β-Amino acid residues: Conformational characterization of an N- and C-protected homo-β-(S)-leucine

Summary An N- and C-protected derivative ofhomo-β-leucine, Fmoc-homo-β-(S)-leucine methyl ester, synthesized from the corresponding proteinogenic parent α-amino acid in enantiopure form has been fully characterized in the solid state by X-ray diffraction analysis. The crystal conformation of this new residue indicates and extended conformation for thishomo-β-residue, with the ϕ torsion angle being more constrained than the μ and ψ angles.     

Physiological price of an induced chemical defense: photosynthesis, respiration, biosynthesis, and growth

A recurring theme in defense allocation theories is that defenses are costly. Most studies that attempt to quantify a cost of defense seek to establish a trade-off between a component of plant fitness and the level of a constitutive defense. Such estimates are ambiguous because they cannot discount the cost of traits that are correlated with defense but are not themselves defensive. We examined the effects of damage-induced synthesis of furanocoumarins, known defense compounds, on the growth of wild parsnip. Plants that had 2% of their leaf area removed accumulated 8.6% less total biomass and 14% less root biomass than intact plants over a 4-week period. We also found that this small amount of leaf damage significantly reduced net photosynthetic rates 0.5 h after damage; the effect was temporary, as photosynthetic rates were no longer significantly different after 48 h. Lastly, we found that increases in respiration rates associated with damage coincided spatially and temporally with increases in furanocoumarin production, and that respiration increases were phenotypically correlated with furanocoumarin production. When damage-induced changes in furanocoumarin content and respiration rates were expressed in glucose equivalents and compared, the energetic cost of furanocoumarin production (12.6 μg glucose cm⁻²) accounted for all of the increase in respiration (12.0 μg glucose cm⁻²). A comparison of other secondary compounds in damaged and intact leaflets revealed that myristicin, a furanocoumarin synergist, is the only other compound aside from furanocoumarins that is inducible. The inducible defense system of wild parsnip thus appears to involve a small subset of secondary compounds. Synthesis of these compounds is tightly linked to damage-induced rates of respiration. Because the negative impact that damage had on the rate of net photosynthesis was short-lived, the impact of damage on growth observed in this study was likely due to the cost of furanocoumarin synthesis elicited by damage rather than the loss of photosynthetic tissue caused by damage. Received: 4 April 1996 / Accepted: 29 August 1996     

Preliminary evidence of endotoxic activity of Bilophila wadsworthia

In this study we describe two properties of the Gram-negative bacterium Bilophila wadsworthia, namely the ability to clot Limulus lysate and the capacity to induce the production of tissue factor-like procoagulant activity by human mononuclear cells in vitro. Although exhibited at a lower degree when compared with those of typical Gram-negative bacteria or Gram-negative endotoxin those activities may account in part for Bilophila's pathogenicity. The capacity indeed to induce fibrin formation through the interaction with mononuclear cells suggests one mechanism by which the microorganism might cause abscess formation in the host. Moreover, since this activity is dependent on the number of Bilophila interacting with mononuclear cells, we hypothesize that this biological activity is closely influenced by growth environment.     

Ubiquitin over-expression promotes E6AP autodegradation and reactivation of the p53/MDM2 pathway in HeLa cells

It has been established that intracellular ubiquitin pools are subject to regulatory constrains. Less certain is the mechanism by which the pool of conjugated ubiquitin shift in parallel with total ubiquitin, and how this type of regulation affects the flux of substrates through the pathway. In this study we demonstrate that ubiquitin over-expression promotes the destabilization of the ubiquitin protein ligase E6AP, by a mechanism involving self-ubiquitination, and the stabilization of p53. These results represent the very first evidence that the levels of a ubiquitin ligase can be regulated in vivo by ubiquitin abundance, supporting the idea that a strict interrelationship between pathway component activities and ubiquitin pool size exists. Interestingly, ubiquitin-induced p53 accumulation did not induce cell-cycle arrest, suggesting that although fluctuations of the intracellular ubiquitin content may actively modulate the level of regulatory proteins, this event is not per se sufficient to elicit a cellular response in terms of proliferation.     

Single-Cell Sequencing of iPSC-Dopamine Neurons Reconstructs Disease Progression and Identifies HDAC4 as a Regulator of Parkinson Cell Phenotypes

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UVA light stimulates the production of cathepsin G and elastase-like enzymes by dermal fibroblasts: a possible contribution to the remodeling of elastotic areas in sun-damaged skin

Solar elastosis is characterized by accumulation of large amounts of material staining similarly to elastin in the dermis. The nature of this material and the process responsible for its accumulation are still unknown. Elastolytic proteases have important functions in the catabolism of the interstitial matrix and can also generate, by the digestion of the interstitial proteins, soluble peptides which can induce collagen and elastin synthesis and deposition. We investigated whether (i) elastolytic enzymes can be detected in samples from sun-exposed and non-exposed skin, and (ii) ultraviolet (UV) rays influence the production of elastolytic activities in cultured dermal fibroblasts. Immunoelectron microscopy showed a positive reaction for neutrophil elastase and cathepsin G in fibroblast-like cells from specimens of sun-exposed areas. Little or no reaction was found in biopsies of sun-protected skin. Fibroblast cultures from sun-exposed skin expressed higher levels of hydrolytic activity against synthetic substrates of elastases and cathepsin G than those obtained from sun-protected areas. Irradiation with UVA strongly stimulated the production of these activities in fibroblasts from sun-protected sites. No significant change was detected in parallel sets of cultures after UVB irradiation. Inhibition experiments indicated that the elastase-like activity expressed by fibroblasts can be attributed to at least two enzymes.     

Allergic inflammatory response to short ragweed allergenic extract in HLA-DQ transgenic mice lacking CD4 gene.

To investigate the role of HLA-DQ molecules and/or CD4(+) T cells in the pathogenesis of allergic asthma, we generated HLA-DQ6 and HLA-DQ8 transgenic mice lacking endogenous class II (Abeta(null)) and CD4 genes and challenged them intranasally with short ragweed allergenic extract (SRW). We found that DQ6/CD4(null) mice developed a strong eosinophilic infiltration into the bronchoalveolar lavage and lung tissue, while DQ8/CD4(null) mice were normal. However, neither cytokines nor eosinophil peroxidase in the bronchoalveolar lavage of DQ6/CD4(null) mice was found. In addition, the airway reactivity to methacholine was elevated moderately in DQ6/CD4(null) mice compared with the high response in DQ/CD4(+) counterparts and was only partially augmented by CD4(+) T cell transfer. The DQ6/CD4(null) mice showed Th1/Th2-type cytokines and SRW-specific Abs in the immune sera in contrast to a direct Th2 response observed in DQ6/CD4(+) mice. The proliferative response of spleen mononuclear cells and peribronchial lymph node cells demonstrated that the response to SRW in DQ6/CD4(null) mice was mediated by HLA-DQ-restricted CD4(-)CD8(-)NK1.1(-) T cells. FACS analysis of PBMC and spleen mononuclear cells demonstrated an expansion of double-negative (DN) CD4(-)CD8(-)TCRalphabeta(+) T cells in SRW-treated DQ6/CD4(null) mice. These cells produced IL-4, IL-5, IL-13, and IFN-gamma when stimulated with immobilized anti-CD3. IL-5 ELISPOT assay revealed that DN T cells were the cellular origin of IL-5 in allergen-challenged DQ6/CD4(null) mice. Our study shows a role for HLA-DQ-restricted CD4(+) and DN T cells in the allergic response.     

DNA and protein content of mouse sperm: Implications regarding sperm chromatin structure

A variety of biochemical and histochemical techniques have been used to compare the composition of chromatin in sperm nuclei isolated from the epididymides of five mouse strains. The DNA content was determined by phosphorus analysis, deoxyribose analysis, absorption spectroscopy at 260 nm, and cytomorphometry following gallocyanine chrome alum staining. All four methods indicate that the mouse sperm nucleus contains approx. 3.3 pg DNA and that the DNA content does not vary significantly among the strains tested. Three different techniques, quantitative amino acid analysis, absorption spectroscopy at 230 nm, and sperm head density analysis in cesium chloride, were used to determine the protein content. Sperm nuclei from each strain of mouse were found to have a protein to DNA ratio of 0.9 and a chromatin protein content of 3 pg/nucleus. Comparisons of the basic proteins by disc gel electrophoresis demonstrate that the sperm nuclei contain only protamine and lack significant levels of somatic histones or transition proteins. The sperm from each strain contained both mouse protamine variants and the relative distribution of the two proteins did not appear to differ among strains. Using this information, we have been able to draw certain conclusions regarding DNA-protamine interactions and the mode of DNA packaging in the sperm nucleus. The most important of these is that the DNA in the mouse sperm nucleus cannot be packaged in nucleosomes. The protamines in sperm chromatin do not function as structural proteins, providing a subunit core around which the DNA is wrapped, but appear to completely neutralize the phosphodiester backbone of the DNA molecule, thereby minimizing the repulsion between neighboring segments of DNA and allowing it to be condensed into a biochemically inactive particle of genetic information.     

Early growth response (EGR)-1 is required for timely cell-cycle entry and progression in hepatocytes after acute carbon tetrachloride exposure in mice

Cell-cycle induction in hepatocytes protects from prolonged tissue damage after toxic liver injury. Early growth response (Egr)-1(-/-) mice exhibit increased liver injury after carbon tetrachloride (CCl(4)) exposure and reduced TNF-α production. Because TNF-α is required for prompt cell-cycle induction after liver injury, here, we tested the hypothesis that Egr-1 is required for timely hepatocyte entry into the cell cycle after CCl(4)-induced liver injury. Acute liver injury was induced by a single injection of CCl(4). Assays were employed to assess indices of the cell cycle in liver after CCl(4) exposure. Bromodeoxyuridine incorporation peaked in wild-type mice at 48 h after CCl(4) but was reduced by 80% in Egr-1(-/-) mice. Proliferating-cell nuclear-antigen immunohistochemistry revealed blocks in cell-cycle entry and progression to DNA synthesis in Egr-1-deficient mice 48 h after CCl(4). Cyclin D, important for G0/G1 progression, was reduced at baseline and 36 h after CCl(4). Cyclin E1, required for G1/S-phase transition, was reduced in Egr-1(-/-) mice 24 and 48 h after CCl(4) exposure and was associated with reduced phosphorylation of the retinoblastoma protein. Proliferation in Egr-1(-/-) mice was delayed, rather than blocked, because indices of cell-cycle progression were restored 72 h after CCl(4) exposure. We concluded that Egr-1 was required for prompt cell-cycle entry (G0- to G1-phase) and G1/S-phase transition after toxic liver injury. These data support the hypothesis that Egr-1 provides hepatoprotection in the CCl(4)-injured liver, attributable, in part, to timely cell-cycle induction and progression.