Biochemistry of Transglutaminases and Cross-Linking in the Skin

Transglutaminase is a calcium-dependent enzyme found widely in nature. It catalyzes the formation of α-(γ-glutamyl)lysine bonds that participate in processes varying from fibrin clot formation to epidermal cell envelope formation. Epidermal transglutaminase is localized to the granular layer of the epidermis. It catalyzes the covalent cross-linking of a soluble cytoplasmic substrate into large polymers to form the cornified envelope that lines the inner membrane of keratinocytes in the stratum corneum, The soluble precursor from epidermis has been named keratolinin, and from keratinocyte culture, it has been named involucrin. Hair follicle transglutaminase is biochemically and immunochemically distinct from its epidermal counters part. It has been localized to the inner root sheath and medulla of the hair follicle, The substrate of hair follicle transglutaminase has been poorly defined but appears to be rich in the amino acid citrulline, Transglutaminase has been shown to be an important marker of normal differentiation. There is a rise in its activity at the time of keratinization, and transglutaminase activity has been shown to be greatly decreased in basal cell epithelioma and in psoriasis. Keratinocyte cell culture has proven most helpful in delineating the processes of normal differentiation and keratinization, since the formation of the cell envelope in culture appears to parallel the formation in vivo

The cc-pV5Z-F12 basis set: reaching the basis set limit in explicitly correlated calculations

We have developed and benchmarked a new extended basis set for explicitly correlated calculations, namely cc-pV5Z-F12. It is offered in two variants, cc-pV5Z-F12 and cc- pV5Z-F12(rev2), the latter of which has additional basis functions on hydrogen not present in the cc-pVnZ-F12 (n=D,T,Q) sequence.A large uncontracted 'reference' basis set is used for benchmarking. cc-pVnZ-F12 (n=D, T, Q, 5) is shown to be a convergent hierarchy. Especially the cc- pV5Z-F12(rev2) basis set can yield the valence CCSD component of total atomization energies (TAEs), without any extrapolation, to an accuracy normally associated with aug-cc-pV{5,6}Z extrapolations. SCF components are functionally at the basis set limit, while the MP2 limit can be approached to as little as 0.01 kcal/mol without extrapolation. The determination of (T) appears to be the most difficult of the three components and cannot presently be accomplished without extrapolation or scaling. (T) extrapolation from cc-pV{T,Q}Z-F12 basis sets, combined with CCSD-F12b/cc-pV5Z-F12 calculations appears to be an accurate combination for explicitly correlated thermochemistry. For accurate work on noncovalent interactions, basis set superposition error with the cc-pV5Z-F12 basis set is shown to be so small that counterpoise corrections can be neglected for all but the most exacting purposes.Comment: Molecular Physics, in press (Nicholas C. Handy memorial issue). DOI preassigne

Inexpensive Ultrahigh Vacuum Heatable/Coolable \u3cem\u3exyz\u3c/em\u3e-Rotary Motion Sample Manipulator

A simple design for a heatable, coolable, rotable sample manipulator, suitable for ultrahigh vacuum (UHV) applications, is described. Highlights of the design include using a combination power/thermocouple feedthrough for heating, cooling, and temperature measurement; and the use of primarily off-the-shelf components available from most UHV components vendors. The described manipulator is capable of sample cooling to ~100 K, sample heating to above 900 K, while maintaining 360o of rotary motion, ~1 in. of x and y motion, and 2 in. of z motion. The apparatus can be assembled for approximately $5500 (all new parts) and uses about 3 l of liquid N2 per day. © 1995 American Institute of Physics

The Near-Infrared Broad Emission Line Region of Active Galactic Nuclei -- I. The Observations

We present high quality (high signal-to-noise ratio and moderate spectral resolution) near-infrared (near-IR) spectroscopic observations of 23 well-known broad-emission line active galactic nuclei (AGN). Additionally, we obtained simultaneous (within two months) optical spectroscopy of similar quality. The near-IR broad emission line spectrum of AGN is dominated by permitted transitions of hydrogen, helium, oxygen, and calcium, and by the rich spectrum of singly-ionized iron. In this paper we present the spectra, line identifications and measurements, and address briefly some of the important issues regarding the physics of AGN broad emission line regions. In particular, we investigate the excitation mechanism of neutral oxygen and confront for the first time theoretical predictions of the near-IR iron emission spectrum with observations.Comment: 45 pages, 17 figures, accepted by ApJ

The Muscle Ankyrin Repeat Proteins CARP, Ankrd2, and DARP Are Not Essential for Normal Cardiac Development and Function at Basal Conditions and in Response to Pressure Overload

Ankrd1/CARP, Ankrd2/Arpp, and Ankrd23/DARP belong to a family of stress inducible ankyrin repeat proteins expressed in striated muscle (MARPs). The MARPs are homologous in structure and localized in the nucleus where they negatively regulate gene expression as well as in the sarcomeric I-band, where they are thought to be involved in mechanosensing. Together with their strong induction during cardiac disease and the identification of causative Ankrd1 gene mutations in cardiomyopathy patients, this suggests their important roles in cardiac development, function, and disease. To determine the functional role of MARPs in vivo, we studied knockout (KO) mice of each of the three family members. Single KO mice were viable and had no apparent cardiac phenotype. We therefore hypothesized that the three highly homologous MARP proteins may have redundant functions in the heart and studied double and triple MARP KO mice. Unexpectedly, MARP triple KO mice were viable and had normal cardiac function both at basal levels and in response to mechanical pressure overload induced by transverse aortic constriction as assessed by echocardiography and hemodynamic studies. Thus, CARP, Ankrd2, and DARP are not essential for normal cardiac development and function at basal conditions and in response to mechanical pressure overload

Nebulette knockout mice have normal cardiac function, but show Z-line widening and up-regulation of cardiac stress markers

Aims: Nebulette is a 109 kDa modular protein localized in the sarcomeric Z-line of the heart. In vitro studies have suggested a role of nebulette in stabilizing the thin filament, and missense mutations in the nebulette gene were recently shown to be causative for dilated cardiomyopathy and endocardial fibroelastosis in human and mice. However, the role of nebulette in vivo has remained elusive. To provide insights into the function of nebulette in vivo, we generated and studied nebulette-deficient (nebl-/-) mice. Methods and results: Nebl-/- mice were generated by replacement of exon 1 by Cre under the control of the endogenous nebulette promoter, allowing for lineage analysis using the ROSA26 Cre reporter strain. This revealed specific expression of nebulette in the heart, consistent with in situ hybridization results. Nebl-/- mice exhibited normal cardiac function both under basal conditions and in response to transaortic constriction as assessed by echocardiography and haemodynamic analyses. Furthermore, histological, IF, and western blot analysis showed no cardiac abnormalities in nebl-/- mice up to 8 months of age. In contrast, transmission electron microscopy showed Z-line widening starting from 5 months of age, suggesting that nebulette is important for the integrity of the Z-line. Furthermore, up-regulation of cardiac stress responsive genes suggests the presence of chronic cardiac stress in nebl-/- mice. Conclusion: Nebulette is dispensable for normal cardiac function, although Z-line widening and up-regulation of cardiac stress markers were found in nebl-/- heart. These results suggest that the nebulette disease causing mutations have dominant gain-of-function effects

Aortic pathology from protein kinase G activation is prevented by an antioxidant vitamin B12 analog.

People heterozygous for an activating mutation in protein kinase G1 (PRKG1, p.Arg177Gln) develop thoracic aortic aneurysms and dissections (TAAD) as young adults. Here we report that mice heterozygous for the mutation have a three-fold increase in basal protein kinase G (PKG) activity, and develop age-dependent aortic dilation. Prkg1R177Q/+ aortas show increased smooth muscle cell apoptosis, elastin fiber breaks, and oxidative stress compared to aortas from wild type littermates. Transverse aortic constriction (TAC)-to increase wall stress in the ascending aorta-induces severe aortic pathology and mortality from aortic rupture in young mutant mice. The free radical-neutralizing vitamin B12-analog cobinamide completely prevents age-related aortic wall degeneration, and the unrelated anti-oxidant N-acetylcysteine ameliorates TAC-induced pathology. Thus, increased basal PKG activity induces oxidative stress in the aorta, raising concern about the widespread clinical use of PKG-activating drugs. Cobinamide could be a treatment for aortic aneurysms where oxidative stress contributes to the disease, including Marfan syndrome

The Luminosities of Protostars in the Spitzer c2d and Gould Belt Legacy Clouds

Motivated by the long-standing "luminosity problem" in low-mass star formation whereby protostars are underluminous compared to theoretical expectations, we identify 230 protostars in 18 molecular clouds observed by two Spitzer Space Telescope Legacy surveys of nearby star-forming regions. We compile complete spectral energy distributions, calculate Lbol for each source, and study the protostellar luminosity distribution. This distribution extends over three orders of magnitude, from 0.01 Lsun - 69 Lsun, and has a mean and median of 4.3 Lsun and 1.3 Lsun, respectively. The distributions are very similar for Class 0 and Class I sources except for an excess of low luminosity (Lbol < 0.5 Lsun) Class I sources compared to Class 0. 100 out of the 230 protostars (43%) lack any available data in the far-infrared and submillimeter (70 um < wavelength < 850 um) and have Lbol underestimated by factors of 2.5 on average, and up to factors of 8-10 in extreme cases. Correcting these underestimates for each source individually once additional data becomes available will likely increase both the mean and median of the sample by 35% - 40%. We discuss and compare our results to several recent theoretical studies of protostellar luminosities and show that our new results do not invalidate the conclusions of any of these studies. As these studies demonstrate that there is more than one plausible accretion scenario that can match observations, future attention is clearly needed. The better statistics provided by our increased dataset should aid such future work.Comment: Accepted for publication in AJ. 21 pages, 10 figures, 4 table