RESUMO
Human kallikrein-related peptidases (KLKs) are a group of 15 secreted serine proteases encoded by the largest contiguous cluster of protease genes in the human genome. KLKs are involved in coordination of numerous physiological functions including regulation of blood pressure, neuronal plasticity, skin desquamation, and semen liquefaction, and thus represent promising diagnostic and therapeutic targets. Until now, quantification of KLKs in biological and clinical samples was accomplished by enzyme-linked immunosorbent assays (ELISA). Here, we developed multiplex targeted mass spectrometry assays for the simultaneous quantification of all 15 KLKs. Proteotypic peptides for each KLK were carefully selected based on experimental data and multiplexed in single assays. Performance of assays was evaluated using three different mass spectrometry platforms including triple quadrupole, quadrupole-ion trap, and quadrupole-orbitrap instruments. Heavy isotope-labeled synthetic peptides with a quantifying tag were used for absolute quantification of KLKs in sweat, cervico-vaginal fluid, seminal plasma, and blood serum, with limits of detection ranging from 5 to 500 ng/ml. Analytical performance of assays was evaluated by measuring endogenous KLKs in relevant biological fluids, and results were compared with selected ELISAs. The multiplex targeted proteomic assays were demonstrated to be accurate, reproducible, sensitive, and specific alternatives to antibody-based assays. Finally, KLK4, a highly prostate-specific protein and a speculated biomarker of prostate cancer, was unambiguously detected and quantified by immunoenrichment-SRM assay in seminal plasma and blood serum samples from individuals with confirmed prostate cancer and negative biopsy. Mass spectrometry revealed exclusively the presence of a secreted isoform and thus unequivocally resolved earlier disputes about KLK4 identity in seminal plasma. Measurements of KLK4 in either 41 seminal plasma or 58 blood serum samples revealed no statistically significant differences between patients with confirmed prostate cancer and negative biopsy. The presented multiplex targeted proteomic assays are an alternative analytical tool to study the biological and pathological roles of human KLKs.
Assuntos
Calicreínas/análise , Sêmen/enzimologia , Soro/enzimologia , Suor/enzimologia , Adulto , Líquidos Corporais/enzimologia , Feminino , Humanos , Marcação por Isótopo , Calicreínas/química , Masculino , Espectrometria de Massas , Peptídeos/química , Peptídeos/metabolismo , ProteômicaRESUMO
Human prolactin-induced protein (PIP) is a major protein found in exocrine fluids such as saliva and sweat. Intriguingly, PIP possesses residues (human PIP (hPIP): PIP (29-63)) that display similarity to the aspartic peptidase candidapepsin. Here, we aimed to determine the effect of PIP as a protease on normal skin structure. Using an adhesive tape-stripping technique, we applied hPIP peptide on the corneocytes of normal-appearing facial skin from infants with eczema and healthy infants and then analyzed the morphological structure of corneocytes with Nile Red fluorescence. We also repeatedly applied the hPIP peptide onto the surface of a three-dimensional (3-D) human skin model and then analyzed any changes to the stratum corneum and epidermis using light microscopy and scanning electron microscopy. In both infant groups, a decrease in hydrophobic lipids from the cornified envelope was observed after treatment with hPIP. The peptide hPIP appeared to digest the fine structure of the stratum corneum and induce a proliferation of epidermal keratinocytes within the 3-D human skin model. Our results suggest that aspartic peptidase of PIP found in sweat or saliva deteriorates the skin barrier in a de novo manner, which potentially leads directly to the proliferation of epidermal keratinocytes without any external antigenic factors.
Assuntos
Proteínas de Transporte/farmacologia , Proliferação de Células/efeitos dos fármacos , Epiderme/efeitos dos fármacos , Epiderme/patologia , Glicoproteínas/farmacologia , Queratinócitos/patologia , Adulto , Estudos de Casos e Controles , Células Cultivadas , Dermatite Atópica/metabolismo , Dermatite Atópica/patologia , Eczema/metabolismo , Eczema/patologia , Epiderme/metabolismo , Humanos , Lactente , Queratinócitos/metabolismo , Queratinócitos/ultraestrutura , Metabolismo dos Lipídeos , Proteínas de Membrana Transportadoras , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Saliva/enzimologia , Suor/enzimologiaRESUMO
The identification of human body fluids or tissues through mRNA-based profiling is very useful for forensic investigations. Previous studies have shown mRNA biomarkers are effective to identify the origin of biological samples. In this study, we selected 16 tissue specific biomarkers to evaluate their specificities and sensitivities for human body fluids and tissues identification, including porphobilinogen deaminase (PBGD), hemoglobin beta (HBB) and Glycophorin A (GLY) for circulatory blood, protamine 2 (PRM2) and transglutaminase 4 (TGM4) for semen, mucin 4 (MUC4) and human beta defensin 1(HBD1) for vaginal secretion, matrix metalloproteinases 7 and 11 (MMP7 and MMP11) for menstrual blood, keratin 4(KRT4) for oral mucosa, loricrin (LOR) and cystatin 6 (CST6) for skin, histatin 3(HTN3) for saliva, statherin (STATH) for nasal secretion, dermcidin (DCD) for sweat and uromodulin (UMOD) for urine. The above mentioned ten common forensic body fluids or tissues were used in the evaluation. Based on the evaluation, a reverse transcription (RT) PCR multiplex assay, XCYR1, which includes 12 biomarkers (i.e., HBB, GLY, HTN3, PRM2, KRT4, MMP11, MUC4, DCD, UMOD, MMP7, TGM4, and STATH) and 2 housekeeping genes [i.e., glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18SrRNA], was developed. This assay was further validated with real casework samples and mock samples (with both single source and mixture) and it was approved that XCYR1 is effective to identify common body fluids or tissues (i.e., circulatory blood, saliva, semen, vaginal secretion, menstrual blood, oral mucosa, nasal secretion, sweat and urine) in forensic casework samples.
Assuntos
Identificação Biométrica/métodos , Líquidos Corporais/química , Genética Forense/métodos , RNA Mensageiro/química , Animais , Análise Química do Sangue , Líquidos Corporais/enzimologia , Líquidos Corporais/metabolismo , Gatos , Bovinos , Galinhas/sangue , Cães , Patos/sangue , Feminino , Peixes/sangue , Marcadores Genéticos , Genótipo , Cabras/sangue , Humanos , Masculino , Mucosa Bucal/química , Mucosa Bucal/enzimologia , Mucosa Bucal/metabolismo , Especificidade de Órgãos , Saliva/química , Saliva/enzimologia , Saliva/metabolismo , Análise do Sêmen , Sensibilidade e Especificidade , Suor/química , Suor/enzimologia , Suor/metabolismo , Urina/químicaRESUMO
Kallikrein-related peptidase-8 (KLK8) is a relatively uncharacterized epidermal protease. Although proposed to regulate skin-barrier desquamation and recovery, the catalytic activity of KLK8 was never demonstrated in human epidermis, and its regulators and targets remain unknown. Herein, we elucidated for the first time KLK8 activity in human non-palmoplantar stratum corneum and sweat ex vivo. The majority of stratum corneum and sweat KLK8 was catalytically active, displaying optimal activity at pH 8.5 and considerable activity at pH 5. We also showed that KLK8 is a keratinocyte-specific protease, not secreted by human melanocytes or dermal fibroblasts. KLK8 secretion increased significantly upon calcium induction of terminal keratinocyte differentiation, suggesting an active role for this protease in upper epidermis. Potential activators, regulators, and targets of KLK8 activity were identified by in vitro kinetic assays using pro-KLK8 and mature KLK8 recombinant proteins produced in Pichia pastoris. Mature KLK8 activity was enhanced by calcium and magnesium ions and attenuated by zinc ions and by autocleavage after Arg(164). Upon screening KLK8 cleavage of a library of FRET-quenched peptides, trypsin-like specificity was observed with the highest preference for (R/K)(S/T)(A/V) at P1-P1'-P2'. We also demonstrated that KLK5 and lysyl endopeptidase activate latent pro-KLK8, whereas active KLK8 targets pro-KLK11, pro-KLK1, and LL-37 antimicrobial peptide activation in vitro. Together, our data identify KLK8 as a new active serine protease in human stratum corneum and sweat, and we propose regulators and targets that augment its involvement in a skin barrier proteolytic cascade. The implications of KLK8 elevation and hyperactivity in desquamatory and inflammatory skin disease conditions remain to be studied.
Assuntos
Epiderme/enzimologia , Calicreínas/metabolismo , Suor/enzimologia , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Biocatálise , Diferenciação Celular , Cumarínicos/metabolismo , Ativação Enzimática , Precursores Enzimáticos/antagonistas & inibidores , Precursores Enzimáticos/química , Precursores Enzimáticos/isolamento & purificação , Precursores Enzimáticos/metabolismo , Células Epidérmicas , Epiderme/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Calicreínas/antagonistas & inibidores , Calicreínas/química , Calicreínas/isolamento & purificação , Queratinócitos/enzimologia , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Inibidores de Proteases/farmacologia , Conformação Proteica , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Our work has identified an aging-related ECTO-NOX activity (arNOX), a hydroquinone oxidase which is cell surface located and generates superoxide. This activity increases with increasing age beginning >30 y. Because of its cell surface location and ability to generate superoxide, the arNOX proteins may serve to propagate an aging cascade both to adjacent cells and to oxidize circulating lipoproteins as significant factors determining atherogenic risk. The generation of superoxide by arNOX proteins is inhibited by Coenzyme Q10 as one basis for an anti-aging benefit of CoQ10 supplementation in human subjects. In a preliminary pilot study, 25 female subjects between 45 and 55 y of age were recruited at Stanford University from the Palo Alto, CA area. Informed consent was obtained. Ten of the subjects received Coenzyme Q10 supplementation of 180 (3 x 60 mg) per day for 28 days. Serum, saliva and perspiration levels of arNOX were determined at 7, 14 and 28 days of CoQ10 supplementation and compared to the initial baseline value. Activity correlated with subject age up to a maximum between age 50 and 55 years of age for saliva and perspiration as well and then declined. With all three sources, the arNOX activity extrapolated to zero at about age 30. Response to Coenzyme Q10 also increased with age being least between ages 45 and 50 and greatest between ages 60 and 65. With all three biofluids, arNOX activity was reduced between 25 and 30% by a 3 x 60 mg daily dose Coenzyme Q10 supplementation. Inhibition was the result of Coenzyme Q10 presence.
Assuntos
NADH NADPH Oxirredutases/metabolismo , Ubiquinona/análogos & derivados , Adulto , Idoso , Envelhecimento , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , NADH NADPH Oxirredutases/efeitos dos fármacos , Projetos Piloto , Saliva/efeitos dos fármacos , Saliva/enzimologia , Suor/efeitos dos fármacos , Suor/enzimologia , Ubiquinona/administração & dosagemRESUMO
Enzymes implicated in cysteine and methionine metabolism such as cystathionine beta-lyase (CBL; EC 4.4.1.8), a pyridoxal-5'-phosphate (PLP)-dependent carbon-sulfur lyase, have been shown to play a central role in the generation of sulfur compounds. This work describes the unprecedented cloning and characterization of the metC-cystathionine beta-lyase from the axillary-isolated strain Staphylococcus haemolyticus AX3, in order to determine its activity and its involvement in amino acid biosynthesis, and in the generation of sulfur compounds in human sweat. The gene contains a cysteine/methionine metabolism enzyme pattern, and also a sequence capable to effect beta-elimination. The recombinant enzyme was shown to cleave cystathionine into homocysteine and to convert methionine into methanethiol at low levels. No odor was generated after incubation of the recombinant enzyme with sterile human axillary secretions; sweat components were found to have an inhibitory effect. These results suggest that the generation of sulfur compounds by Staphylococci and the beta-lyase activity in human sweat are mediated by enzymes other than the metC gene or by the concerted activities of more than one enzyme.
Assuntos
Liases/metabolismo , Staphylococcus haemolyticus/enzimologia , Sequência de Aminoácidos , Cistationina/metabolismo , Cisteína/química , Cisteína/metabolismo , Humanos , Liases/classificação , Liases/genética , Masculino , Metionina/química , Metionina/metabolismo , Filogenia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Staphylococcus haemolyticus/genética , Especificidade por Substrato , Compostos de Sulfidrila/metabolismo , Suor/química , Suor/enzimologiaRESUMO
Recently, in Italy, a murder and a putative sexual violence was accomplished on a child. A bodily fluids mixture on the child's underwear between the victim (female) and the suspect (male) was ascertained by short tandem repeat (STR) DNA typing and, due to the absence of seminal fluid, saliva from the suspect and urine from the child was hypothesized. In order to investigate the possibility of specifically and rapidly detecting saliva stains both alone and mixed with other bodily fluids, we used a quantitative spectrophotometric technique, named Amylase test, for the detection of alpha-amylases. We determined alpha-amylase activity and reaction kinetic curves in several samples collected from the child's underwear. In order to confirm our intuition, we first tested saliva, perspiration, and urine, singularly and in mixtures; second, several forensic stains including saliva, perspiration, urine stains, saliva/perspiration, and saliva/urine mixture stains were tested. Evaluating alpha-amylase activity values and time-course curves' behavior of alpha-amylase reactions we were able to recognize successfully, in all cases, the presence of saliva and to distinguish it specifically from other bodily fluids containing alpha-amylase. A further confirmation of our result was provided by STR DNA typing on several areas of the underwear: a clear correlation between alpha-amylases activity and male DNA was detected on all the samples evaluated.
Assuntos
Medicina Legal/métodos , Saliva/enzimologia , alfa-Amilases/análise , Criança , Abuso Sexual na Infância/diagnóstico , Vestuário , Colorimetria , DNA/isolamento & purificação , Feminino , Humanos , Masculino , Reação em Cadeia da Polimerase , Suor/enzimologia , Sequências de Repetição em Tandem , Urina/químicaRESUMO
The protein pattern of healthy human eccrine sweat was investigated and 10 major proteins were detected from which apolipoprotein D, lipophilin B, and cathepsin D (CatD) were identified for the first time in human eccrine sweat. We focused our studies on the function of the aspartate protease CatD in sweat. In vitro digestion experiments using a specific fluorescent CatD substrate showed that CatD is enzymatically active in human sweat. To identify potential substrates of CatD in human eccrine sweat LL-37 and DCD-1L, two antimicrobial peptides present in sweat, were digested in vitro with purified CatD. LL-37 was not significantly digested by CatD, whereas DCD-1L was cleaved between Leu(44) and Asp(45) and between Leu(29) and Glu(30) almost completely. The DCD-1L-derived peptides generated in vitro by CatD were also found in vivo in human sweat as determined by surface-enhanced laser desorption/ionization (SELDI) mass spectrometry. Furthermore, besides the CatD-processed peptides we identified additionally DCD-1L-derived peptides that are generated upon cleavage with a 1,10-phenanthroline-sensitive carboxypeptidase and an endoprotease. Taken together, proteolytic processing generates 12 DCD-1L-derived peptides. To elucidate the functional significance of postsecretory processing the antimicrobial activity of three CatD-processed DCD-1L peptides was tested. Whereas two of these peptides showed no activity against Gram-positive and Gram-negative bacteria, one DCD-1L-derived peptide showed an even higher activity against Escherichia coli than DCD-1L. Functional analysis indicated that proteolytic processing of DCD-1L by CatD in human sweat modulates the innate immune defense of human skin.
Assuntos
Catepsina D/metabolismo , Glândulas Écrinas/metabolismo , Peptídeos/metabolismo , Suor , Sequência de Aminoácidos , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Peptídeos/genética , Análise Serial de Proteínas , Processamento de Proteína Pós-Traducional , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Suor/química , Suor/enzimologiaRESUMO
An ELISA method for the detection of salivary amylase in dried stains using a monoclonal anti-human salivary amylase antibody was developed. Studies demonstrated the assay to be sensitive down to 0.0002 Sigma units and showed a linear response between absorbance and salivary amylase activity between 0.002 and 0.2 units. The assay showed no cross reactivity with either commercially purchased human pancreatic or bacterial amylase. Sample studies utilizing swabs from several human body fluids showed that 100% of all saliva containing swabs (sixteen of sixteen) and 13% of non-saliva human body fluid swabs (eight of sixty-three) showed a net absorbance with the method. Of these eight non-saliva swabs yielding a net absorbance, none exceeded a salivary amylase activity of 0.003 units. In contrast, only three of the sixteen saliva-containing swabs (swabs produced from saliva diluted 1:5, 1:6, and 1:10, respectively) showed an activity below 0.2 units. Of these swabs, the 1:100 dilution showed the lowest activity (0.048). This value is still more than ten times that of the non-saliva containing swab with the highest activity.
Assuntos
Amilases/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Medicina Legal , Saliva/enzimologia , Anticorpos Monoclonais , Análise Química do Sangue , Fezes/enzimologia , Feminino , Conteúdo Gastrointestinal/enzimologia , Humanos , Masculino , Pâncreas/enzimologia , Sêmen/enzimologia , Suor/enzimologia , Urina/química , Vagina/enzimologia , Vagina/metabolismoRESUMO
We confirmed for the first time, both biochemically and immunologically, the existence of deoxyribonuclease I (DNase I) in human liquid sweat. Isoelectric focusing of sweat samples on polyacrylamide gels (pH 3.5 to 5), followed by dried agarose film overlay detection, was used to determine the phenotypes of sweat DNase I. Because this detection method not only had high sensitivity, but also high band resolution, it was possible to determine DNase I types from sweat samples of 50 to 100 microL. Pretreatment of sweat samples with sialidase was essential for typing to enhance markedly the sensitivity accompanied by simplification of the isozyme pattern. The DNase I types in all sweat samples were consistently related to the types found in corresponding blood, urine, and semen samples. DNase I typing could, therefore, provide a novel discriminant characteristic in the forensic examination of sweat.
Assuntos
Desoxirribonuclease I/análise , Suor/enzimologia , Biomarcadores/análise , Biomarcadores/sangue , Biomarcadores/urina , Desoxirribonuclease I/sangue , Desoxirribonuclease I/urina , Humanos , Masculino , Fenótipo , Polimorfismo Genético , Sêmen/enzimologiaRESUMO
The presence of urokinase-type plasminogen activator (uPA) in human eccrine sweat has not been reported previously. Clean sweat was obtained from the upper trunk and arms of subjects which had been painted with white petrolatum to minimize epidermal contamination. Sweat was concentrated x 50 by ultrafiltration, and its PA activity determined by the two-step assay method (conversion of plasminogen to plasmin with the subsequent assay of plasmin activity using the substrate S-2251). PA activity was detectable in nine of 17 subjects by this method, which probably represents an underestimate of the true activity because of possible loss of the enzyme during concentration. Scraped (crude) sweat samples contained less PA activity. Sephacryl S-200 gel chromatography of the PA-positive pooled sweat showed a major peak of PA activity at M(r) 55,000. Gelatin-polyacrylamide enzymography revealed a major PA band at M(r) 55,000 and a minor band at 33,000. Sweat PA activity was 94% inhibited by epidermal PA inhibitor and anti-uPA IgG, but not by anti-tPA IgG. We conclude that the PA activity in sweat is derived from the sweat gland and is most likely of the urokinase type. The physiological significance of sweat uPA remains to be determined.
Assuntos
Suor/enzimologia , Ativador de Plasminogênio Tipo Uroquinase/análise , Adolescente , Adulto , Cromatografia em Gel , Glândulas Écrinas/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , MasculinoAssuntos
Calicreínas/análise , Suor/enzimologia , Adulto , Feminino , Humanos , Calicreínas/urina , Masculino , Esforço Físico , Valores de ReferênciaRESUMO
Attempts were made to purify and characterize cysteine proteinases in human eccrine sweat and further clarify their origin. Benzoyl-DL-arginine-beta-naphthylamide (BANA) and L-leucine beta-naphthylamide (LeuNA) hydrolases in thermally induced sweat were sequentially purified by Sephacryl S-200 chromatography and chromatofocusing, which yielded two major peaks of BANA hydrolase activity, BANA-I and BANA-II. Both enzymes are cysteine proteinases as evidenced by stimulation of enzymic activity by dithiothreitol and ethylenediaminetetraacetic acid and its inhibition by iodoacetic acid, (PCMB), and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane (E-64). Unlike BANA-II, BANA-I showed an additional aminopeptidase activity, an affinity to concanavalin A-Sepharose but no affinity to organomercurial sepharose and failed to hydrolyze benzyloxycarbonyl-phenylalanyl-arginine 4-methyl 7-coumarylamide (Z-Phe-Arg-NMec), a specific substrate for cathepsin B, which is poorly sensitive to leupeptin [inhibitor constant (Ki) = 1 X 10(-5) M] and relatively heat resistant. These and other characteristics such as its isoelectric points (PI) (= 5.8) and the Km for Arg-NMec (0.1 mM) and BANA (0.71 mM) all support the possibility that BANA-I is closely related to cathepsin H. In contrast, BANA-II is sensitive to Zn2+, leupeptin (Ki = 5.5 X 10(-9) M), is not adsorbed by concanavalin A- (Con-A)Sepharose, but is bound to organomercurial sepharose. It has a specificity to Z-Phe-Arg-NMec but not to Arg-NMec, has the molecular weight of 27, PI of 5.2, the pH optima for BANA (6.0), and the Km for BANA of 3.3 mM and the Km for Z-Phe-Arg-NMec of 0.1 mM. These features resemble those of liver cathepsin B. Leupeptin-sensitive BANA hydrolase was observed in the glandular extract of isolated sweat glands, which was increased after stimulation with methacholine and isoproterenol in vitro. The data are consistent with the notion that cathepsins B- and H-like enzymes are present in eccrine sweat and the former may be derived from the sweat gland.
Assuntos
Glândulas Écrinas/metabolismo , Endopeptidases/isolamento & purificação , Glândulas Sudoríparas/metabolismo , Suor/enzimologia , Benzoilarginina-2-Naftilamida/metabolismo , Cromatografia de Afinidade , Cromatografia em Gel , Cisteína Endopeptidases , Ditiotreitol/farmacologia , Ácido Edético/farmacologia , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , Isoenzimas/isolamento & purificação , Leucina/análogos & derivados , Leucina/metabolismo , Leupeptinas/farmacologia , Peso MolecularRESUMO
Thermally induced human eccrine sweat was collected both by simple scraping (SS) and by a polyethylene sweat collector that covered the Vaseline-coated skin of the back to minimize epidermal contamination (CS, clean sweat). Using synthetic chromogenic Kabi S-series substrates, we demonstrated a variety of proteases in both SS and CS, the former being higher than the latter to varying extents. The higher enzyme activity in SS is mainly due to epidermal contamination because abundant protease activity was eluted from the nonperspiring stripped and unstripped skin in vivo and from the stripped stratum corneum itself. The electrophoretic separation of sweat protein has shown that CS contains at least 7 and SS 15 gelatinolytic proteinases. Although some CS proteinases could be derived from the sweat duct, the sweat secretory coil itself is responsible for at least two proteinases at 78 and 25 kilodaltons. The identity and function of these enzymes remain to be studied.
Assuntos
Peptídeo Hidrolases/metabolismo , Suor/enzimologia , Caseínas/metabolismo , Eletroforese , Epiderme/enzimologia , Gelatina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Inibidores de Proteases/farmacologia , Especificidade por SubstratoAssuntos
Glândulas Apócrinas/enzimologia , Fibras Colinérgicas/enzimologia , Colinesterases/metabolismo , Glândulas Écrinas/enzimologia , Mamíferos/metabolismo , Glândulas Sudoríparas/enzimologia , Suor/enzimologia , Acetilcolinesterase/metabolismo , Fosfatase Alcalina/metabolismo , Comunicação Animal , Animais , Animais Domésticos , Animais Selvagens , Glândulas Apócrinas/citologia , Glândulas Apócrinas/inervação , Glândulas Écrinas/citologia , Glândulas Écrinas/inervação , HistocitoquímicaRESUMO
The presence of renin or renin-like activity (RLA) was demonstrated in human eccrine sweat incubated with purified sheep angiotensinogen, using rat bioassay and angiotensin I radioimmunoassay. Following cholinergic stimulation, sweat RLA was found to range between 0 (unmeasurable) and 266 ng/ml.h, i.e. RLA-values of sweat can be about 10 times higher than those of plasma. Therefore, renin synthesis in sweat glands could be assumed. RLA following activation of beta-adrenergic receptors by the administration of isoprenaline (Aludrin) did not exceed the mean values obtained by cholinergic activation. After beta-adrenergic receptor blockade by propranolol (Dociton), RLA became unmeasurably low. Higher RLA-values were found after local injection of dibutyryl-c-AMP (90--210 ng/ml.h). The results indicate a beta-adrenergic regulation of RLA-release in human sweat glands. Human sweat glands appear to be useful for studying extrarenal renin release.
Assuntos
Endopeptidases/metabolismo , Renina/metabolismo , Suor/enzimologia , Animais , Bioensaio , Pressão Sanguínea/efeitos dos fármacos , Carbacol/farmacologia , Humanos , Cinética , Ratos , Suor/efeitos dos fármacosRESUMO
UNLABELLED: The concentration of gamma-glutamyl transpeptidase was determined in eccrine, thermal sweat of 56 healthy men (ages 20--60 years) and 48 healthy women (ages 17--55 years). Samples of sweat were collected from the chest and back. RESULTS: The concentrations of gamma-glutamyl transpeptidase showed great individual variations. The sex specific comparison of the concentrations of gamma-glutamyl transpeptidase revealed that the women excreted in the sweat from the chest and the back nearly the double amount of this enzyme. The gamma-glutamyl transpeptidase concentrations determined in the sweat from the chest of the examined men and women were three times higher as compared with those excreted simultaneous from the back of the same persons. In the group of the women was this differences statistically significant.
Assuntos
Suor/enzimologia , gama-Glutamiltransferase/metabolismo , Adulto , Dorso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Taxa Secretória , TóraxRESUMO
Using bioassay method (rat blood pressure technique) as well as the radioimmunoassay, renin-like activity (RLA) was measured in eccrine sweat of patients with cystic fibrosis of the pancreas (CF) and of controls. Sweat-formation was induced by pilocarpine-iontophoresis or by local injection of carbamylcholine (Doryl). RLA-values between O (not measurable) and 460 ng/ml.h were measured. With increasing sweat flow-rate a tendency to lower RLA-values was detected. No significant difference was observed between CF and controls. From the observation that RLA of sweat is up to 30 times higher than that of plasma, it is concluded that RLA is probably released not from plasma but from the sweat glands themselves, where it is stored or synthesized.
Assuntos
Fibrose Cística/enzimologia , Renina/análise , Suor/enzimologia , Adolescente , Adulto , Criança , Pré-Escolar , Humanos , Glândulas Sudoríparas/enzimologiaAssuntos
Cerebrosídeo Sulfatase/metabolismo , Saliva/enzimologia , Venenos de Serpentes/análise , Sulfatases/metabolismo , Suor/enzimologia , Lágrimas/enzimologia , Animais , Ritmo Circadiano , Humanos , Masculino , Glândula Parótida/metabolismo , Proteínas/análise , Ratos , Glândula Sublingual/metabolismo , Glândula Submandibular/metabolismo , Fatores de TempoRESUMO
Eccrine sweat collected from the human skin surface contains at least five different esterases. One of them is a cholinesterase. A non-specific carboxylesterase with the electrophoretic mobility of an alpha-globulin appears to be a serum protein. Besides this, there are two isoenzymes of human origin migrating with the same electrophoretic mobility as gamma-globulins. These two isoenzymes are immunologically identical with a non-specific carboxylesterase occurring in numerous organs and body fluids. Lipase activity could not be demonstrated.