The evolution of developmental mechanisms

Over the past two to three decades, developmental biology has demonstrated that all multicellular organisms in the animal kingdom share many of the same molecular building blocks and many of the same regulatory genetic pathways. Yet we still do not understand how the various organisms use these molecules and pathways to assume all the forms we know today. Evolutionary developmental biology tackles this problem by comparing the development of one organism to another and comparing the genes involved and gene functions to understand what makes one organism different from another. In this review, we revisit a set of seven concepts defined by Lewis Wolpert (fate maps, asymmetric division, induction, competence, positional information, determination, and lateral inhibition) that describe the characters of many developmental systems and supplement them with three additional concepts (developmental genomics, genetic redundancy, and genetic networks). We will discuss examples of comparative developmental studies where these concepts have guided observations on the advent of a developmental novelty. Finally, we identify a set of evolutionary frameworks, such as developmental constraints, cooption, duplication, parallel and convergent evolution, and homoplasy, to adequately describe the evolutionary properties of developmental systems.     

ATF-2 cooperates with Smad3 to mediate TGF-beta effects on chondrocyte maturation

This study demonstrates that ATF-2 cooperates with Smad3 to regulate the rate of chondrocyte maturation in response to TGF-beta. ATF-2 was rapidly phosphorylated in chick embryonic cephalic sternal chondrocytes following treatment with TGF-beta, and the effect was dependent upon p38 kinase activity. Transient transfection of both wild-type ATF-2 or Smad3 activated the TGF-beta-responsive reporter, p3TP-Lux, and synergistic effects were observed with ATF-2 and Smad3 coexpression. The effect of Smad3 and ATF-2 alone and in combination on chondrocyte maturation was examined in cultures simultaneously infected with RCAS viruses expressing different viral envelope proteins. When expressed alone, wild-type ATF-2 or Smad3 both inhibit colX expression and partially mimic the effects of exogenous TGF-beta. However, in combination the effects were additive and similar to the inhibitory effects of TGF-beta on colX expression. Loss of function experiments using dominant negative ATF-2 or Smad3 partially blocked the inhibitory effect of TGF-beta on colX, while together the blockade was complete. Similar effects were observed with another TGF-beta-responsive gene, PTHrP. However, the induction of colX by BMP-2 was not affected by overexpression of either wild-type or dominant negative ATF-2, indicating specificity for TGF-beta signaling. In contrast, although TGF-beta does not activate CRE/CREB signaling, dominant negative CREB enhanced colX expression in control and in TGF-beta and BMP-2-treated cultures. Thus, ATF-2 regulates chondrocyte maturation as a direct target of TGF-beta signaling while CREB regulates differentiation by targeting genes independent of the individual signaling effects of TGF-beta or BMP-2.     

Purification of bioethanol effluent in an UASB reactor system with simultaneous biogas formation

In this study, the prospect of using an Upflow Anaerobic Sludge Blanket (UASB) reactor for detoxification of process water derived from bioethanol production has been investigated. The bioethanol effluent (BEE) originated from wet oxidized wheat straw fermented by Saccharomyces cerevisiae and Thermoanaerobacter mathranii A3M4 to produce ethanol from glucose and xylose, respectively. In batch experiments the methane potential of BEE was determined to 529 mL-CH(4)/g-VS. In batch degradation experiments it was shown that the presence of BEE had a positive influence on the removal of the inhibitors 2-furoic acid, 4-hydroxyacetophenone, and acetovanillone as compared to conversion of the inhibitors as sole substrate in synthetic media. Furthermore, experiments were carried out treating BEE in a laboratory-scale UASB reactor. The results showed a Chemical Oxygen Demand (COD) removal of 80% (w/w) at an organic loading rate of 29 g-COD/(L. d). GC analysis of the lignocellulosic related potentially inhibitory compounds 2-furoic acid, vanillic acid, homovanillic acid, acetovanillone, syringic acid, acetosyringone, syringol, 4-hydroxybenzoic acid, and 4-hydroxybenzaldehyde showed that all of these compounds were removed from the BEE in the reactor. Implementation of a UASB purification step was found to be a promising approach to detoxify process water from bioethanol production allowing for recirculation of the process water and reduced production costs.     

Heme and pH-dependent stability of an anionic horseradish peroxidase

Horseradish peroxidase A1 thermal stability was studied by steady-state fluorescence, circular dichroism and differential scanning calorimetry at pH values of 4, 7 and 10. Changes in the intrinsic protein probes, tryptophan fluorescence, secondary structure, and heme group environment are not coincident. The T(m) values measured from the visible CD data are higher than those measured from Trp fluorescence and far-UV CD data at all pH values showing that the heme cavity is the last structural region to suffer significant conformational changes during thermal denaturation. However ejection of the heme group leads to an irreversible unfolding behavior at pH 4, while at pH 7 and 10 refolding is still observed. This is putatively correlated with the titration state of the heme pocket. Thermal transitions of HRPA1 showed scan rate dependence at the three pH values, showing that the denaturation process was kinetically controlled. The denaturation process was interpreted in terms of the classic scheme, N<-->U-->D and fitted to far-UV CD ellipticity. A good agreement was obtained between the experimental and theoretical T(m) values and percentages of irreversibility. However the equilibrium between N and U is probably more complex than just a two-state process as revealed by the multiple T(m) values.     

Spontaneous tandem-base mutations (TBM) show dramatic tissue,age, pattern and spectrum specificity

To supplement a previous analysis of spontaneous tandem-base mutations (TBM) in the lacI gene of Big Blue((R)) mice, 2658 additional mutants were sequenced from 13 tissues and 44 spontaneous TBM were identified (tripling the sample size). Previous findings were confirmed and generalized and several new observations were made. TBM differ from single and other double mutations in that TBM frequency varies dramatically with tissue type. In certain tissues, most notably male germ cells, no TBM are observed despite screening as many as 26 million plaque forming units. TBM are most frequent in kidney and liver (3.45 and 2x10(-6), respectively), accounting for 7.6 and 4.8% of all mutational events in kidney and liver, respectively. There is a trend for elevated TBM frequency in thymic lymphomas in p53-deficient mice. TBM are more frequent in old age in both liver and kidney. TBM differ from single mutations and other double mutations because they display a marked difference in pattern and dramatic tissue specificity for target sequence. Five of the 78 possible TBM outcomes comprise 79% of those observed, and mutations at GG/CC predominate. TBM in mice were compared with TBM found in human mutation databases. TBM are also rare in the human germline (one in 5133 germline mutations reported in five human mutation databases). In general, the types of somatic TBM are similar in mice and humans except for an excess of TG/CA to CA/TG TBM in humans (TBM related to ultraviolet light-induced skin cancer were excluded). TBM may be the result of unknown mechanisms that may have some similarities in mice and humans.     

Dapsone for the treatment of doxorubicin extravasation injury in the rat

Doxorubicin is the most common antitumor drug implicated in serious extravasation injuries. Progressive tissue necrosis may lead to intense pain, chronic ulceration, and disfiguring tissue loss. This progressive necrosis is analogous to that seen with brown recluse spider bites, where dapsone is an established mode of therapy, minimizing the area of tissue loss by a proposed antiinflammatory mechanism. The backs of 50 Lewis rats were injected intradermally with 1 mg of doxorubicin in 1 cc of saline to simulate an extravasation injury. The rats were divided into five groups for treatment with oral dapsone 50 mg/kg/day: 10 were controls (no treatment), 10 were started the day before injury, 10 were started the day of injury, 10 were started the day after injury, and 10 were started 1 week after injury. The area of ulceration was calculated by planimetry. The data suggest that dapsone has little positive effect on healing extravasation ulcers.     

Purification and characterization of the complex I from the respiratory chain of Rhodothermus marinus

The rotenone sensitive NADH: menaquinone oxidoreductase (NDH-I or complex I) from the thermohalophilic bacterium Rhodothermus marinus has been purified and characterized. Three of its subunits react with antibodies against 78, 51, and 21.3c kDa subunits of Neurospora crassa complex I. The optimum conditions for NADH dehydrogenase activity are 50°C and pH 8.1, and the enzyme presents a K _M of 9 M for NADH. The enzyme also displays NADH:quinone oxidoreductase activity with two menaquinone analogs, 1,4-naphtoquinone (NQ) and 2,3-dimethyl-1,4-naphtoquinone (DMN), being the last one rotenone sensitive, indicating the complex integrity as purified. When incorporated in liposomes, a stimulation of the NADH:DMN oxidoreductase activity is observed by dissipation of the membrane potential, upon addition of CCCP. The purified enzyme contains 13.5 ± 3.5 iron atoms and 3.7 menaquinone per FMN. At least five iron—sulfur centers are observed by EPR spectroscopy: two [2Fe–2S]^2+/1+ and three [4Fe–4S]^2+/1+ centers. By fluorescence spectroscopy a still unidentified chromophore was detected in R. marinus complex I.     

A novel mutation in PTPRC interferes with splicing and alters the structure of the human CD45 molecule

CD45, encoded by the protein tyrosine phosphatase receptor type C ( PTPRC) gene, is essentially involved in maturation, activation, and migration of immune cells. Lack of CD45 results in severe immunodeficiency, and alterations of the receptor may result in autoimmunity. Here, we describe a novel mutation in PTPRCas a cause of variant CD45 expression in humans. Several members of a multiple sclerosis multiplex family showed expression of CD45RA on memory T cells and monocytes. The variant expression pattern was linked to the PTPRCgene by DNA microsatellite studies. DNA analysis identified a novel point mutation in exon 4 (position 59 C-->A) in all family members with variant CD45 expression, but not in donors with normal CD45 expression. The mutation interferes with alternative splicing and alters amino acid sequence (H-->Q), interfering with antibody binding to the CD45RA domain. Overall, we describe the first mutation in PTPRCthat interferes with splicing and results in surface expression of a structurally altered CD45 molecule in humans.     

In vitro and in vivo invasiveness of different pulsed-field gel electrophoresis types of Listeria monocytogenes

The virulence of different pulsed-field gel electrophoresis (PFGE) types of Listeria monocytogenes was examined by monitoring their ability to invade Caco-2 cells. Strains belonging to seven different PFGE types originating from both foods and humans were included. No significant differences in invasiveness were detected between strains isolated from humans and those isolated from food. Strains belonging to PFGE type 1 expressed a significantly lower ability to invade cells compared to strains belonging to other PFGE types. Although strains of PFGE type 2 also seemed to invade at a low level, this was not significant in the present study. PFGE types 1 and 2 as well as type 14 are more frequently found in food than the four other PFGE types examined and moreover have a relatively low prevalence in humans compared to their prevalence in food. Thus, the hypothesis that some PFGE types are less virulent than others is supported by this study showing that certain PFGE types of L. monocytogenes commonly found in food are less invasive than others to Caco-2 cells. In contrast to the differences in invasion, identical intracellular growth rates between the different PFGE types were observed. In vivo studies of the actual ability of the strains to invade the liver and spleen of cimetidine-treated rats following an oral dose of 10(9) L. monocytogenes cells were performed for isolates of PFGE types 1, 2, 5, and 15. After 2 days, equal amounts of bacteria were observed in the liver and spleen of the rats for any of the PFGE types tested.     

Cell-intrinsic and cell-extrinsic cues regulating lineage decisions in multipotent neural crest-derived progenitor cells

Multipotent stem cells must generate various differentiated cell types in correct number and sequence during neural development. In the peripheral nervous system (PNS), this involves the formation of postmigratory progenitor cell types which maintain multipotency and are able to give rise to neural and non-neural cells in response to instructive growth factors. We propose that fate restrictions in such progenitor cells are controlled by the combinatorial interaction of different extracellular signals, including community effects in response to both neurogenic and gliogenic factors. In addition, distinct progenitor cell types display intrinsic differences which modulate their response to the extracellular environment. Thus, a progenitor cell is apparently able to integrate multiple intrinsic and extrinsic cues and thereby to choose fates appropriate for its location. Fate analysis of genetically modified progenitor cells will help to identify the molecules involved. This approach appears promising given the identification of multipotent progenitor cells from the mouse PNS and the availability of genetics in the mouse system.