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Angiotensin converting enzyme-1 (ACE) has been implicated in sleep regulation and nociception. In a secondary, per-protocol analysis, we investigated the effect of a 12-week aerobic exercise program on plasma ACE activity (primary outcome variable), migraine clinical outcomes, and psychometric scores between migraine and control, non-headache participants. Fifty-nine participants (migraine: n=31 and control: n=28) gave signed consent form and were per-protocol analyzed. At baseline, there were no differences between groups for ACE activity. After the intervention period, the ACE activity increased in the migraine exercise group compared to control waitlist group [mean difference (95% CI) = 33.8 nM.min– 1.mg–1 (1.0, 66.5), p = 0.02]. Among patients, the migraine exercise group showed greater numeric reduction in the number of sleep deprivation-triggered attacks compared to migraine waitlist group (-21 vs -8, respectively), and lower insomnia scores [mean difference (95% CI) = -0.625 (-996, -254), p = 0.001]. There was an inverse correlation between BECK-II insomnia domain scores and ACE activity (r = -0.53, p = 0.035). This study suggests that aerobic exercise training increases plasma ACE activity with possible implication on sleep regulation in migraine patients.

Actinomycetes are versatile about their metabolism, displaying high capacity to produce bioactive metabolites. Enzymes from actinomycetes represent new opportunities for industrial applications. However, proteases from actinomycetes are poorly described by literature. Thereby, to verify proteolytic potential of actinomycetes, the present study aimed the investigation of bacterial isolates from Caatinga and Atlantic Forest rhizosphere. Fluorescence Resonance Energy Transfer (FRET) peptide libraries were adopted for the evaluations, since they are faster and more qualitative methods, if compared to others described by most reports. A total of 52 microorganisms were inoculated in different culture media (PMB, PDA, BHI, SCA and R2A), temperatures (12, 20, 30, 37, 45 and 60 °C), and saline conditions (0 to 4 M NaCl), during 7 days. The actinomycetes named as AC 01, 02 and 52 were selected and show enzymatic abilities under the peptide probes Abz-KLRSSKQ-EDDnp and Abz-KLYSSKQ-EDDnp, achieving enhanced performance at 30 °C. Biochemical parameters were established, showing a predominance of alkaline proteases with activity under saline conditions. Secreted proteases hydrolyzed preferentially polar uncharged residues (Y and N) and positively charged groups (R). PMSF and EDTA inhibited the proteins, a characteristic of serine (AC 01 e 02) and metalloproteases (AC 52). All selected strains belonged to Streptomyces genera. In summary, actinomycete strains with halophilic proteolytic abilities were selected, which improve possibilities for their use in detergent formulations, food processing, waste management and industrial bioconversion. It is important to highlight that this is the first report using FRET libraries for proteolytic screening from Caatinga and Atlantic Forest actinobacteria.

Human tissue Kallikrein-related peptidases (hKLKs) are serine proteases distributed in several tissues that are involved in several biological processes. In skin, many are responsible for skin desquamation in the Stratum Corneum (SC) of the epidermis, specially hKLK5, hKLK7, hKLK6, hKLK8, and hKLK14. In SC, hKLKs cleave proteins of corneodesmosomes, an important structure responsible to maintain corneocytes attached. As part of skin desquamation, hKLKs are also involved in skin diseases with abnormal desquamation and inflammation, such as Atopic Dermatitis (AD), psoriasis, and the rare disease Netherton Syndrome (NS). Many studies point to hKLK overexpression or overactive in skin diseases, and they are also part of the natural skin inflammation process, through the PAR2 cleavage pathway. Therefore, the control of hKLK activity may offer successful treatments for skin diseases, improving the quality of life in patients. Diseases like AD, Psoriasis, and NS have an impact on social life, causing pain, itchy and mental disorders. In this review, we address the molecular mechanisms of skin desquamation, emphasizing the roles of human tissue Kallikrein-related peptidases, and the promising therapies targeting the inhibition of hKLKs.

Carboxypeptidase M (CPM) is a glycosylphosphatidylinositol anchored enzyme that plays an important role in the kallikrein–kinin system (KKS). CPM catalytic domain hydrolyzes Arg from C-terminal peptides (i.e., bradykinin and kallidin), generating des-Arg-kinins, the agonists of B1 receptor (B1R). It is known that CPM and kinin B1R are co-localized in the plasma membrane microdomains, where they interact with each other, facilitating receptor signaling. We hypothesized here that this CPM-B1R interaction could also affect the activity of the enzyme. Thus, in this work, we evaluated the impact of B1R presence or absence on CPM activity and expression, using primary culture of microvascular endothelial cells from wild-type, kinin B1R knockout mice (B 1 −/− ), and transgenic rats overexpressing B1 receptor exclusively in the endothelium. In addition, HEK293T cells, as wells as B 1 −/− primary culture of endothelial cells, both transfected with B1R, were also used. CPM expression and activity were downregulated in cells of knockout mice compared to control and this reduction was rescued after B1R transfection. Cells overexpressing B1R presented higher levels of CPM mRNA, protein, and activity. This profile was reverted by pre-incubation with the B1R antagonist, R715, in highly expressing receptor cells. Our data show that kinin B1R positively modulates both CPM expression and activity, suggesting that CPM-B1R interaction in membrane microdomains might affect enzyme activity, beyond interfering in receptors signaling. This work highlights the interactions among different components of KKS and contributes to a better understanding of its patho-physiological role.

STUDY OBJECTIVES: The pathogenesis of inflammatory bowel disease (IBD) is not well understood, and sleep disorders may be potential triggers for IBD. Thus, an evaluation of the sleep characteristics, fatigue symptoms, and cytokine levels was performed in patients with IBD during periods of active disease and remission. METHODS: A total of 20 participants presenting with Crohn's disease or ulcerative colitis, with active disease (n = 7) or in remission (n = 13), underwent home polysomnography (H-PSG). Pittsburgh Sleep Quality Index (PSQI) and Modified Fatigue Impact Scale (MFIS) were applied, in addition to the evaluation of interleukin (IL)-6, IL-10, and tumor necrosis factor alpha (TNF-α) serum levels. Exploratory analysis, t test and Mann-Whitney U test were used. RESULTS: The mean sleep latency in patients with active disease was 133.07 minutes and 106.79 minutes in those in remission. The sleep efficiency and sleep fragmentation in patients with active disease and those in remission were 80.90% and 84.20% (median), and 76.36/min and 69.82/min (mean), respectively, although the H-PSG parameters did not differ between the groups. The PSQI scores indicated poor sleep quality (global score above 5) in all participants with IBD, and the participants with active disease presented more symptoms of fatigue (P = .032). IL-6 and TNF-α average levels were higher in the participants with disease remission, although with a larger dispersion of the data. CONCLUSIONS: No significant difference in the H-PSG characteristics was observed between the patients with IBD with active disease and those in remission; however, the perception of the participants with IBD showed significant effect on the sleep quality and fatigue symptoms. CITATION: Paixão DL, Poyares D, de Paula MS, Duarte JW, Castelo PM, Ambrogini-Júnior O, Miszputen SJ, Oshima CT, Chagas JR, Paiotti AP. Evaluation of home polysomnography findings, quality of sleep, and fatigue in inflammatory bowel disease: a case series. J Clin Sleep Med. 2019;15(1):39–45.

Intermittent hypoxia (IH) is a feature of obstructive sleep apnea (OSA), a condition highly associated with hypertension‐related cardiovascular diseases. Repeated episodes of IH contribute to imbalance of angiogenic growth factors in the hypertrophic heart, which is key in the progression of cardiovascular complications. In particular, the interaction between vascular endothelial growth factor (VEGF) and the kallikrein‐kinin system (KKS) is essential for promoting angiogenesis. However, researchers have yet to investigate experimental models of IH that reproduce OSA, myocardial angiogenesis, and expression of KKS components. We examined temporal changes in cardiac angiogenesis in a mouse IH model. Adult male C57BI/6 J mice were implanted with Matrigel plugs and subjected to IH for 1–5 weeks with subsequent weekly histological evaluation of vascularization. Expression of VEGF and KKS components was also evaluated. After 3 weeks, in vivo myocardial angiogenesis and capillary density were decreased, accompanied by a late increase of VEGF and its type 2 receptor. Furthermore, IH increased left ventricular myocardium expression of the B2 bradykinin receptor, while reducing mRNA levels of B1 receptor. These results suggest that in IH, an unexpected response of the VEGF and KKS systems could explain the reduced capillary density and impaired angiogenesis in the hypoxic heart, with potential implications in hypertrophic heart malfunction., The imbalance between VEGF and B1R‐B2R expressions may contribute to the diminished myocardial angiogenesis in the intermittent hypoxia. Our results indicate a potential role of the reduced capillary density and impaired angiogenesis in the hypertrophic heart with potential sleep apnea implications. ​

Obesity is assumed to be a major cause of human essential hypertension; however, the mechanisms responsible for weight-related increase in blood pressure (BP) are not fully understood. The prevalence of hypertension induced by obesity has grown over the years, and the role of the renin-angiotensin-aldosterone system (RAAS) in this process continues to be elucidated. In this scenario, the ob/ob mice are a genetic obesity model generally used for metabolic disorder studies. These mice are normotensive even though they present several metabolic conditions that predispose them to hypertension. Although the normotensive trait in these mice is associated with the poor activation of sympathetic nervous system by the lack of leptin, we demonstrated that ob/ob mice present massively increased aminopeptidase A (APA) activity in the circulation. APA enzyme metabolizes angiotensin (ANG) II into ANG III, a peptide associated with intrarenal angiotensin type 2 (AT2) receptor activation and induction of natriuresis. In these mice, we found increased ANG-III levels in the circulation, high AT2 receptor expression in the kidney, and enhanced natriuresis. AT2 receptor blocking and APA inhibition increased BP, suggesting the ANG III-AT2 receptor axis as a complementary BP control mechanism. Circulating APA activity was significantly reduced by weight loss independently of leptin, indicating the role of fat tissue in APA production. Therefore, in this study we provide new data supporting the role of APA in BP control in ob/ob mouse strain. These findings improve our comprehension about obesity-related hypertension and suggest new tools for its treatment. NEW & NOTEWORTHY In this study, we reported an increased angiotensin III generation in the circulation of ob/ob mice caused by a high aminopeptidase A activity. These findings are associated with an increased natriuresis found in these mice and support the role of renin-angiotensin-aldosterone system as additional mechanism regulating blood pressure in this genetic obese strain.

Thimet oligopeptidase (TOP, EC 3.4.24.15) and neurolysin (NEL, EC 3.4.24.16) are closely related zinc-dependent metalo-oligopeptidases, which take part in the metabolism of oligopeptides (from 5 to 17 amino acid residues) inside and outside cells. Both peptidases are ubiquitously distributed in tissues. TOP is one of the main intracellular peptide-processing enzymes being important for the antigen selection in the MHC Class I presentation route, while NEL function has been more associated with the extracellular degradation of neurotensin. Despite efforts being made to develop specific inhibitors for these peptidases, the most used are: CPP-Ala-Ala-Tyr-PABA, described by Orlowski et al. in 1988, and CPP-Ala-Aib-Tyr-PABA (JA-2) that is an analog more resistant to proteolysis, which development was made by Shrimpton et al. in 2000. In the present work, we describe other analogs of these compounds but, with better discriminatory capacity to inhibit specifically NEL or TOP. The modifications introduced in these new analogs were based on a key difference existent in the extended binding sites of NEL and TOP: the negatively charged Glu469 residue of TOP corresponds to the positively charged Arg470 residue of NEL. These residues are in position to interact with the residue at the P1′ and/or P2′ of their substrates (mimicked by the Ala-Ala/P1′-P2′ residues of the CPP-Ala-Ala-Tyr-PABA). Therefore, exploring this single difference, the following compounds were synthesized: CPP-Asp-Ala-Tyr-PABA, CPP-Arg-Ala-Tyr-PABA, CPP-Ala-Asp-Tyr-PABA, CPP-Ala-Arg-Tyr-PABA. Confirming the predictions, the replacement of each non-charged residue of the internal portion Ala-Ala by a charged residue Asp or Arg resulted in compounds with higher selectivity for NEL or TOP, especially due to the electrostatic attraction or repulsion by the NEL Arg470 or TOP Glu469 residue. The CPP-Asp-Ala-Tyr-PABA and CPP-Ala-Asp-Tyr-PABA presented higher affinities for NEL, and, the CFP-Ala-Arg-Tyr-PABA showed higher affinity for TOP.

Individualized dosing is often required in pharmacotherapy, particularly for pediatric and geriatric patients and adjustment of drugs that demand dose adaptation. This study aimed to evaluate critical quality attributes (CQAs) of doses obtained by distinct approaches for achieving individual dosing. Approaches were evaluated as follows: subdivision of tablets by splitter and hand (haloperidol) and delivery by plastic dropper bottle (haloperidol), glass dropper bottle (clonazepam), dosing cup (sodium valproate), and dosing syringe (carbamazepine), including brand name, generic, and similar marketed products. Measuring devices were packaged with their respective product. Drug content uniformity was assessed to each substance according to pharmacopeial methods. Tablets subdivided by splitter had the poorest performance among all approaches, in which doses ranged around 60% of the labeled amount (Acceptance Value = 58.1 and RSD = 23.2%). The greatest performances were observed for the dosing syringe which fulfilled all the requirements for dose precision and for the glass dropper bottle. There were significant differences in dose delivery between manufacturers of the same medicine when measuring the same volume or number of drops. High drug content variability is extremely harmful to pharmacotherapy and may result in therapeutic failure or toxicity. It is crucial that measuring devices and scoring of tablets be checked for functionality and standardized for different manufacturers of the same medicine. Part of the approaches for achieving individual dosing did not meet the quality needs for drug content and uniformity. Yet, our findings show that more accurate and precise dosing can be accessed when using the dosing syringe and glass dropper bottle.

The maintenance of blood-brain barrier (BBB) integrity is essential for providing a suitable environment for nervous tissue function. BBB disruption is involved in many central nervous system diseases, including epilepsy. Evidence demonstrates that BBB breakdown may induce epileptic seizures, and conversely, seizure-induced BBB disruption may cause further epileptic episodes. This study was conducted based on the premise that the impairment of brain tissue during the triggering event may determine the organization and functioning of the brain during epileptogenesis, and that BBB may have a key role in this process. Our purpose was to investigate in rats the relationship between pilocarpine-induced status epilepticus (SE), and BBB integrity by determining the time course of the BBB opening and its subsequent recovery during the acute phase of the pilocarpine model. BBB integrity was assessed by quantitative and morphological methods, using sodium fluorescein and Evans blue (EB) dyes as markers of the increased permeability to micromolecules and macromolecules, respectively. Different time-points of the pilocarpine model were analyzed: 30 min after pilocarpine injection and then 1, 5, and 24 h after the SE onset. Our results show that BBB breakdown is a dynamic phenomenon and time-dependent, i.e., it happens at specific time-points of the acute phase of pilocarpine model of epilepsy, recovering in part its integrity afterwards. Pilocarpine-induced changes on brain tissue initially increases the BBB permeability to micromolecules, and subsequently, around 5 h after SE, the BBB breakdown to macromolecules occurs. After BBB breakdown, EB dye is captured by damaged cells, especially neurons, astrocytes, and oligodendrocytes. Although the BBB permeability to macromolecules is restored 24 h after the start of SE, the leakage of micromolecules persists and the consequences of BBB degradation are widely disseminated in the brain. Our findings reveal the existence of a temporal window of BBB dysfunction in the acute phase of the pilocarpine model that is important for the development of therapeutic strategies that could prevent the epileptogenesis.

Thimet oligopeptidase (THOP1) is thought to be involved in neuropeptide metabolism, antigen presentation, neurodegeneration, and cancer. Herein, the generation of THOP1 C57BL/6 knockout mice (THOP1&minus, /&minus, ) is described showing that they are viable, have estrus cycle, fertility, and a number of puppies per litter similar to C57BL/6 wild type mice (WT). In specific brain regions, THOP1-/- exhibit altered mRNA expression of proteasome beta5, serotonin 5HT2a receptor and dopamine D2 receptor, but not of neurolysin (NLN). Peptidomic analysis identifies differences in intracellular peptide ratios between THOP1-/- and WT mice, which may affect normal cellular functioning. In an experimental model of multiple sclerosis THOP1-/- mice present worse clinical behavior scores compared to WT mice, corroborating its possible involvement in neurodegenerative diseases. THOP1-/- mice also exhibit better survival and improved behavior in a sepsis model, but also a greater peripheral pain sensitivity measured in the hot plate test after bradykinin administration in the paw. THOP1-/- mice show depressive-like behavior, as well as attention and memory retention deficits. Altogether, these results reveal a role of THOP1 on specific behaviors, immune-stimulated neurodegeneration, and infection-induced inflammation.

Introduction: Product switching followed by suspected adverse events are common and unsettling for antiepileptic drugs. The objective of this case study was to describe the investigation performed after report of suspected therapeutic failure in pediatric patients following a switch to a different valproate manufacturer and identify strategies concerning medication management for improving therapeutic outcomes. Case description: It was reported that different pediatric patients’ condition changed (agitation/ seizures) after refilling the same drug prescription (sodium valproate syrup) from a different manufacturer. Medical staff reported a suspected therapeutic failure and some units of the product batch associated with the problem were seized by the local Post-marketing Surveillance Service for investigation of potential quality deviations. The seized units were evaluated by the State’s Surveillance Laboratory, nevertheless, drug potency was found to be 98.7%. Conclusion: We consider that the reported event could be associated with aspects of medication use, i.e. potential dose measurement deviations resulting from remaining of residual liquid in the cup or eventual delay at prescription refilling process and consequential - even though brief - pharmacotherapy discontinuity. Patient education and counseling by pharmacists are essential for preventing drug-related problems and enhancing positive outcomes of pharmacotherapy. Article Type: Case Study

Objective To assess through a systematic review of the literature if the practice of splitting tablets containing psychoactive/psychotropic medications for medical or economic reasons would result in the expected doses. Data Sources A MEDLINE and PsycInfo comprehensive search of English-language publications from January 1999 to December 2015 was conducted using the terms describing tablet splitting (tablet splitting, split tablets, tablet subdivision, divided tablets, and half tablets) and psychoactive substances (psychoactive medicines, psychotropic medicines, antidepressants, anxiolytics, anticonvulsants, antipsychotics, and antiparkinsonian agents). An additional supplementary search included the references from the articles found. Study Selection/Data Extraction Studies were included if splitting content was directly related to psychoactive medications and examined the effect of tablet splitting on drug uniformity, weight uniformity, and adherence of psychoactive drugs. Articles were systematically reviewed and examined regarding the study design, methodology, and results of the study. A total of 125 articles were screened, and 13 were selected. Results Tablet splitting implications are extensive, yet substantial deviations from the ideal weight, potency, and dose uniformity are more prone to be important to patient safety. The uneven division of tablets might result in the administration of different doses than what was prescribed, causing under- or overdosing, which might be relevant depending on the drug. In 55% of the cases, splitting psychoactive drugs was satisfactory. Conclusions It cannot be generalized that splitting psychoactive drugs compromises dose accuracy, thus tablet splitting might still be employed in cases in which the advantages outweigh the disadvantages. It is recommended that alternatives be adopted to prevent the disadvantages related to tablet splitting.

Obstructive sleep apnea (OSA) is a frequent sleeping breathing disorder associated with cognitive impairments. Neprilysin (NEP) is responsible for degrading several substrates related to cognition; however, the effect of chronic intermittent hypoxia (CIH) on NEP is still unknown. This study aimed to evaluate the expression and activity of NEP in cognitive-related brain structures of rats submitted to CIH. Western blot, qRT-PCR and enzyme activity assay, demonstrated that a six-week intermittent hypoxia increased NEP expression and activity, selectively in temporal cortex, but not in the hippocampus and frontal cortex. The increase in NEP activity and expression was reverted followed by two weeks recovery in normoxia. These data show that CIH protocol increases the expression and activity of NEP selectively in the temporal cortex. Additional mechanisms must be investigated to elucidate the effects of CIH in cognition.

Serum amyloid A (SAA) was recently associated with metabolic endotoxemia, obesity and insulin resistance. Concurrently, insufficient sleep adversely affects metabolic health and is an independent predisposing factor for obesity and insulin resistance. In this study we investigated whether sleep loss modulates SAA production. The serum SAA concentration increased in C57BL/6 mice subjected to sleep restriction (SR) for 15 days or to paradoxical sleep deprivation (PSD) for 72 h. Sleep restriction also induced the upregulation of Saa1.1/Saa2.1 mRNA levels in the liver and Saa3 mRNA levels in adipose tissue. SAA levels returned to the basal range after 24 h in paradoxical sleep rebound (PSR). Metabolic endotoxemia was also a finding in SR. Increased plasma levels of SAA were also observed in healthy human volunteers subjected to two nights of total sleep deprivation (Total SD), returning to basal levels after one night of recovery. The observed increase in SAA levels may be part of the initial biochemical alterations caused by sleep deprivation, with potential to drive deleterious conditions such as metabolic endotoxemia and weight gain.

The consequences of sleep deprivation on memory, cognition, nociception, stress, and endocrine function are related to the balance of neuropeptides, with peptidases being particularly essential. Thimet oligopeptidase (THOP1) is a metallopeptidase implicated in the metabolism of many sleep-related peptides, including angiotensin I, gonadotropin releasing hormone (GnRH), neurotensin, and opioid peptides. In the present study, we evaluated the effect of sleep deprivation and sleep recovery in male rats on THOP1 expression and specific activity in the central nervous system. In the striatum and hypothalamus, THOP1 activity decreased following sleep deprivation and a recovery period. Meanwhile, THOP1 activity and immunoexpression increased in the hippocampal dentate gyrus during the sleep recovery period. Changes in THOP1 expression after sleep deprivation and during sleep recovery can potentially alter the processing of neuropeptides. In particular, processing of opioid peptides may be related to the known increase in pain sensitivity in this model. These results suggest that THOP1 may be an important player in the effects of sleep deprivation., Biochemistry; Neuroscience; Cognitive neuroscience; Sleep; Enzymology; Peptides; Gonadotropin releasing hormone; Neuropeptides; Peptidases; Sleep loss; Striatum; Memory; Opioid peptides; Angiotensin 1-7

Obesity is assumed to be a major cause of human essential hypertension; however, the mechanisms responsible for weight-related increase in blood pressure (BP) are not fully understood. The prevalence of hypertension induced by obesity has grown over the years, and the role of the renin-angiotensin-aldosterone system (RAAS) in this process continues to be elucidated. In this scenario, the

Obstructive sleep apnea (OSA) has been associated with oxidative stress and various cardiovascular consequences, such as increased cardiovascular disease risk. Quantitative real-time PCR is frequently employed to assess changes in gene expression in experimental models. In this study, we analyzed the effects of chronic intermittent hypoxia (an experimental model of OSA) on housekeeping gene expression in the left cardiac ventricle of rats. Analyses via four different approaches-use of the geNorm, BestKeeper, and NormFinder algorithms; and 2−ΔCt (threshold cycle) data analysis-produced similar results: all genes were found to be suitable for use, glyceraldehyde-3-phosphate dehydrogenase and 18S being classified as the most and the least stable, respectively. The use of more than one housekeeping gene is strongly advised. RESUMO A apneia obstrutiva do sono (AOS) tem sido associada ao estresse oxidativo e a várias consequências cardiovasculares, tais como risco aumentado de doença cardiovascular. A PCR quantitativa em tempo real é frequentemente empregada para avaliar alterações na expressão gênica em modelos experimentais. Neste estudo, analisamos os efeitos da hipóxia intermitente crônica (um modelo experimental de AOS) na expressão de genes de referência no ventrículo cardíaco esquerdo de ratos. Análises a partir de quatro abordagens - uso dos algoritmos geNorm, BestKeeper e NormFinder e análise de dados 2−ΔCt (ciclo limiar) - produziram resultados semelhantes: todos os genes mostraram-se adequados para uso, sendo que gliceraldeído-3-fosfato desidrogenase e 18S foram classificados como o mais e o menos estável, respectivamente. A utilização de mais de um gene de referência é altamente recomendada.

Some central effects of cocaine administration seem to be related to angiotensin II (Ang II) or its metabolites. Nonetheless, it is still an open question whether or not the levels of angiotensin I-converting enzyme (ACE), the main Ang II generating enzyme, are modified by cocaine administration. To evaluate the effect of acute and subchronic cocaine administration on ACE activity and mRNA expression, male rats were randomly assigned to saline or cocaine group. Acute and subchronic cocaine administration induced a significant increase in ACE activity and mRNA expression in the frontal cortex and striatum but not in the hippocampus. These results suggest that some of the Ang II related effects of cocaine upon the central nervous system can be mediated by changes on the expression and activity of ACE in the striatum and frontal cortex.

Angiotensin-converting enzyme (ACE) is an ectoprotein able to modulate the activity of a plethora of compounds, among them angiotensin I and bradykinin. Despite several decades of research, new aspects of the mechanism of action of ACE have been elucidated, expanding our understanding of its role not only in cardiovascular regulation but also in different areas. Recent findings have ascribed an important role for ACE/kinin B 2 receptor heterodimerization in the pharmacological properties of the receptor. In this work, we tested the hypothesis that this interaction also affects ACE enzymatic activity. ACE catalytic activity was analyzed in Chinese hamster ovary cell monolayers coexpressing the somatic form of the enzyme and the receptor coding region using as substrate the fluorescence resonance energy transfer peptide Abz-FRK(Dnp)P-OH. Results show that the coexpression of the kinin B 2 receptor leads to an augmentation in ACE activity. In addition, this effect could be blocked by the B 2 receptor antagonist icatibant. The hypothesis was also tested in endothelial cells, a more physiological system, where both proteins are naturally expressed. Endothelial cells from genetically ablated kinin B 2 receptor mice showed a decreased ACE activity when compared with wild-type mice cells. In summary, this is the first report showing that the ACE/kinin B 2 receptor interaction modulates ACE activity. Taking into account the interplay among ACE, ACE inhibitors, and kinin receptors, we believe that these results will shed new light into the arena of the controversial search for the mechanism controlling these interactions.

Two proteins with phospholipase A(2) (PLA(2)) activity were purified to homogeneity from Bothrops leucurus (white-tailed-jararaca) snake venom through three chromatographic steps: Conventional gel filtration on Sephacryl S-200, ion-exchange on Q-Sepharose and reverse phase on Vydac C4 HPLC column. The molecular mass for both enzymes was estimated to be approximately 14 kDa by SDS-PAGE. The N-terminal sequences (48 residues) show that one enzyme presents lysine at position 48 and the other an aspartic acid in this position, and therefore they were designated blK-PLA(2) and blD-PLA(2) respectively. blK-PLA(2) presented negligible levels of PLA(2) activity as compared to that of blD-PLA(2). The PLA(2) activity of both enzymes is Ca(2+)-dependent. blD-PLA(2) did not have any effect upon platelet aggregation induced by arachidonic acid, ADP or collagen, but strongly inhibits coagulation and is able to stimulate Ehrlich tumor growth but not angiogenesis.

Obstructive sleep apnea syndrome is mainly characterized by intermittent hypoxia (IH) during sleep, being associated with several complications. Exposure to IH is the most widely used animal model of sleep apnea, short-term IH exposure resulting in cognitive and neuronal impairment. Pigment epithelium-derived factor (PEDF) is a hypoxia-sensitive factor acting as a neurotrophic, neuroprotective, and antiangiogenic agent. Our study analyzed performance on learning and cognitive tasks, as well as PEDF gene expression and PEDF protein expression in specific brain structures, in rats exposed to long-term IH.Male Wistar rats were exposed to IH (oxygen concentrations of 21-5%) for 6 weeks-the chronic IH (CIH) group-or normoxia for 6 weeks-the control group. After CIH exposure, a group of rats were allowed to recover under normoxic conditions for 2 weeks (the CIH+N group). All rats underwent the Morris water maze test for learning and memory, PEDF gene expression and PEDF protein expression in the hippocampus, frontal cortex, and temporal cortex being subsequently assessed.The CIH and CIH+N groups showed increased PEDF gene expression in the temporal cortex, PEDF protein expression remaining unaltered. PEDF gene expression and PEDF protein expression remained unaltered in the frontal cortex and hippocampus. Long-term exposure to IH did not affect cognitive function.Long-term exposure to IH selectively increases PEDF gene expression at the transcriptional level, although only in the temporal cortex. This increase is probably a protective mechanism against IH-induced injury.A síndrome da apneia obstrutiva do sono caracteriza-se principalmente por episódios de hipóxia intermitente (HI) durante o sono e associa-se a diversas complicações. A exposição à HI é o mais usado modelo animal de apneia do sono, e protocolos de curta duração causam diversos prejuízos cognitivos e neuronais.Ratos Wistar foram expostos a HI (21-5% de oxigênio) durante 6 semanas - o grupo HI crônica (HIC) - ou a normóxia durante 6 semanas - o grupo controle. Após a exposição à HIC, um grupo de ratos foi exposto a normóxia durante 2 semanas (o grupo HIC+N). Todos os animais foram submetidos ao labirinto aquático de Morris para avaliação de memória e aprendizado; avaliou-se também a expressão do geneOs grupos HIC e HIC+N apresentaram um aumento de expressão do geneA exposição de longa duração à HI aumenta seletivamente a expressão do gene

The reactive center loop (RCL) of serpins plays an essential role in the inhibition mechanism acting as a substrate for their target proteases. Changes within the RCL sequence modulate the specificity and reactivity of the serpin molecule. Recently, we reported the construction of α1-antichymotrypsin (ACT) variants with high specificity towards human kallikrein 2 (hK2) [Cloutier SM, Kundig C, Felber LM, Fattah OM, Chagas JR, Gygi CM, Jichlinski P, Leisinger HJ & Deperthes D (2004) Eur J Biochem271, 607–613] by changing amino acids surrounding the scissile bond of the RCL and obtained specific inhibitors towards hK2. Based on this approach, we developed highly specific recombinant inhibitors of human kallikrein 14 (hK14), a protease correlated with increased aggressiveness of prostate and breast cancers. In addition to the RCL permutation with hK14 phage display-selected substrates E8 (LQRAI) and G9 (TVDYA) [Felber LM, Borgono CA, Cloutier SM, Kundig C, Kishi T, Chagas JR, Jichlinski P, Gygi CM, Leisinger HJ, Diamandis EP & Deperthes D (2005) Biol Chem386, 291–298], we studied the importance of the scaffold, serpins α1-antitrypsin (AAT) or ACT, to confer inhibitory specificity. All four resulting serpin variants ACTE8, ACTG9, AATE8 and AATG9 showed hK14 inhibitory activity and were able to form covalent complex with hK14. ACT inhibitors formed more stable complexes with hK14 than AAT variants. Whereas E8-based inhibitors demonstrated a rather relaxed specificity reacting with various proteases with trypsin-like activity including several human kallikreins, the two serpins variants containing the G9 sequence showed a very high selectivity for hK14. Such specific inhibitors might prove useful to elucidate the biological role of hK14 and/or its implication in cancer.

Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and pro-teins at neutral pH. CPM plays an important role in the control ofpeptide hormones and growth factor activity on the cell surface. Thepresent study was carried out to clone and express human CPM in theyeast Pichia pastoris in order to evaluate the importance of thisenzyme in physiological and pathological processes. The cDNA forthe enzyme was amplified from total placental RNA by RT-PCR andcloned in the vector pPIC9, which uses the methanol oxidase promoterand drives the expression of high levels of heterologous proteins in P.pastoris . The cpm gene, after cloning and transfection, was integratedinto the yeast genome, which produced the active protein. The recom-binant protein was secreted into the medium and the enzymaticactivity was measured using the fluorescent substrate dansyl-Ala-Arg.The enzyme was purified by a two-step protocol including gel filtra-tion and ion-exchange chromatography, resulting in a 1753-foldpurified active protein (16474 RFU mg protein

The reactive site loop of serpins undoubtedly defines in part their ability to inhibit a particular enzyme. Exchanges in the reactive loop of serpins might reassign the targets and modify the serpin-protease interaction kinetics. Based on this concept, we have developed a procedure to change the specificity of known serpins. First, reactive loops are very good substrates for the target enzymes. Therefore, we have used the phage-display technology to select from a pentapeptide phage library the best substrates for the human prostate kallikrein hK2 [Cloutier, S.M., Chagas, J.R., Mach, J.P., Gygi, C.M., Leisinger, H.J.Deperthes, D. (2002) Eur. J. Biochem. 269, 2747-2754]. Selected substrates were then transplanted into the reactive site loop of alpha1-antichymotrypsin to generate new variants of this serpin, able to inhibit the serine protease. Thus, we have developed some highly specific alpha1-antichymotrypsin variants toward human kallikrein 2 which also show high reactivity. These inhibitors might be useful to help elucidate the importance of hK2 in prostate cancer progression.

Human glandular kallikrein 2 (hK2) is a trypsin-like serine protease expressed predominantly in the prostate epithelium. Recently, hK2 has proven to be a useful marker that can be used in combination with prostate specific antigen for screening and diagnosis of prostate cancer. The cleavage by hK2 of certain substrates in the proteolytic cascade suggest that the kallikrein may be involved in prostate cancer development; however, there has been very little other progress toward its biochemical characterization or elucidation of its true physiological role. In the present work, we adapt phage substrate technology to study the substrate specificity of hK2. A phage-displayed random pentapeptide library with exhaustive diversity was generated and then screened with purified hK2. Phages displaying peptides susceptible to hK2 cleavage were amplified in eight rounds of selection and genes encoding substrates were transferred from the phage to a fluorescent system using cyan fluorescent protein (derived from green fluorescent protein) that enables rapid determination of specificity constants. This study shows that hK2 has a strict preference for Arg in the P1 position, which is further enhanced by a Ser in P'1 position. The scissile bonds identified by phage display substrate selection correspond to those of the natural biological substrates of hK2, which include protein C inhibitor, semenogelins, and fibronectin. Moreover, three new putative hK2 protein substrates, shown elsewhere to be involved in the biology of the cancer, have been identified thus reinforcing the importance of hK2 in prostate cancer development.

Corneal avascularization is essential for normal vision. Several antiangiogenic factors were identified in cornea such as endostatin and angiostatin. Cathepsin V, which is highly expressed in the cornea, can hydrolyze human plasminogen to release angiostatin fragments. Herein, we describe a detailed investigation of the expression profile of cathepsins B, L, S and V in the human cornea and the role of cysteine peptidases in modulating angiogenesis both in vitro and in vivo. We used various methodological tools for this purpose, including real-time PCR, SDS-PAGE, western blotting, catalytic activity assays, cellular assays and induction of corneal neovascularity in rabbit eyes. Human corneal enzymatic activity assays revealed the presence of cysteine proteases that were capable of processing endogenous corneal plasminogen to produce angiostatin-like fragments. Comparative real-time analysis of cathepsin B, L, S and V expression revealed that cathepsin V was the most highly expressed, followed by cathepsins L, B and S. However, cathepsin V depletion revealed that this enzyme is not the major cysteine protease responsible for plasminogen degradation under non-pathological conditions. Furthermore, western blotting analysis indicated that only cathepsins B and S were present in their enzymatically active forms. In vivo analysis of angiogenesis demonstrated that treatment with the cysteine peptidase inhibitor E64 caused a reduction in neovascularization. Taken together, our results show that human corneal cysteine proteases are critically involved in angiogenesis.

Objective Thimet oligopeptidase (TOP) is a metalloprotease that has been associated with peptide processing in several nervous system structures, and its substrates include several peptides such as bradykinin, amyloid beta (Aβ), and major histocompatibility complex (MHC) class I molecules. As shown previously by our research group, patients with temporal lobe epilepsy (TLE) have a high level of kinin receptors as well as kallikrein, a kinin-releasing enzyme, in the hippocampus. Methods In this study, we evaluated the expression, distribution, and activity of TOP in the hippocampus of patients with TLE and autopsy-control tissues, through reverse-transcription polymerase chain reaction (RT-PCR), enzymatic activity, Western blot, and immunohistochemistry. In addition, hippocampi of rats were analyzed using the pilocarpine-induced epilepsy model. Animals were grouped according to the epilepsy phases defined in the model as acute, silent, and chronic. Results Increased TOP mRNA expression, decreased protein levels and enzymatic activity were observed in tissues of patients, compared to control samples. In addition, decreased TOP distribution was also visualized by immunohistochemistry. Similar results were observed in tissues of rats during the acute phase of epilepsy model. However, increased TOP mRNA expression and no changes in immunoreactivity were found in the silent phase, whereas increased TOP mRNA expression and increased enzymatic activity were observed in the chronic phase. Significance The results show that these alterations could be related to a failure in the mechanisms involved in clearance of inflammatory peptides in the hippocampus, suggesting an accumulation of potentially harmful substances in nervous tissue such as Aβ, bradykinin, and antigenic peptides. These accumulations could be related to hippocampal inflammation observed in TLE subjects.

The Trypanosoma cruzi cysteine protease cruzipain contains a 130-amino-acid C-terminal extension, in addition to the catalytic domain. Natural cruzipain is a complex of isoforms, because of the simultaneous expression of several genes, and the presence of either high mannose-type, hybrid monoantennary-type or complex biantenary-type oligosacharide chains at Asn255 of the C-terminal extension. Cruzipain and its recombinant form without this extension (cruzain) were studied comparatively in this work. S2 to S2′ subsite specificities of these enzymes were examined using four series of substrates derived from the internally quenched fluorescent peptide Abz-KLRFSKQ-EDDnp (Abz, ortho-aminobenzoic acid; EDDnp, N-(2,4-dinitrophenyl)-ethylenediamine). Large differences in the kinetic parameters were not observed between the enzymes; however, Km values were consistently lower for the hydrolysis of most of the substrates by cruzain. No difference in the pH–activity profile between the two enzymes was found, but in 1 m NaCl cruzipain presented a kcat value significantly higher than that of cruzain. The activation energy of denaturation for the enzymes did not differ significantly; however, a negative entropy value was observed for cruzipain denaturation whereas the value for cruzain was positive. We determined the individual rate constants (k1, substrate diffusion; k−1, substrate dissociation; k2, acylation; k3, deacylation) and the respective activation energies and entropies for hydrolysis of Abz-KLRFSKQ-EDDnp determining the temperature dependence of the Michaelis–Menten parameters kcat/Km and kcat as previously described [Ayala, Y.M. & Di Cera, E. (2000) Protein Sci.9, 1589–1593]. Differences between the two enzymes were clearly detected in the activation energies E1 and E−1, which are significantly higher for cruzipain. The corresponding ΔS1 and ΔS−1 were positive and significantly higher for cruzipain than for cruzain. These results indicate the presence of a larger energy barrier for cruzipain relating to substrate diffusion and dissociation, which could be related to the C-terminal extension and/or glycosylation state of cruzipain.

It has been shown that lysosomal cysteine proteinases, specially cathepsin B, has been implicated in a variety of diseases involving tissue remodeling states, such as inflammation, parasite infection, and tumor metastasis, by degradation of extracellular matrix components. Recently, we have shown that heparin and heparan sulfate bind to papain specifically; this interaction induces an increase of its alpha-helix content and stabilizes the enzyme structure even at alkaline pH (Almeida, P. C., Nantes, I. L., Rizzi, C. C. A., Judice, W. A. S., Chagas, J. R., Juliano, L., Nader, H. B., and Tersariol, I. L. S. (1999) J. Biol. Chem. 274, 30433-30438). In the present work, a combination of circular dichroism analysis, affinity chromatography, cathepsin B mutants, and fluorogenic substrate assays were used to characterize the interaction of human cathepsin B with glycosaminoglycans. The nature of the cathepsin B-glycosaminoglycans interaction was sensitive to the charge and type of polysaccharide. Like papain, heparin and heparan sulfate bind cathepsin B specifically, and this interaction reduces the loss of cathepsin B alpha-helix content at alkaline pH. Our data show that the coupling of cathepsin B with heparin or heparan sulfate can potentiate the endopeptidase activity of the cathepsin B, increasing 5-fold the half-life (t(12)) of the enzyme at alkaline pH. Most of these effects are related to the interaction of heparin and heparan sulfate with His(111) residue of the cathepsin B occluding loop. These results strongly suggest that heparan sulfate may be an important binding site for cathepsin B at cell surface, reporting a novel physiological role for heparan sulfate proteoglycans.

The ability of the prodomains of trypanosomal cysteine proteinases to inhibit their active form was studied using a set of 23 overlapping 15-mer peptides covering the whole prosequence of congopain, the major cysteine proteinase of Trypanosoma congolense. Three consecutive peptides with a common 5-mer sequence YHNGA were competitive inhibitors of congopain. A shorter synthetic peptide consisting of this 5-mer sequence flanked by two Ala residues (AYHNGAA) also inhibited purified congopain. No residue critical for inhibition was identified in this sequence, but a significant improvement in K i value was obtained upon N-terminal elongation. Procongopain-derived peptides did not inhibit lysosomal cathepsins B and L but did inhibit native cruzipain (from Dm28c clone epimastigotes), the major cysteine proteinase of Trypanosoma cruzi, the proregion of which also contains the sequence YHNGA. The positioning of the YHNGA inhibitory sequence within the prosegment of trypanosomal proteinases is similar to that covering the active site in the prosegment of cysteine proteinases, the three-dimensional structure of which has been resolved. This strongly suggests that trypanosomal proteinases, despite their long C-terminal extension, have a prosegment that folds similarly to that in related mammal and plant cysteine proteinases, resulting in reverse binding within the active site. Such reverse binding could also occur for short procongopain-derived inhibitory peptides, based on their resistance to proteolysis and their ability to retain inhibitory activity after prolonged incubation. In contrast, homologous peptides in related cysteine proteinases did not inhibit trypanosomal proteinases and were rapidly cleaved by these enzymes.

Peptide substrates with intramolecularly quenched fluorescence that reproduce the rat kininogen sequences at both ends of the bradykinin moiety were synthesized and used to investigate the kinin-releasing properties of five rat tissue kallikreins (rK1, rK2, rK7, rK9, rK10). Substrates derived from rat H- and L-kininogen were cleaved best by rK1, especially that including the N-terminal insertion site of bradykinin, Abz-TSVIRRPQ-EDDnp(Abz = O-aminobenzoyl, EDDnp = ethylenediamine 2,4-dinitrophenyl), which was cleaved at the R-R bond with a k(cat)/Km of 12400 mM(-1) s(-1). Replacement of the P2' residue Pro by Val in Abz-TSVIRRPQ-EDDnp gave a far less specific substrate that was rapidly hydrolysed by all five rat kallikreins and human kallikrein hK1. Peptidyl-N-methyl coumarylamide substrates, which lack prime residues, also had low specificities. The importance of the P2' residue for rK1 specificity was further demonstrated using a human-kininogen-derived substrate that included the N-terminal insertion site of bradykinin (Abz-LMKRP-EDDnp). This was cleaved at the M-K bond by hK1 (kallidin-releasing site), but at the K-R bond (bradykinin-releasing site) by rK1. Competition experiments with Abz-TSVIRRPQ-EDDnp, which is resistant to most kallikreins, and Abz-TSVIRRVQ-EDDnp, a general kallikrein substrate, demonstrated that the former competitively inhibited hydrolysis by rK9 and hK1, with Ki values similar to the Km values for the substrate. Thus Pro in P2' does not prevent the peptide binding to the enzyme active site, but impairs cleavage of the scissile bond. The T-kininogen-derived substrate with the T-kinin C-terminal sequence (Abz-FRLVR-EDDnp) was cleaved by rK10 (k(cat)/Km = 2310 mM(-1) s(-1)) and less rapidly by rK1, rK7 and hK1, at the R-L bond, while that corresponding to the N-terminal (Abz-ALDMMISRP-EDDnp) of T-kinin was resistant to all five kallikreins used, suggesting that none has T-kininogenase activity. But this substrate was hydrolysed by a semipurified sample of submandibular gland extract. Another kallikrein, identified as kallikrein rK3, was isolated from this fraction and shown to hydrolyze Abz-ALDMMISRP-EDDnp; rK3 also specifically released T-kinin from purified T1/T2-kininogen after HPLC fractionation. Injection of purified rK3 and of Abz-ALDMMISRP-EDDnp-cleaving fractions into the circulation of anesthesized rats caused transient falls in blood pressure, as did purified rK1 but none of the other purified rat or human kallikreins. This effect occurred via activation of the kinin system since it was blocked by Hoe140, a kinin receptor antagonist.

Many studies indicate that thimet oligopeptidase (EC3.4.24.15; TOP) can be implicated in the metabolism of bioactive peptides, including dynorphin 1–8, α-neoendorphin, β-neoendorphin and GnRH. Furthermore, the higher levels of this peptidase are found in neuroendocrine tissue and testis. In the present study, we have evaluated the effect of acute cocaine administration in male rats on TOP specific activity and mRNA levels in prosencephalic brain areas related with the reward circuitry; ventral striatum, hippocampus, and frontal cortex. No significant differences on TOP specific activity were detected in the hippocampus and frontal cortex of cocaine treated animals compared to control vehicle group. However, a significant increase in activity was observed in the ventral striatum of cocaine treated-rats. The increase occurred in both, TOP specific activity and TOP relative mRNA amount determined by real time RT-PCR. As TOP can be implicated in the processing of many neuropeptides, and previous studies have shown that cocaine also alters the gene expression of proenkephalin and prodynorphin in the striatum, the present findings suggest that TOP changes in the brain could play important role in the balance of neuropeptide level correlated with cocaine effects.

Proteases are essential either for the release of neuropeptides from active or inactive proteins or for their inactivation. Neuropeptides have a fundamental role in sleep-wake cycle regulation and their actions are also likely to be regulated by proteolytic processing. Using fluorescence resonance energy transfer substrates, specific protease inhibitors and real-time PCR we demonstrate changes in angiotensin I-converting enzyme (ACE) expression and proteolytic activity in the central nervous system in an animal model of paradoxical sleep deprivation during 96 h (PSD). Male rats were distributed into five groups (PSD, 24 h, 48 h and 96 h of sleep recovery after PSD and control). ACE activity and mRNA levels were measured in hypothalamus, hippocampus, brainstem, cerebral cortex and striatum tissue extracts. In the hypothalamus, the significant decrease in activity and mRNA levels, after PSD, was only totally reversed after 96 h of sleep recovery. In the brainstem and hippocampus, although significant, changes in mRNA do not parallel changes in ACE specific activity. Changes in ACE activity could affect angiotensin II generation, angiotensin 1–7, bradykinin and opioid peptides metabolism. ACE expression and activity modifications are likely related to some of the physiological changes (cardiovascular, stress, cognition, metabolism function, water and energy balance) observed during and after sleep deprivation.

Six intramolecularly quenched fluorogenic peptides related to the sequences Phe 8 to His 13 , His 6 to His 13 , and Tyr 4 to His 13 of the human angiotensinogen, containing o -aminobenzoyl (Abz) and ethylenediamine dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acids residues, were synthesized by classical solution methods. The Leu-Val is the only bond of all obtained peptides that was hydrolyzed by human renin with different degrees of purity and was resistant to hydrolysis by pig renin and cathepsin D. The hydrolysis of Abz-His-Pro-Phe-His-Leu-Val-Ile-His-EDDnp by human renin was inhibited by a highly specific transition-state analog of angiotensinogen (IC 50 = 7.8 × 10 −9 m ), described by K. Iizuka et al. (1990, J. Med. Chem. 33, 2707–2714). Therefore, specific and sensitive substrates for the continuous assay of human renin in which as little as 70 μGU of human renin could be detected by Abz-Phe-His-Leu-Val-Ile-His-EDDnp were described. The optimal pHs of hydrolysis of the substrates were in the range 4 to 6.

Five intramolecularly quenched fluorogenic substrates for arginyl hydrolases with the sequence Abz-Phe-Arg-X-Y-EDDnp (X = Arg or Ser; Y = Val, Pro, or Arg) were synthesized by classical solution methods. Kinetics of their hydrolysis by tissue and plasma kallikreins, trypsin, and thrombin characterized Abz-Phe-Arg-Ser-Arg-EDDnp as a specific and sensitive substrate for the continuous assay of tissue kallikreins while Abz-Phe-Arg-Arg-Pro-EDDnp was the best substrate for human plasma kallikrein. The five peptides were poor substrates for trypsin and resistant to thrombin.

Summary A novel quenched fluorescence substrate, QF-ERP7 (Abz-G-G-F-L-R-R-V-EDDn), structurally related to enkephalins, proved to be suitable for assaying the endooligopeptidase A (E.C.3.4.22.19) activity. The enzyme only splits the L-R bond (Km 1.75 μM, Kcat 8.25 s−1), a reaction efficiently blocked by anti-endooligopeptidase A antibodies and by inhibitor and alternative substrates of the enzyme. Evidences based on the action of inhibitors and on the analysis of QF-ERP7 fragments demonstrated that endooligopeptidase A contributes with 100% of the QF-ERP7 cleaving activity found in the cytosol of rabbit brain homogenates and with 85% of that recovered in the membrane fraction. Homologous substrates, Abz-G-G-F-L-R-R-EDDn and Abz-G-G-F-L-R-EDDn, were resistant to hydrolysis. The convenience and sensitivity of the fluorimetric assay based on the QF-ERP7 moiety offers several advantages compared with previously described painstaking procedures for endooligopeptidase A activity measurements, what will certainly contribute to further our understanding of the role of this enzyme on the peptide hormone metabolism.

Post-exercise hypotension is an important event for blood pressure regulation, especially in hypertensive individuals. Although post-exercise hypotension is a well-known phenomenon, the mechanism responsible is still unclear. The kallikrein-kinin system is involved in blood pressure control, but its role in post-exercise hypotension has not yet been investigated. Thus, the purpose of this study was to investigate the involvement of the vasodilators bradykinin and des-Arg9-BK and kallikrein activity in post-exercise hypotension promoted by 35 min of cycle ergometer (CE) or circuit weight-training (CWT) bouts in normotensive and hypertensive individuals. A significant decrease in mean arterial pressure at 45 and 60 min after CE and 45 min after CWT was observed in normotensive individuals. Hypertensive values of mean arterial pressure were significantly reduced at 45 and 60 min after CE and at 60 min after CWT. Before exercise, plasma bradykinin concentrations and kallikrein activity were higher in hypertensive compared to normotensive volunteers. Kinin levels increased in the groups evaluated at the end of the training period and 60 min post-exercise. These data suggest that the kallikrein-kinin system may be involved in post-exercise hypotension in normotensive and hypertensive individuals subjected to CE and CWT bouts.

The humanKLK14gene is one of the newly identified serine protease genes belonging to the human kallikrein family, which contains 15 members.KLK14, like all other members of the human kallikrein family, is predicted to encode for a secreted serine protease already found in various biological fluids. This new kallikrein is mainly expressed in prostate and endocrine tissues, but its function is still unknown. Recent studies have demonstrated thatKLK14gene expression is up-regulated in prostate and breast cancer tissues, and that higher expression levels correlate with more aggressive tumors. In this work, we used phage-display substrate technology to study the substrate specificity of hK14. A phage-displayed random pentapeptide library with exhaustive diversity was screened with purified recombinant hK14. Highly specific and sensitive substrates were selected from the library. We show that hK14 has dual activity, trypsin- and chymotrypsin-like, with a preference for cleavage after arginine residues. A SwissProt database search with selected sequences identified six potential human protein substrates for hK14. Two of them, laminin α-5 and collagen IV, which are major components of the extracellular matrix, have been demonstrated to be hydrolyzed efficiently by hK14.

New organotellurium(IV) compounds with specific cysteine protease inhibitory activity were synthesized. Serine and aspartic protease activity were not affected by any of these compounds. All Te(IV) compounds tested exhibited high specific second-order constant for cathepsin B inactivation. Tellurium(IV) compound 6 was the best inhibitor of the series, showing a second-order constant of 36,000 M(-1)s(-1). This value is about 100-fold higher than the second-order rate for cysteine protease inactivation shown by the historic Te(IV) compound AS 101 (1). The inhibition was irreversible and time and concentration dependent; no saturation kinetics were observed, suggesting a direct bimolecular reaction. The results described in this paper show that the new organotellurium(IV) compounds are powerful inhibitors of cathepsin B, constituting promising potential anti-metastatic agents.

There are few reports concerning the biological role and the mechanisms of interaction between proteinases and carbohydrates other than those involved in clotting. It has been shown that the interplay of enzymes and glycosaminoglycans is able to modulate the activity of different proteases and also to affect their structures. From the large number of proteases belonging to the well-known protease families and also the variety of carbohydrates described as widely distributed, only few events have been analyzed more deeply. The term family is used to describe a group of proteases in which every member shows an evolutionary relationship to at least one other protease. This relationship may be evident throughout the entire sequence, or at least in that part of the sequence responsible for catalytic activity. The majority of proteases belong to the serine, cysteine, aspartic or metalloprotease families. By considering the existing limited proteolysis process, in addition to the initial idea that the proteinases participate only in digestive processes, it is possible to conclude that the function of the enzymes is strictly limited to the cleavage of intended substrates since the destruction of functional proteins would result in normal tissue damage. In addition, the location as well as the eventual regulation of protease activity promoted by glycosaminoglycans can play an essential role in the development of several physiopathological conditions. Universidade de Mogi das Cruzes Centro Interdisciplinar de Investigação Bioquímica Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de Biofísica UNIFESP, EPM, Depto. de Biofísica SciELO