error.     

Complementary roles for Nkx6 and Nkx2 class proteins in the establishment of motoneuron identity in the hindbrain

The genetic program that underlies the generation of visceral motoneurons in the developing hindbrain remains poorly defined. We have examined the role of Nkx6 and Nkx2 class homeodomain proteins in this process, and provide evidence that these proteins mediate complementary roles in the specification of visceral motoneuron fate. The expression of Nkx2.2 in hindbrain progenitor cells is sufficient to mediate the activation of Phox2b, a homeodomain protein required for the generation of hindbrain visceral motoneurons. The redundant activities of Nkx6.1 and Nkx6.2, in turn, are dispensable for visceral motoneuron generation but are necessary to prevent these cells from adopting a parallel program of interneuron differentiation. The expression of Nkx6.1 and Nkx6.2 is further maintained in differentiating visceral motoneurons, and consistent with this the migration and axonal projection properties of visceral motoneurons are impaired in mice lacking Nkx6.1 and/or Nkx6.2 function. Our analysis provides insight also into the role of Nkx6 proteins in the generation of somatic motoneurons. Studies in the spinal cord have shown that Nkx6.1 and Nkx6.2 are required for the generation of somatic motoneurons, and that the loss of motoneurons at this level correlates with the extinguished expression of the motoneuron determinant Olig2. Unexpectedly, we find that the initial expression of Olig2 is left intact in the caudal hindbrain of Nkx6.1/Nkx6.2 compound mutants, and despite this, all somatic motoneurons are missing. These data argue against models in which Nkx6 proteins and Olig2 operate in a linear pathway, and instead indicate a parallel requirement for these proteins in the progression of somatic motoneuron differentiation. Thus, both visceral and somatic motoneuron differentiation appear to rely on the combined activity of cell intrinsic determinants, rather than on a single key determinant of neuronal cell fate.     

Bioconversion of nitriles by Candida guilliermondii CCT 7207 cells immobilized in barium alginate

Nitrile degradation by Candida guilliermondii CCT 7207 using free and immobilized cell systems was compared. Different specific growth rates were observed for immobilized (mumax=0.021 h(-1)) and the free cells (mumax=0.029 h(-1)). The maximum specific rate of acetic acid formation was 0.387 h(-1) and 0.266 h(-1) for free and immobilized cells, respectively. Cell adhesion to the support materials was confirmed by scanning electron microscopy. When immobilized, the yeast was able to use high nitrile and amide concentrations (aliphatic and aromatic) as nitrogen sources. The results suggest that C. guilliermondii CCT 7207 presents a physiological pattern potentially useful for the bioremediation of polluted environments or for the bioproduction of amides and organic acid of high commercial value.     

Screening and characterization of Bacillus thuringiensis isolates from Brazil for the presence of coleoptera-specific cry genes

Forty-three Bacillus thuringiensis isolates from Brazil and 3 from Argentina were screened, using the polymerase chain reaction (PCR), for various coleoptera-specific cry genes. Seven isolates produced specific and/or nonspecific DNA fragments in a PCR reaction with primers specific for two coleopteran cry genes and 4 of these produced DNA fragments with primers specific for 7 known coleopteran cry genes. These isolates showed, by electron microscopy, the presence of spherical crystals. They also showed proteins of around 70 kDa which were immunologically similar to the Cry3Aa protein from B. thuringiensis subsp. tenebrionis. The 3 isolates from Argentina were toxic to T. molitor, and although no isolate from Brazil showed toxicity, they might show toxicity to another insect species.     

Deletion of follicle-stimulating hormone (FSH) receptor residues encoded by exon one decreases FSH binding and signaling in the rat

The rat FSH receptor (rFSHR) shares considerable homology with the rat LH receptor (rLHR), yet binds human FSH (hFSH) with high fidelity, suggesting that the binding determinant encoded by the rFSHR gene shares no homology with the analogous rLHR primary sequence, thereby affording specificity of ligand binding. Two such regions of primary sequence have been previously identified and studied by peptide challenge tests and immunoneutralization studies. We therefore implemented site-directed mutagenesis to delete the regions S9-N30 and D300-F315 of the mature rFSHR sequence. The mutant receptor (DeltarFSHR) cDNAs were expressed in insect cells. The large deletion DeltarFSHRS9-N30 and a smaller deletion, DeltarFSHRS9-S18, did not bind (125)I-hFSH. However, DeltarFSHRK19-R29 and DeltarFSHRD300-F315 bound (125)I-hFSH with an affinity indistinguishable from wild-type rFSHR. The deletion mutants DeltarFSHR S9-N30 or DeltarFSHRS9-S18 were not detectable on the cell surface by flow cytometry unless cells were sheared. Although (125)I-hFSH binding to DeltarFSHRK19-R29 was normal, this form of the receptor was defective for signal transduction whereas DeltarFSHRD300-F315 was not. Furthermore, neither region seems to be a specificity determinant, since their removal did not result in high-affinity binding of hCG to DeltarFSHR.     

Definition of minimal domains of interaction within the recombination-activating genes 1 and 2 recombinase complex

During V(D)J recombination, recognition and cleavage of the recombination signal sequences (RSSs) requires the coordinated action of the recombination-activating genes 1 and 2 (RAG1/RAG2) recombinase complex. In this report, we use deletion mapping and site-directed mutagenesis to determine the minimal domains critical for interaction between RAG1 and RAG2. We define the active core of RAG2 required for RSS cleavage as aa 1-371 and demonstrate that the C-terminal 57 aa of this core provide a dominant surface for RAG1 interaction. This region corresponds to the last of six predicted kelch repeat motifs that have been proposed by sequence analysis to fold RAG2 into a six-bladed beta-propeller structure. Residue W317 within this sixth repeat is shown to be critical for mediating contact with RAG1 and concurrently for stabilizing binding and directing cleavage of the RSS. We also show that zinc finger B (aa 727-750) of RAG1 provides a dominant interaction domain for recruiting RAG2. In all, the data support a model of RAG2 as a multimodular protein that utilizes one of its six faces for establishing productive contacts with RAG1.     

The effect of stirring and seeding on the AcPheLeuNH(2) synthesis and crystallization in a reversed micellar system

The present work describes the enzymatic synthesis and simultaneous crystallization of the dipeptide AcPheLeuNH(2) by alpha-chymotrypsin in a reversed micellar system of tetradecyltrimethylammonium bromide (TTAB)/heptane/octanol/carbonate buffer. The low solubility of the dipeptide in the micellar solution led to the formation and growth of needle-like crystals during the synthesis as soon as supersaturation was achieved. The crystallization process then followed a typical pattern, proceeding in three phases: nucleation, de-supersaturation, and re-equilibrium of saturation. Crystallization was followed by visual observation with an optical microscope, and the increase of crystal number and size was confirmed. Experiments showed that the supersaturation concentration decreases with the addition of AcPheLeuNH(2) seeds before the reaction, and also with a decrease of the stirring speed. It was also observed that the increase of both seed concentration and stirring advances the start of crystallization, so that the dipeptide is more quickly removed from solution. The consequent decrease in its loss through hydrolysis causes an increase in its yield. Both stirring and seeding could constitute important generic strategies for promoting crystallization of more soluble dipeptides during their synthesis in similar reversed micellar systems.     

The influence of environmental characteristics on the distribution of ciliates (Protozoa, Ciliophora) in an urban stream of southeast Brazil

The aim of this research was to study the ciliated protozoa community at three sampling stations that receive different levels of domestic sewage along the S?o Pedro Stream in the municipality of Juiz de Fora, Minas Gerais, Brazil, in order to determine the influence of organic pollution on this community and to assess the feasibility of using ciliates as water quality indicators. Four physical-chemical parameters of the water samples were evaluated: dissolved oxygen concentration, electrical conductivity, pH and temperature. The sediment was obtained manually, using dredges with capacity of 300 mL, at each collection point. Point 1 was located in a rural region that receives a low sewage load, while Points 2 and 3 were located in populated regions receiving high sewage loads. We found 22 ciliate species, of which 18 are included in the saprobic system and are considered bioindicators. These showed beta-mesosaprobic environments at Point 1 and alfa-mesosaprobic to polisaprobic environments at Points 2 and 3. The low levels of dissolved oxygen and the high electrical conductivity values at Points 2 and 3, together with the strong similarity between the ciliate taxocenoses of these points and the weak similarity between Point 1 and the other two, confirm the high sewage loads received at the latter two points. The combination of the biological indicators and physical-chemical analyses therefore proved itself to be an efficient method of evaluating water quality, and has excellent potential to support decisions on the conservation of headwaters and recuperation of degraded environments in lotic systems.     

Cellular senescence and autophagy of myoepithelial cells are involved in the progression of in situ areas of carcinoma ex-pleomorphic adenoma to invasive carcinoma. An in vitro model

During tumor invasion, benign myoepithelial cells of carcinoma ex-pleomorphic adenoma (CXPA) surround malignant epithelial cells and disappear. The mechanisms involved in the death and disappearance of these myoepithelial cells were investigated via analysis of the expression of regulatory proteins for apoptosis, autophagy and cellular senescence in an in situ in vitro model. Protein expression relating to apoptosis (Bax, Bcl-2, Survivin), autophagy (Beclin-1, LC3B) and cellular senescence (p21, p16) was evaluated using indirect immunofluorescence. β-galactosidase expression was assessed via histochemistry. Biopsies of CXPA (ex vivo) allowed immunhistochemical evaluation of p21 and p16, whilst LC3B, p21 and p16 protein expression was analyzed by western blotting. In the in vitro model, the myoepithelial cells were positive for LC3B (cytoplasm) and p21 (nucleus), whilst in vivo positivity for p21 and p16 was observed. In vitro, β-galactosidase activity increased in the myoepithelial cells over time. Western blotting analysis revealed an increased LC3B, p16 and p21 expression in the myoepithelial cells with previous contact with the malignant cells when compared with those without contact. The investigation of behavior of benign myoepithelial cells in ductal areas of CXAP revealed that the myoepithelial cells are involved in the autophagy-senescence phenotype that subsequently leads to their disappearance.