Shape, variance and integration during craniogenesis: contrasting marsupial and placental mammals

Studies of morphological integration can provide insight into developmental patterns, even in extinct taxa known only from skeletal remains, thus making them an important tool for studies of evolutionary development. However, interpreting patterns of integration and assessing their significance for organismal evolution requires detailed understanding of the developmental interactions that shape integration and how those interactions change through ontogeny. Thus far, relatively little comparative data have been produced for this important topic, and the data that do exist are overwhelmingly from humans and their close relatives or from laboratory models such as mice. Here, we compare data on shape, variance and integration through postnatal ontogeny for a placental mammal, the least shrew, Cryptotis parva, and a marsupial mammal, the gray short-tailed opossum, Monodelphis domestica. Cranial variance decreased dramatically from early to late ontogeny in Cryptotis, but remained stable through ontogeny in Monodelphis, potentially reflecting functional constraints related to the short gestation and early ossification of oral bones in marsupials. Both Cryptotis and Monodelphis showed significant changes in cranial integration through ontogeny, with a mixture of increased, decreased and stable levels of integration in different cranial regions. Of particular note is that Monodelphis showed an unambiguous decrease in integration of the oral region through ontogeny, potentially relating to their early ossification. Selection at different stages of development may have markedly different effects if patterns of integration change substantially through ontogeny. Our results suggest that high integration of the oral region combined with functional constraints for suckling during early postnatal ontogeny may drive the stagnant variance observed in Monodelphis and potentially other marsupials.     

Hereditary hemochromatosis protein, HFE, interaction with transferrin receptor 2 suggests a molecular mechanism for mammalian iron sensing

HFE and transferrin receptor 2 (TFR2) are membrane proteins integral to mammalian iron homeostasis and associated with human hereditary hemochromatosis. Here we demonstrate that HFE and TFR2 interact in cells, that this interaction is not abrogated by disease-associated mutations of HFE and TFR2, and that TFR2 competes with TFR1 for binding to HFE. We propose a new model for the mechanism of iron status sensing that results in the regulation of iron homeostasis.     

Molecular and functional interactions between Escherichia coli nucleoside-diphosphate kinase and the uracil-DNA glycosylase Ung

Escherichia coli nucleoside-diphosphate kinase (Ndk) catalyzes nucleoside triphosphate synthesis and maintains intracellular triphosphate pools. Mutants of E. coli lacking Ndk exhibit normal growth rates but show a mutator phenotype that cannot be entirely attributed to the absence of Ndk catalytic activity or to an imbalance in cellular triphosphates. It has been suggested previously that Ndk, similar to its human counterparts, possesses nuclease and DNA repair activities, including the excision of uracil from DNA, an activity normally associated with the Ung and Mug uracil-DNA glycosylases (UDGs) in E. coli. Here we have demonstrated that recombinant Ndk purified from wild-type E. coli contains significant UDG activity that is not intrinsic, but rather, is a consequence of a direct physical and functional interaction between Ung and Ndk, although a residual amount of intrinsic UDG activity exists as well. Co-purification of Ung and Ndk through multicolumn low pressure and nickel-nitrilotriacetic acid affinity chromatography suggests that the interaction occurs in a cellular context, as was also suggested by co-immunoprecipitation of endogenous Ung and Ndk from cellular extracts. Glutathione S-transferase pulldown and far Western analyses demonstrate that the interaction also occurs at the level of purified protein, suggesting that it is specific and direct. Moreover, significant augmentation of Ung catalytic activity by Ndk was observed, suggesting that the interaction between the two enzymes is functionally relevant. These findings represent the first example of Ung interacting with another E. coli protein and also lend support to the recently discovered role of nucleoside-diphosphate kinases as regulatory components of multiprotein complexes.     

Assessment of compressive modulus, hydraulic permeability and matrix content of trypsin-treated nucleus pulposus using quantitative MRI

A clinical strength MRI and intact bovine caudal intervertebral discs were used to test the hypotheses that (1) mechanical loading and trypsin treatment induce changes in NMR parameters, mechanical properties and biochemical contents; and (2) mechanical properties are quantitatively related to NMR parameters. MRI acquisitions, confined compression stress-relaxation experiments, and biochemical assays were applied to determine the NMR parameters (relaxation times T1 and T2, magnetization transfer ratio (MTR) and diffusion trace (TrD)), mechanical properties (compressive modulus H(A0) and hydraulic permeability k(0)), and biochemical contents (H(2)O, proteoglycan and total collagen) of nucleus pulposus tissue from bovine caudal discs subjected to one of two injections and one of two mechanical loading conditions. Significant correlations were found between k(0) and T1 (r=0.75,p=0.03), T2 (r=0.78, p=0.02), and TrD (r=0.85, p=0.007). A trend was found between H(A0) and TrD (r=0.56, p=0.12). However, loading decreased these correlations (r=0.4, p=0.2). The significant effect of trypsin treatment on mechanical properties, but not on NMR parameters, may suggest that mechanical properties are more sensitive to the structural changes induced by trypsin treatment. The significant effect of loading on T1 and T2, but not on H(A0) or k(0), may suggest that NMR parameters are more sensitive to the changes in water content enhanced by loading. We conclude that MRI offers promise as a sensitive and non-invasive technique for describing alterations in material properties of intervertebral disc nucleus, and our results demonstrate that the hydraulic permeability correlated more strongly to the quantitative NMR parameters than did the compressive modulus; however, more studies are necessary to more precisely characterize these relationships.     

Small molecule RPE65 antagonists limit the visual cycle and prevent lipofuscin formation

The accumulation of the lipofuscin fluorophores in retinal pigment epithelial (RPE) cells leads to the blinding degeneration characteristic of Stargardt disease and related forms of macular degeneration. RPE lipofuscin, including the fluorophore A2E, forms in large part as a byproduct of the visual cycle. Inhibiting visual cycle function with small molecules is required to prevent the formation of the retinotoxic lipofuscins. This in turn requires identification of rate-limiting steps in the operation of the visual cycle. Specific, non-retinoid isoprenoid compounds are described here, and shown through in both in vitro and in vivo experiments, to serve as antagonists of RPE65, a protein that is essential for the operation of the visual cycle. These RPE65 antagonists block regeneration of 11-cis-retinal, the chromophore of rhodopsin, thereby demonstrating that RPE65 is at least partly rate-limiting in the visual cycle. Furthermore, chronic treatment of a mouse model of Stargardt disease with the RPE65 antagonists abolishes the formation of A2E. Thus, RPE65 is also on the rate-limiting pathway to A2E formation. These nontoxic isoprenoid RPE65 antagonists are candidates for the treatment of forms of macular degeneration wherein lipofuscin accumulation is an important risk factor. These antagonists will also be used to probe the molecular function of RPE65 in vision.     

Oxidative stress promotes mutant huntingtin aggregation and mutant huntingtin-dependent cell death by mimicking proteasomal malfunction

Huntington's disease (HD) is a familial neurodegenerative disorder caused by an abnormal expansion of CAG repeats in the coding region of huntingtin gene. A major hallmark of HD is the proteolytic production of N-terminal fragments of huntingtin containing polyglutamine repeats that form ubiquitinated aggregates in the nucleus and cytoplasm of the affected neurons. However, the mechanism by which the mutant huntingtin causes neurodegeneration is not well understood. Here, we found that oxidative stimuli enhance the polyglutamine-expanded truncated N-terminal huntingtin (mutant huntingtin) aggregation and mutant huntingtin-induced cell death. Oxidative stimuli also lead to rapid proteasomal dysfunction in the mutant huntingtin expressing cells as compared to normal glutamine repeat expressing cells. Overexpression of Cu/Zn superoxide dismutase (SOD1), Hsp40 or Hsp70 reverses the oxidative stress-induced proteasomal malfunction, mutant huntingtin aggregation, and death of the mutant huntingtin expressing cells. Finally, we show the higher levels of expression of SOD1 and DJ-1 in the mutant huntingtin expressing cells. Our result suggests that oxidative stress-induced proteasomal malfunction might be linked with mutant huntingtin-induced cell death.     

Infrared spectroscopic study of the structural and functional properties of the Na/H antiporter MjNhaP1 from Methanococcus jannaschii

In this study, structural, functional, and mechanistic properties of the Na⁺/H⁺ antiporter MjNhaP1 from Methanococcus jannaschii were analyzed by infrared spectroscopic techniques. Na⁺/H⁺ antiporters are generally responsible for the regulation of cytoplasmic pH and Na⁺ concentration. MjNhaP1 is active in the pH range between pH 6 and pH 6.5; below and above it is inactive.The secondary structure analysis on the basis of ATR-IR spectra provides the first insights into the structural changes between inactive (pH 8) and active (pH 6) state of MjNhaP1. It results in decreased ordered structural elements with increasing the pH-value i.e. with inactivation of the protein. Analysis of temperature-dependent FTIR spectra indicates that MjNhaP1 in the active state exhibits a much higher unfolding temperature in the spectral region assigned to α-helical segments. In contrast, the temperature-induced structural changes for β-sheet structure are similar for inactive and active state. Consequently, this structure element is not the part of the activation region of the protein. The surface accessibility of the protein was analyzed by following the extent of H/D exchange. Due to higher content of unordered structural elements a higher accessibility for amide protons is observed for the inactive as compared to the active state of MjNhaP1. Altogether, the results present the active state of MjNhaP1 as the state with ordered structural elements which exhibit high thermal stability and increased hydrophobicity.     

Hydroxylation of l-proline to cis-3-hydroxy-l-proline by recombinant Escherichia coli expressing a synthetic l-proline-3-hydroxylase gene

A synthetic gene encoding a Streptomyces l-proline-3-hydroxylase was constructed and used to produce the hydroxylase protein in recombinant Escherichia coli. A fermentation process for growth of this recombinant E. coli for enzyme production was scaled-up to 250 L. A biotransformation process was developed using cell suspensions of the recombinant E. coli and subsequently scaled-up to 10 L for conversion of l-proline to cis-3-hydroxy-l-proline. A reaction yield of 85 M% and d.e. of 99.9% was obtained for cis-3-hydroxy-l-proline.