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Nonenzymatic glycation of collagen has long been associated with the progressive secondary complications of diabetes. How exactly such random glycations result in impaired tissues is still poorly understood. Because of the slow turnover rate of most fibrillar collagens, they are more susceptible to accumulate time-dependent glycations and subsequent advanced glycation end-products. The latter are believed to include cross-links that stiffen host tissues. However, diabetic animal models have also displayed weakened tendons with reduced stiffness. Strikingly, not a single experimentally identified specific molecular site of glycation in a collagen has been reported. Here, using targeted MS, we have identified partial fructosyl-hydroxylysine glycations at each of the helical domain cross-linking sites of type I collagen that are elevated in tissues from a diabetic mouse model. Glycation was not found at any other collagen lysine residues. Type I collagen in mouse tendons is cross-linked intermolecularly by acid-labile aldimine bonds formed by the addition of telopeptide lysine aldehydes to hydroxylysine residues at positions α1(I)Lys87, α1(I)Lys930, α2(I)Lys87, and α2(I)Lys933 of the triple helix. Our data reveal that site-specific glycations of these specific lysines may significantly impair normal lysyl oxidase-controlled cross-linking in diabetic tendons. We propose that such N-linked glycations can hinder the normal crosslinking process, thus altering the content and/or placement of mature cross-links with the potential to modify tissue material properties. [ABSTRACT FROM AUTHOR]

Tandem mass spectrometry was applied to tissues from targeted mutant mouse models to explore the collagen substrate specificities of individual members of the prolyl 3-hydroxylase (P3H) gene family. Previous studies revealed that P3h1 preferentially 3-hydroxylates proline at a single site in collagen type I chains, whereas P3h2 is responsible for 3-hydroxylating multiple proline sites in collagen types I, II, IV, and V. In screening for collagen substrate sites for the remaining members of the vertebrate P3H family, P3h3 and Sc65 knock-out mice revealed a common lysine under-hydroxylation effect at helical domain cross-linking sites in skin, bone, tendon, aorta, and cornea. No effect on prolyl 3-hydroxylation was evident on screening the spectrum of known 3-hydroxyproline sites from all major tissue collagen types. However, collagen type I extracted from both Sc65-/- and P3h3-/- skin revealed the same abnormal chain pattern on SDS-PAGE with an overabundance of a γ112 crosslinked trimer. The latter proved to be from native molecules that had intramolecular aldol cross-links at each end. The lysine under-hydroxylation was shown to alter the divalent aldimine cross-link chemistry of mutant skin collagen. Furthermore, the ratio of mature HP/LP cross-links in bone of both P3h3-/- and Sc65-/- mice was reversed compared with wild type, consistent with the level of lysine under-hydroxylation seen in individual chains at cross-linking sites. The effect on cross-linking lysines was quantitatively very similar to that previously observed in EDS VIA human and Plod1-/- mouse tissues, suggesting that P3H3 and/or SC65 mutations may cause as yet undefined EDS variants. [ABSTRACT FROM AUTHOR]

The purpose of this study was to compare the rotational profile of adults with higher versus lower foot progression angles (FPA). Tibial torsion, femoral torsion, hip internal and external rotation and FPA data were collected on one hundred two healthy volunteers. Data were stratified into Low, Mid, and High FPA groups to study relationships among the components of the rotational profile and if there were group differences in the magnitude of torsion angles. Direction of torsion was also studied as an attempt to look at the incidence of offsetting between the tibia and femur. Tibial torsion was significantly higher in the High FPA group (p<0.001) and was significantly related to the FPA (p≤0.01). Femoral torsion was related to hip internal rotation (p<0.05). Offsetting tibial and femoral torsions occurred in 59% of subjects with low FPA and 56% of subjects with high FPA. All subjects in the low FPA group with offsetting had tibial torsion that followed the FPA and the femurs that were directed opposite. In the High FPA group, 68% of subjects with offsetting had tibias that followed the FPA. Stratifying the data by FPA revealed there were significant differences in tibial torsion among the groups and provided evidence that tibial torsion influences the direction and magnitude of the FPA. Offsetting torsions between the tibia and femur were more common in people with higher and lower FPA and had clearer patterns where the tibia tended to follow the direction of the FPA. [ABSTRACT FROM AUTHOR]

Myopia, the leading cause of visual impairment worldwide, results from an increase in the axial length of the eyeball. Mutations in LEPREL1, the gene encoding prolyl 3-hydroxylase-2 (P3H2), have recently been identified in individuals with recessively inherited nonsyndromic severe myopia. P3H2 is a member of a family of genes that includes three isoenzymes of prolyl 3-hydroxylase (P3H), P3H1, P3H2, and P3H3. Fundamentally, it is understood that P3H1 is responsible for converting proline to 3-hydroxyproline. This limited additional knowledge also suggests that each isoenzyme has evolved different collagen sequence-preferred substrate specificities. In this study, differences in prolyl 3-hydroxylation were screened in eye tissues from P3h2-null (P3h2n/n) and wild-type mice to seek tissue-specific effects due the lack of P3H2 activity on post-translational collagen chemistry that could explain myopia. The mice were viable and had no gross musculoskeletal phenotypes. Tissues from scleraandcornea (type I collagen) and lens capsule (type IV collagen) were dissected from mouse eyes, and multiple sites of prolyl 3-hydroxylation were identified by mass spectrometry. The level of prolyl 3-hydroxylation at multiple substrate sites from type I collagen chains was high in sclera, similar to tendon. Almost every known site of prolyl 3-hydroxylation in types I and IV collagen from P3h2n/n mouse eye tissues was significantly under-hydroxylated compared with their wild-type littermates. We conclude that altered collagen prolyl 3-hydroxylation is caused by loss of P3H2. We hypothesize that this leads to structural abnormalities in multiple eye tissues, but particularly sclera, causing progressive myopia. [ABSTRACT FROM AUTHOR]

Collagen triple helices are stabilized by 4-hydroxyproline residues. No function is known for the much less common 3-hydroxyproline (3Hyp), although genetic defects inhibiting its formation cause recessive osteogenesis imperfecta. To help understand the pathogenesis, we used mass spectrometry to identify the sites and local sequence motifs of 3Hyp residues in fibril-forming collagens from normal human and bovine tissues. The results confirm a single, essentially fully occupied 3Hyp site (A1) at Pro986 in A-clade chains α1(I), α1(II), and α2(V). Two partially modified sites (A2 and A3) were found at Pro944 in α1(II) and α2(V) and Pro707 in α2(I) and α2(V), which differed from A1 in sequence motif. Significantly, the distance between sites 2 and 3, 237 residues, is close to the collagen D-period (234 residues). A search for additional D-periodic 3Hyp sites revealed a fourth site (A4) at Pro470 in α2(V), 237 residues N-terminal to site 3. In contrast, human and bovine type III collagen contained no 3Hyp at any site, despite a candidate proline residue and recognizable A1 sequence motif. A conserved histidine in mammalian a1(III) at A1 may have prevented 3-hydroxylation because this site in chicken type III was fully hydroxylated, and tyrosine replaced histidine. All three B-clade type V/XI collagen chains revealed the same three sites of 3Hyp but at different loci and sequence contexts from those in A-clade collagen chains. Two of these B-clade sites were spaced apart by 231 residues. From these and other observations we propose a fundamental role for 3Hyp residues in the ordered self-assembly of collagen supramolecular structures. [ABSTRACT FROM AUTHOR]

In biological systems, iron exerts 2 contrasting effects. The chemical reactivity of iron is essential for the biological activities of proteins such as hemoglobin, ribonucleotide reductase, the cytochromes, and aconitases. However, free iron in a cell has the propensity to generate free radicals which can damage cellular components containing proteins, lipids, and nucleic acids. To maintain the balance between iron as an essential nutrient and iron as a potential cytotoxin, a number of biological protective mechanisms have evolved. As shown in the thalassemias, iron imbalance can have devastating effects on human health. Recently, several new proteins have been described that play critical roles in iron regulation including the master regulator of iron metabolism (hepcidin). In this review, we discuss the new knowledge that has arisen from studies in yeast and in humans, and we show how these studies are shedding new light on some well-known human disorders. [Copyright &y& Elsevier]

In the normal human prostate, undifferentiated proliferative cells reside in the basal layer and give rise to luminal secretory cells. There are, however, few epithelial cell lines that have a basal cell phenotype and are able to differentiate. We set out to develop a cell line with these characteristics that would be suitable for the study of the early stages of prostate epithelial cell differentiation. We produced a matched pair of conditionally immortalized prostate epithelial and stromal cell lines derived from the same patient. The growth of these cells is temperature dependent and differentiation can be induced following a rise in culture temperature. Three-dimensional co-cultures of these cell lines elicited gland-like structures reminiscent of prostatic acini. cDNA microarray analysis of the epithelial line demonstrated changes in gene expression consistent with epithelial differentiation. These genes may prove useful as markers for different prostate cell types. The cell lines provide a model system with which to study the process of prostatic epithelial differentiation and stromal–epithelial interactions. This may prove to be useful in the development of differentiation-targeted prostate cancer therapies. [ABSTRACT FROM AUTHOR]

Because of its unique physical and chemical properties, rat tail tendon collagen has long been favored for crystallographic and biochemical studies of fibril structure. In studies of the distribution of 3-hydroxyproline in type I collagen of rat bone, skin, and tail tendon by mass spectrometry, the repeating sequences of Gly-Pro-Pro (GPP) triplets at the C terminus of α1(I) and α2(I) chains were shown to be heavily 3-hydroxylated in tendon but not in skin and bone. By isolating the tryptic peptides and subjecting them to Edman sequence analysis, the presence of repeating 3-hydroxyprolines in consecutive GPP triplets adjacent to 4-hydroxyproline was confirmed as a unique feature of the tendon collagen. A 1960s study by Piez et al. (Piez, K. A., Eigner, E. A., and Lewis, M. S. (1963) Biochemistry 2, 58-66) in which they compared the amino acid compositions of rat skin and tail tendon type I collagen chains indeed showed 3-4 residues of 3Hyp in tendon α1(I) and α2(I) chains but only one 3Hyp residue in skin α1(I) and none in α2(I). The present work therefore confirms this difference and localizes the additional 3Hyp to the GPP repeat at the C terminus of the triple-helix. We speculate on the significance in terms of a potential function in contributing to the unique assembly mechanism and molecular packing in tendon collagen fibrils and on mechanisms that could regulate 3-hydroxylation at this novel substrate site in a tissue-specific manner. [ABSTRACT FROM AUTHOR]

Abstract: The results of recent research indicate that agonist replacement may be a viable option in the treatment of cocaine dependence. For example, d-amphetamine and modafinil have shown promise in managing cocaine dependence in preliminary clinical trials. The aim of this study was to determine the physiological and subject-rated effects of acute intranasal cocaine doses during chronic atomoxetine treatment. Atomoxetine was chosen because it produces pharmacological and subject-rated effects similar to those of prototypical stimulants and thus may also be a viable agonist replacement therapy. To this end, seven cocaine-dependent subjects were maintained on doses of atomoxetine (0mg [lead in], 5, 10, 20 and 0mg [washout], four times daily) for 3–5 days prior to completing experimental sessions in which ascending doses of intranasal cocaine (4, 20, 40 and 60mg) were administered. Cocaine produced prototypical cardiovascular and subject-rated effects. Atomoxetine attenuated the systolic pressure increasing effects and enhanced the heart rate increasing effects of cocaine, but was otherwise devoid of effects. These results indicate that cocaine is well tolerated during atomoxetine maintenance. Further research is needed to better determine the effects of atomoxetine and cocaine combinations. [Copyright &y& Elsevier]

• Nonpregnant and pregnant compound-null mice deficient in class i (decorin and biglycan) and class II (lumican) SLRPs demonstrate inappropriate assembly of collagen fibrils and elastic fibers in the cervix. • Decorin and biglycan influence the uniformity of collagen fibril structure and spacing and their loss impacts the mechanical properties of the cervix during pregnancy. • Formation of mature hydroxylysylpyridinoline (HP) collagen cross-links is independent of decorin and biglycan interactions in the cervical ECM. • Synergistic regulatory functions of class I and class II SLRPs ensure ECM homeostasis through all stages of cervical remodeling. The cervix undergoes rapid and dramatic shifts in collagen and elastic fiber structure to achieve its disparate physiological roles of competence during pregnancy and compliance during birth. An understanding of the structure-function relationships of collagen and elastic fibers to maintain extracellular matrix (ECM) homeostasis requires an understanding of the mechanisms executed by non-structural ECM molecules. Small-leucine rich proteoglycans (SLRPs) play key functions in biology by affecting collagen fibrillogenesis and regulating enzyme and growth factor bioactivities. In the current study, we evaluated collagen and elastic fiber structure-function relationships in mouse cervices using mice with genetic ablation of decorin and/or biglycan genes as representative of Class I SLRPs, and lumican gene representative of Class II SLRP. We identified structural defects in collagen fibril and elastic fiber organization in nonpregnant mice lacking decorin, or biglycan or lumican with variable resolution of defects noted during pregnancy. The severity of collagen and elastic fiber defects was greater in nonpregnant mice lacking both decorin and biglycan and defects were maintained throughout pregnancy. Loss of biglycan alone reduced tissue extensibility in nonpregnant mice while loss of both decorin and biglycan manifested in decreased rupture stretch in late pregnancy. Collagen cross-link density was similar in the Class I SLRP null mice as compared to wild-type nonpregnant and pregnant controls. A broader range in collagen fibril diameter along with an increase in mean fibril spacing was observed in the mutant mice compared to wild-type controls. Collectively, these findings uncover functional redundancy and hierarchical roles of Class I and Class II SLRPs as key regulators of cervical ECM remodeling in pregnancy. These results expand our understating of the critical role SLRPs play to maintain ECM homeostasis in the cervix. [ABSTRACT FROM AUTHOR]

Abstract: Auditory response profiles for a group of ten healthy young and ten healthy elderly subjects, evoked by implicit memory and delayed verbal recognition tasks, were evaluated to determine if effects of stimulus repetition could be identified in the superior temporal gyrus (STG) and prefrontal cortical regions. We hypothesized that effects of stimulus repetition should occur both early in time and at early levels of the nervous system (STG) followed by later effects in prefrontal regions. Magnetoencephalographic (MEG) responses were recorded using a whole-head MEG system and automated, multi-start analysis methods were applied to the data in order to characterize the temporal response profiles from distributed but focal, cortical regions engaged in memory-related tasks. The findings revealed a main effect of age for early activity (∼50 ms) in STG which appeared to be nonspecific for Old/New words and an Age × Task interaction for late activity (∼100–800 ms) in STG which was specific to Old/New words. Although the behavioral performance measures did not reveal traditional effects of response priming, the MEG measures did reveal a reduction in amplitude with stimulus repetition in young subjects. The elderly did not reveal a reduction in amplitude concomitant with stimulus repetition for either the global attributes of words or for specific Old/New words. Long duration effects of stimulus repetition noted in the present study raise the possibility that results from sensory gating, mismatch negativity and P300 paradigms may represent a continuum of stimulus repetition effects. Two of these paradigms evoke greater enhancement to novel or infrequent stimuli, or rather, greater reduction of amplitude with repetition. [Copyright &y& Elsevier]

Objective: The role of the ipsilateral cortex in proximal muscle control in normal human subjects is still under debate. One clinical finding, rapid recovery of proximal muscle relative to distal muscle use following stroke, has led to the suggestion that the ipsilateral as well as the contralateral motor cortex may be involved in normal proximal muscle control. The primary goal of this project was to identify contralateral and ipsilateral motor cortex activation associated with proximal muscle movement in normal subjects using magnetoencephalography (MEG).Methods: We developed protocols for a self-paced bicep motor task and a deltoid, electrical-stimulation somatosensory task. The MEG data were analyzed using automated multi-dipole spatiotemporal modeling techniques to localize the sources and characterize the associated timing of these sources.Results: Reliable contralateral primary motor and somatosensory sources localized to areas consistent with the homunculus. Ipsilateral M1 activation was only found in 2/12 hemispheres.Conclusions: Robust contralateral motor cortex activation and sparse ipsilateral motor cortex activation suggest that the ipsilateral motor cortex is not involved in normal proximal muscle control.Significance: The results suggest that proximal and distal muscle control is similar in normal subjects in the sense that proximal muscle control is primarily governed by the contralateral motor cortex. [Copyright &y& Elsevier]

Receptors of the integrin family are largely confined to the basal layer of keratinocytes, both in human epidermis and in stratified cultures of human keratinocytes. However, suprabasal integrin expression is observed during epidermal wound healing and in psoriatic lesions. We have investigated potential stimuli of suprabasal expression. Addition of transforming growth factor-β (TGF-β), interferon-γ (IFN-γ or tumor necrosis factor-α (TNF-α) to keratinocytes cultured with a 3T3 feeder layer did not induce suprabasal expression. The cytokines caused small changes in the levels of α2β1 or α3β1 on the surface of basal keratinocytes but had no significant effect on the proportion of cells adhering to fibronectin, type IV collagen, and laminin, and did not cause changes in the mobility of integrin subunits on polyacrylamide gels. Injection of TNF-α or IFN-γ intradermally into healthy human volunteers induced an inflammatory response but did not induce suprabasal integrin expression. However, we did observe transient suprabasal Integrin expression when keratinocytes were grown on a dermal equivalent consisting of fibroblasts in a collagen gel. One week after raising the cultures to the air-liquid interface, β1 integrins were found in all the viable cell layers, with suprabasal cells co-expressing integrins and involucrin; 1 week later integrins were confined to the basal layer. Addition of TGF-β, IFN-γ, or TNF-α to the dermal equivalents neither induced nor inhibited suprabasal integrin expression. We conclude that suprabasal integrin expression is not induced by the inflammatory cytokines tested, and instead may reflect the proliferation/differentiation status of the epidermis. [ABSTRACT FROM AUTHOR]