Effects of aging and xerosis on the amino acid composition of human skin.

Amino acid compositions of skin samples from young and old subjects and from age-matched donors with dry skin syndrome (xerosis) were examined. The amino acid contents of the free amino acid (FAA) fraction, soluble hydrolysate (SH) fraction, and whole cell hydrolysate (WCH) were determined. The greatest differences were observed between the FAA compositions of the young and old normal subjects. Xerosis did not appear to affect the amino acid compositions of samples from young subjects as much as old subjects. Overall, the effect of aging on the amino acid contents was more pronounced than the effect of xerosis. The amino acid composition of the FAA showed a high degree of similarity to filaggrin, whereas the WCH showed a similarity to keratin.

Transgenic mouse model of the mild dominant form of osteogenesis imperfecta.

Osteogenesis imperfecta type I is a mild, dominantly inherited, connective tissue disorder characterized by bone fragility. Mutations in type I collagen account for all known cases. In Mov-13 mice, integration of a murine retrovirus within the first intron of the alpha 1(I) collagen gene results in a null allele blocked at the level of transcription. This study demonstrates that mutant mice heterozygous for the null allele are a model of osteogenesis imperfecta type I. A defect in type I collagen production is associated with dominant-acting morphological and functional defects in mineralized and nonmineralized connective tissue and with progressive hearing loss. The model provides an opportunity to investigate the effect of a reduced amount of type I collagen on the structure and integrity of extracellular matrix. It also may represent a system in which therapeutic strategies to strengthen connective tissue can be developed.

Chondroitin proteoglycans from squid skin. Isolation, characterization and immunological studies.

Two populations of proteochondroitins were isolated from 4 M guanidine hydrochloride extracts of squid skin by a combination of ion exchange, gel chromatography and density gradient centrifugation. The proteoglycans, Mr 4.8 x 10(5) and 2.8 x 10(5), contained four and two chondroitin chains respectively and unusual oligosaccharides with uronic acid and sulphate groups, and had different amino acid and neutral sugar composition. The chondroitin chains isolated after alkaline borohydride treatment contained varying amounts of glucose, galactose, mannose, fucose and xylose, most likely as branches. Both proteoglycans were antigenic to the rabbit and showed considerable cross-reactivity as assessed by competition experiments using the ELISA technique. The proteoglycans reacted neither with exogenous hyaluronic acid nor with each other to form aggregates.

Cutaneous interstitial fluid protein concentrations in the inflammatory syndrome: pharmacological consequences.

Concentrations of alpha 1 acid glycoprotein, albumin, transferrin, haptoglobin, immunoglobulins G, A, M and apolipoprotein B were measured in serum and suction blister fluid from a group of individuals presenting a biologically proven inflammatory syndrome, and from a control group. Protein values in suction blister fluid did not change from the 2nd to the 3rd h after the beginning of blister formation. The ratio of the concentration of proteins in blister fluid and serum did not differ significantly between the groups. However, a 25% decrease in blister fluid albumin and a 100% increase in blister fluid alpha 1 acid glycoprotein, recorded in the inflammatory group, were worth noting, since they possibly influence the tissular distribution of some protein-binding drugs. Finally, an inverse relationship was established between the blister fluid/serum concentration ratio and the respective molar mass of each protein.

Dermal vascular IgA deposits in IgA nephropathy secondary to alcohol abuse.

Skin, kidney and liver samples were investigated from 103 people with evidence of alcohol abuse at forensic autopsy. The diagnosis of alcohol abuse was based on clinical history, post mortem blood alcohol level and liver pathology. The findings confirmed the frequent widespread vascular IgA deposition in individuals with evidence of alcohol abuse. The frequency of IgA deposition in superficial small vessels of the dermis was not significantly different between individuals with IgA nephritis (16/85) and without such pathology (7/38). Based on these results, it can be inferred that had a pre-mortem skin biopsy been performed, it would not have had clinical usefulness in predicting the renal disease.

A critical crosslink region in human-bone-derived collagen type I. Specific cleavage site at residue Leu95.

Collagen was extracted from human adult bone by limited pepsin digestion and collagen types were purified by consecutive salt precipitation first under neutral and then under acid conditions. In SDS/PAGE, all collagen type I preparations showed a protein band [alpha 1s(I)] migrating between alpha 1(I) and alpha 2(I) as well as a band [alpha 2s(I)] migrating in front of alpha 2(I). The collagenous nature of the pepsin-stable alpha 1s(I) protein was clearly demonstrated by digestion with human-leucocyte-derived collagenase, immunoblotting with antibodies against collagen type I and amino acid analysis. Partial amino acid sequencing of alpha 1(I) and alpha 1s(I) identified alpha 1s(I) as a shortened alpha 1(I) chain due to a specific cleavage site between residues Leu95 and Asp96 which is in close vicinity to the hydroxylysine-derived crosslink at position 87. In circular dichroism, the proportion of thermally labile collagen molecules was proportional to the amount of shortened alpha 1(I) and alpha 2(I) chains, respectively. The melting temperature was found to be 36 +/- 0.5 degrees C as judged from circular dichroism and susceptibility to proteolysis. Our data provide clear evidence that a shortened alpha 1-derived collagen chain can be extracted from human adult bone whereas it is hardly found in human skin. The unique cleavage site might provide important information about the collagen I molecule embedded in the calcified matrix of human bone.

Essential fatty acid nutrition of the American alligator (Alligator mississippiensis).

The essential fatty acid (EFA) nutrition of young American alligators (Alligator mississippiensis) was examined by feeding a variety of fats/oils with potential EFA activity. Over a 12-wk period, alligators fed diets containing 2.5 or 5.0% chicken liver oil grew longer and heavier and converted feed to body mass more efficiently than alligators fed other fat/oil combinations that lacked or contained only trace amounts of arachidonic acid [20:4(n-6)]. Alligators fed an EFA-deficient diet (containing only coconut fat as the dietary fat) were the slowest-growing animals and converted feed to body mass least efficiently. However, over a 41-wk feeding period, alligators fed this diet showed no obvious external signs of deficiency other than being reduced in size and unthrifty. Fatty acid composition of heart, liver, muscle, skin and adipose tissue lipids was influenced markedly by dietary fat composition. Tissues varied significantly in response to dietary fat composition. Heart lipids contained the lowest levels of short- and medium-chain fatty acids and the highest levels of arachidonic acid. Arachidonic acid levels were less influenced by diet than were levels of other 20- and 22-carbon polyunsaturated fatty acids. Radiotracer studies indicated that linoleic acid was converted to arachidonic acid in the liver. Nevertheless, tissue arachidonic acid levels also appeared to be maintained by concentration from dietary sources and selective conservation. It appears that a dietary source of arachidonic acid may be required for a maximum rate of growth.

In situ detection of basic fibroblast growth factor by highly specific antibodies.

Basic fibroblast growth factor (bFGF) is thought to be of major importance for fibrosis and angiogenesis. Despite intensive studies dealing with the biochemistry and multiple biologic effects of bFGF, the cellular distribution is virtually unknown. Therefore, using the indirect immunoperoxidase technique, we examined the effect of bFGF on a large pattern of normal, inflammatory, and tumorous human tissues. Staining was performed on cryostat sections with a highly specific affinity-purified antiserum. In normal tissues, especially those of the thymus and placenta, mainly dendritic cells contained the growth factor. High levels of bFGF were also detected in basal cells and gland epithelial cells of skin biopsies. A conspicuous expression was observed in chronic inflammatory tissues corresponding to a generally pronounced proliferation of fibroblasts and endothelial cells in these situations. Tumors revealed a very heterogenous staining pattern. In some lesions, bFGF was predominantly present in infiltrating and endothelial cells. In several, neoplasms tumor cells exhibited an intensive staining. In some, especially vascular tumors, bFGF could not be detected. From the staining results it is concluded that angiogenesis is not simply controlled by the presence of bFGF but is mediated by a balance of several angiogenic inducers and inhibitors.

[Biological features of the skin of the pika, Ochotona rufescens rufescens--concentration of histamine in the skin].

The quantity of histamine and the number of mast cells in the skin of the pika were measured and compared with rabbits, guinea pigs and rats. The ranking of regional histamine levels in the skin of the pika was: perianal region greater than abdomen greater than interscapular region = back greater than lumbus greater than head greater than auricle, and the average value of the 7 regions was 22.6 micrograms/g. The level of histamine in the 6 regions, except the auricle, was 2-5 times that of rabbits and guinea pigs. In the auricle of each of the 4 kinds of animal (pika, rabbit, guinea pig and rat), the levels were almost identical. With respect to histamine levels, those in the pika resembled those in rats. The number of mast cells in the skin of the pika was less than in rats, and was greater than that in rabbits and guinea pigs. The average value was 9.9/mm2.

Increased glycation and pigmentation of collagen in aged and young parabiotic rats and mice.

Changes of non-enzymatic collagen glycosylation were followed in 2- and 24-month-old rats and mice and in parabiotic animals of the same age. With advancing age increased glycation of collagen was observed in both old male Wistar rats and white mice. Further it was demonstrated that both aortal and skin collagen of young animals is rapidly non-enzymatically glycosylated in the common milieu created by parabiotic animals and the proportion of non-enzymatically incorporated glucose approaches in the young counterparts the level found in old individuals. Similar trends as with non-enzymatic glycosylation were found with a fluorescent (370/440 nm) product present in both categories of collagen preparations. This fluorescence was higher in old animals and was considerably increased in the young counterpart of the parabiotic couple 6 weeks after operation. The nature of the fluorescent product appears different from pyridinoline and remains to be elucidated.

A method for the sequence analysis of dermatan sulphate.

We are attempting to develop methods for the sequencing of glycosaminoglycans from their reducing end. Here we describe a procedure for the analysis of dermatan sulphate from pig skin. The glycosaminoglycan is released from its parent proteoglycan by exhaustive proteolysis by using both endo- and exo-peptidases. The amino group of the residual serine residue is conjugated with a p-hydroxyphenyl group, which in turn is iodinated with 125I (the Bolton-Hunter reagent, BHR). The ion-exchange-purified end-labelled dermatan sulphate is then degraded partially or completely by various enzymic or chemical means to yield fragments extending from the labelled serine residue to the point of cleavage. The various products are separated by gradient PAGE, detected by autoradiography and quantified by videodensitometry. Complete digestion with chondroitin ABC lyase affords the labelled fragment delta HexA-GalNAc(-SO4)-GlcA-Gal-Gal-Xyl-Ser(-BHR). The structure was confirmed by sequential degradation from the non-reducing end by chondroitin AC lyase, HgCl2, and beta-galactosidase. Periodate oxidation cleaves most of the Xyl even without treatment with alkaline phosphatase, showing that Xyl is not substituted with phosphate. Results from partial and selective periodate oxidation indicate that most of the non-sulphated IdoA residues are located towards the non-reducing end. Partial or complete digestions with testicular hyaluronidase (in the presence of an excess of beta-glucuronidase) or chondroitin AC lyase identify the positions of GlcA residues. The results confirm that HexA next to Gal is always GlcA. Moreover, GlcA is common in the first three disaccharide repeats. Results with testicular hyaluronidase indicate that the distribution of clustered GlcA-GalNAc repeats is periodic and peaks at positions 1-3, 8-9 and around 25. Although there must be chains that contain IdoA in nearly all of the available positions, regions that have not been fully processed during biosynthesis are markedly non-random.

Association of EGF receptor expression with proliferating cells and of ras p21 expression with differentiating cells in various skin tumours.

The localization of DNA replicating cells, epidermal growth factor (EGF) receptor-expressing cells and ras oncogene product p21 (p-21ras) positive cells were examined in various skin tumours to elucidate the role of EGF receptor and p21ras in the epidermis. Normal skin, keratoacanthoma (KA), solar keratosis (SK), Bowen's disease (BD), squamous cell carcinoma (SCC), basal cell carcinoma (BCC) and extramammary Paget's disease (PD) were studied. EGF receptors were seen in proliferating layers, where DNA replicating cells localize, but p21ras was found in the more differentiated layers. We conclude that EGF receptor expression is closely associated with cellular proliferation, but p21ras may play a role in the differentiation of cells in various skin tumours.

Properties and nature of a cysteine proteinase inhibitor located in keratohyalin granules of rat epidermis.

The pI 4.7, 14.5 kDa hematoxylin-stainable protein (HSP) from rat epidermis inhibited the activities of the cysteine proteinases papain, ficin, cathepsins B, H and L with similar inhibitory characteristics as recombinant cystatin-alpha. Proteinases of other classes were not inhibited. The inhibitory activity of HSP was heat stable in the wide pH range of 3.0-10.0. Polyclonal antibodies against HSP cross-reacted with cystatin-alpha and the molecular mass of HSP was similar to that of cystatin-alpha, though its isoelectric point was different. The in vivo location of both HSP and cystatin-alpha is on keratohyalin granules in epidermis as detected by indirect immunofluorescence technique using individual antibodies. Therefore it is highly probable that HSP is a cystatin-alpha derivative or a very similar proteinase inhibitor belonging to a family of cystatins.

Immunohistologic abnormalities of the microfibrillar-fiber system in the Marfan syndrome.

BACKGROUND: Indirect-immunofluorescence studies of skin and cultured dermal fibroblasts from patients with the Marfan syndrome demonstrate apparent deficiency of one element of connective tissue--the microfibrillar-fiber system--in assays using specific antibodies against fibrillin, a major microfibrillar protein. This study was designed to test whether these immunostaining abnormalities are consistent and diagnostic features of the disease. METHODS: We studied patients with either the Marfan syndrome or various other inherited connective-tissue disorders and normal subjects according to a single-blind protocol in which coded samples of skin, fibroblast cultures, or both were analyzed without knowledge of the clinical diagnosis and classified as "Marfan" or "non-Marfan" before the sample codes were broken. RESULTS: Of the 27 patients with the Marfan syndrome, 24 were correctly identified by the decreased content of microfibrillar fibers in their skin, cultured fibroblasts, or both; in contrast, 19 of 25 patients with other heritable disorders of connective tissue and all 13 normal subjects were correctly classified as "non-Marfan" by these assays (P less than 0.001). CONCLUSIONS: These results document consistent, relatively specific abnormalities of microfibrillar fibers in the Marfan syndrome. The biomechanical incompetence of these structural elements, due to quantitative or qualitative abnormalities, may account for the pleiotropic clinical manifestations of the disease. Therefore, various defects in the expression, structure, assembly, or degradation of the constituent structural glycoprotein (or glycoproteins) of microfibrils may be implicated in the causation of the Marfan syndrome.

In the absence of a downstream element, the apolipoprotein E gene is expressed at high levels in kidneys of transgenic mice.

Human apolipoprotein (apo) E gene constructs with 30 or 5 kilobases of 5'-flanking and 1.5 kilobases of 3'-flanking regions were used to create transgenic mice. High levels of human apoE mRNA were present in the transgenic kidney, but none was detected in the liver, which is normally the major source of apoE. When a construct with 5 kilobases of 5'- and 23 kilobases of 3'-flanking regions was used, only trace levels of human apoE mRNA were detected in the kidney, whereas high levels were found in the liver. These results indicated that regulatory elements downstream of the human apoE gene interacted with the transcription initiation complex to stimulate gene expression in the liver while suppressing expression in the kidney. In each case, human apoE was secreted into the plasma. The source of human apoE in the transgenic kidney was the epithelial cells lining the proximal tubule and Bowman's capsule.

Neuropeptides in skin from patients with atopic dermatitis: an immunohistochemical study.

The distribution and localization of several neuropeptides were investigated in the lichenified lesions of 11 patients with atopic dermatitis using indirect immunofluorescence. Substance P-positive nerve fibres were observed in most of the cases of atopic dermatitis, but not in normal controls. Somatostatin immunoreactive nerves were not found in the skin of atopic dermatitis, whereas a normal pattern of immunoreactivity could be detected in most of the healthy subjects. Neuropeptide Y-positive dendritic epidermal cells were observed in lesional skin from patients with atopic dermatitis, but not in controls. Calcitonin gene-related peptide and vasoactive intestinal polypeptide immunoreactivity in patients with atopic dermatitis did not differ from that in healthy subjects. With galanin antiserum a diffuse intracellular staining was observed in the epidermis of both atopic patients and controls, while no positive staining was found with either neurotensin or neurokinin A antibodies in either group. These findings suggest a possible involvement of some neuropeptides in the pathomechanisms of atopic dermatitis.

Partial characterization of dermatan sulfate by proton nuclear magnetic resonance spectroscopy.

The degree of galactosamine N-acetylation, iduronic acid composition, and total uronic acid/hexosamine ratios of the three dermatan sulfates of human skin, DS18, DS28, and DS35 (M. O. Longas et al. (1987) Carbohydr. Res. 159, 127-136), were determined by Fourier transform, proton nuclear magnetic resonance (FT 1H NMR) spectroscopy. Analysis of DS of varying ages was conducted at 400 MHz and 60 degrees C. Chemical shifts for H-1, H-2, H-4, and H-5 of L-IdUA were independent of those for the respective protons of D-GalNAc and D-GlcUA. The resonance intensities of H-1 and acetamido methyl protons of D-GalNac did not display the expected 1:3 ratios. Therefore, their integration values were employed to estimate the percentage N-acetylation (N-CH3/3 H-1) which was corroborated chemically. The L-IdUA content, relative to total uronic acid, was calculated from signal intensities of H-1 of L-IdUA and D-GlcUA and ascertained by quantitative chemical methods. Total uronic acid/hexosamine ratios were determined from both 1H NMR spectroscopy and chemical analyses. The data show the following N-acetylation (N-CH3/3 H-1) of galactosamine in DS:DS18, 61-72% between 17 and 60 years, unaffected by senescence; DS28, 78-86% with no age-related trend; DS35, 101% at 19 years. Furthermore, in all ages investigated, the percentage (wt/wt) L-IdUA relative to total uronic acid was 42-44% for DS18 and 37-40% for DS28. At age 19 years, DS35 had a 29% (wt/wt) L-IdUA. The total uronic acid/hexosamine ratios for DS18 and DS28 varied from 1.40:1.0 to 1.70:1.0 irrespective of age.

Human type VI collagen: isolation and characterization of alpha 1 and alpha 2 chains by two-dimensional preparative gel electrophoresis.

Two 140 kDa collagenous glycoproteins were isolated from 5 M guanidinium chloride extracts of human uterine leiomyoma by two-dimensional preparative gel electrophoresis. The glycoproteins represented the major concanavalin A binding fraction of the extract and were also present in adult human skin. On two-dimensional gel electrophoresis the glycoproteins appeared as elongated spots, indicating variations of their isoelectric points from 5 to 6. These glycoproteins were disulfide-bonded components of high molecular mass protein and, after reduction, became sensitive to collagenase treatment that generated peptides corresponding in size to those of the noncollagenous domains of type VI collagen. Antisera raised against these purified glycoproteins reacted with either pepsin-derived alpha 1(VI) or pepsin-derived alpha 2(VI) chains but not with alpha 3(VI) chain of human type VI collagen. Reciprocally, these glycoproteins reacted with monoclonal antibodies against type VI collagen. These results indicate that the glycoproteins represent the integral alpha 1 and alpha 2 chains of type VI collagen. The globular domains of alpha 1(VI) and alpha 2(VI) chains remaining after collagenase treatment appeared on two-dimensional gel electrophoresis as elongated spots, suggesting that the noncollagenous portions determine the well known microheterogeneity of the molecule. The differences in isoelectric points between and within alpha chains may facilitate the formation of microfibrillar network.