Ultraviolet-A absorbance analysis in thin porcine corneas pre-and post-crosslinking.

PURPOSE: To determine the absorbance coefficient of the thin porcine cornea to ultraviolet-A radiation (365 nm) submitted for crosslinking. METHODS: This in vitro, benchtop experiment using cadaver tissue study analyzed 12 porcine corneal lamellas, which were obtained using a microkeratome after mechanical de-epithelization and separated into three thickness groups: 180, 300, and 360 µm. The corneal thickness values were measured by anterior-segment optical coherence tomography. All lamellas had ultraviolet-A (365 nm) absorbance measured with a 96-well plate spectrophotometer using an ultraviolet transparent microplate before riboflavin instillation and preand post-crosslinking according to the Dresden protocol. RESULTS: The ultraviolet absorbance profiles of the 180, 300, and 360 µm groups were obtained as α-coefficients of 12.85, 76.55, and 120.27, respectively. A theoretical formula was calculated though a statistical analysis that demonstrated the correlation between stromal lamellar thickness and ultraviolet absorbance. CONCLUSIONS: Corneal thickness and ultraviolet-A spectral absorbance of corneal lamellas showed linear correlation. These findings can potentially contribute to the optimization of ultraviolet-A application during crosslinking, making the treatment of corneas with thickness <400 µm safe and personalized energy delivery for each corneal thickness.

A novel metalloproteinase-derived cryptide from Bothrops cotiara venom inhibits angiotensin-converting enzyme activity.

Snake venoms are primarily composed of proteins and peptides, which selectively interact with specific molecular targets, disrupting prey homeostasis. Identifying toxins and the mechanisms involved in envenoming can lead to the discovery of new drugs based on natural peptide scaffolds. In this study, we used mass spectrometry-based peptidomics to sequence 197 peptides in the venom of Bothrops cotiara, including a novel 7-residue peptide derived from a snake venom metalloproteinase. This peptide, named Bc-7a, features a pyroglutamic acid at the N-terminal and a PFR motif at the C-terminal, homologous to bradykinin. Using FRET (fluorescence resonance energy transfer) substrate assays, we demonstrated that Bc-7a strongly inhibits the two domains of angiotensin converting enzyme (Ki < 1 µM). Our findings contribute to the repertoire of biologically active peptides from snake venoms capable of inhibiting angiotensin-converting enzyme (ACE), beyond current known structural motifs and precursors. In summary, we report a novel snake venom peptide with ACE inhibitory activity, suggesting its potential contribution to the hypotensive effect observed in envenomation.

Ultraviolet-A absorbance analysis in thin porcine corneas pre-and post-crosslinking

ABSTRACT Purpose: To determine the absorbance coefficient of the thin porcine cornea to ultraviolet-A radiation (365 nm) submitted for crosslinking. Methods: This in vitro, benchtop experiment using cadaver tissue study analyzed 12 porcine corneal lamellas, which were obtained using a microkeratome after mechanical de-epithelization and separated into three thickness groups: 180, 300, and 360 μm. The corneal thickness values were measured by anterior-segment optical coherence tomography. All lamellas had ultraviolet-A (365 nm) absorbance measured with a 96-well plate spectrophotometer using an ultraviolet transparent microplate before riboflavin instillation and preand post-crosslinking according to the Dresden protocol. Results: The ultraviolet absorbance profiles of the 180, 300, and 360 μm groups were obtained as α-coefficients of 12.85, 76.55, and 120.27, respectively. A theoretical formula was calculated though a statistical analysis that demonstrated the correlation between stromal lamellar thickness and ultraviolet absorbance. Conclusions: Corneal thickness and ultraviolet-A spectral absorbance of corneal lamellas showed linear correlation. These findings can potentially contribute to the optimization of ultraviolet-A application during crosslinking, making the treatment of corneas with thickness <400 μm safe and personalized energy delivery for each corneal thickness.

Spectrophotometric Analysis of Different Polymethyl Methacrylate Filters and their Importance in the Implantation of Corneal Rings.

PURPOSE: This study evaluated the luminous behavior applied to materials used in intraocular surgeries. METHODS: Discs of the different products were delivered in 19.00 mm × 3.00 mm. Each sample was fixed on support keeping it perpendicular to the spectrophotometer beam. Later, their analyses were carried out in the air/PMMA ratio. The graphs of individual profiles of the measurements along the length were constructed according to each of the filters from the spectrophotometric analysis. In addition, descriptive statistics of transmittance and absorbance for each wavelength presented were correlated for each filter. RESULTS: It is possible to observe that the minimum absorption measure was found in the Red Filter, especially in the blue and green light spectrum. CONCLUSION: Using filters in PMMA materials appears to improve visual quality in corneal implants, especially the red filter, due to greater absorbance of light leading to fewer light scattering phenomena through corneal rings. However, further studies comparing the effects of different filters on Intracorneal rings should be carried out to elucidate this field of study.

Biocompatibility of polyvinyl alcohol/trisodium trimetaphosphate as vitreous substitute in experimental vitrectomy model in rabbits.

Synthetic hydrogels have been proposed as vitreous substitutes recently. This study aims to evaluate the biocompatibility of polyvinyl alcohol (PVA) crosslinked with trisodium trimetaphosphate (SMTP) hydrogel in rabbit vitrectomized eyes. Seven animals were submitted to pars plana vitrectomy and the vitreous was replaced by PVA/SMTP hydrogel. Optical coherence tomography, fluorescein angiogram, clinical, and electrophysiological (ERG) examinations were analyzed at baseline, on postoperative days 7 and 30. The fellow eye was used as the control group. Hydrogel opacification was observed and ERG recordings were reduced in the hydrogel group in rod response, b-wave cone response and flicker. A histological analysis showed retinal disorganization, presence of multinucleated cells, and intraretinal hydrogel particles. The PVA/SMTP hydrogel showed poor biocompatibility. Novel biomaterials compounds should be analyzed in vivo.

Characterization of PVA/glutaraldehyde hydrogels obtained using Central Composite Rotatable Design (CCRD).

Hydrogels are made from natural or synthetic polymers and, currently, they have many biomedical applications. In this work, the conditions for obtaining a hydrogel with similar physicochemical characteristics to the vitreous humor were defined using polyvinyl alcohol (PVA) and glutaraldehyde (GLUT) as cross-linker. The concentration of PVA and GLUT were modified, and their effect was analyzed in terms of the refractive index, density, and dynamic viscosity. The hydrogel which was obtained using 3.98% (w/V) of PVA, 3.13 mL (1.57 g) of GLUT in 100 mL, and the initial pH of 7.2 showed similar characteristics to the vitreous humor (density = 1.0174 ± 0.0050 g mL-1 , dynamic viscosity = 3.7425 ± 0.1800 mPa s and refractive index = 1.3410 ± 0.0010). The hydrogels were further investigated by rheological measurements, infrared spectroscopy, differential scanning calorimetry, X-ray diffraction and determination of swelling degree. The reticulation with GLUT promoted an increase in viscosity and glass transition temperature. On the other hand, it stimulated a decrease in the swelling degree, crystallinity, melting temperature, and intensity of the band related to the -OH bond, compared with the PVA without reticulation. The reticulated hydrogel displayed Newtonian behavior and a higher apparent viscosity than the PVA. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1558-1566, 2018.

Paramagnetic bradykinin analogues as substrates for angiotensin I-converting enzyme: Pharmacological and conformation studies.

This study uses EPR, CD, and fluorescence spectroscopy to examine the structure of bradykinin (BK) analogues attaching the paramagnetic amino acid-type Toac (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 3, 7, and 9. The data were correlated with the potencies in muscle contractile experiments and the substrate properties towards the angiotensin I-converting enzyme (ACE). A study of the biological activities in guinea pig ileum and rat uterus indicated that only Toac0-BK partially maintained its native biological potency among the tested peptides. This and its counterpart, Toac3-BK, maintained the ability to act as ACE substrates. These results indicate that peptides bearing Toac probe far from the ACE cleavage sites were more susceptible to hydrolysis by ACE. The results also emphasize the existence of a finer control for BK-receptor interaction than for BK binding at the catalytic site of this metallodipetidase. The kinetic kcat/Km values decreased from 202.7 to 38.9µM-1min-1 for BK and Toac3-BK, respectively. EPR, CD, and fluorescence experiments reveal a direct relationship between the structure and activity of these paramagnetic peptides. In contrast to the turn-folded structures of the Toac-internally labeled peptides, more extended conformations were displayed by N- or C-terminally Toac-labeled analogues. Lastly, this work supports the feasibility of monitoring the progress of the ACE-hydrolytic process of Toac-attached peptides by examining time-dependent EPR spectral variations.

Conditions for obtaining polyvinyl alcohol/trisodium trimetaphosphate hydrogels as vitreous humor substitute.

Hydrogels are polymeric materials with numerous medical and biological applications because of their physicochemical properties. In this context, the conditions were defined for obtaining a hydrogel with characteristics similar to the vitreous humor using polyvinyl alcohol (PVA) and trisodium trimetaphosphate (STMP). The concentration of PVA (X1 ), PVA/STMP ratio (X2 ), and initial pH (X3 ) were modified, and their effect was analyzed in terms of the refractive index (Y1 ), density (Y2 ), dynamic viscosity (Y3 ), and final pH (Y4 ). The results demonstrated that X1 interferes with Y1 , Y2 , and Y3 , and X2 interferes with Y2 and Y3 . The best condition for obtaining a hydrogel with characteristics similar to the vitreous humor was 4.2586% PVA (wt/wt), STMP/PVA ratio of 1:6.8213 (wt/wt), and initial pH of 9.424. DSC, ATR-FTIR, swelling degree, and AFM analysis confirmed the PVA reticulation with STMP. Furthermore, STMP increased the glass transition temperature and decreased the water uptake of ∼50% of the hydrogels, which can be explained by the crosslinking of PVA chains. Infrared spectroscopy revealed a decrease of hydroxyl bonds and confirmed the reticulation between PVA and STMP. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1386-1395, 2016.

Angiotensin-converting enzyme in pericardial fluid: comparative study with serum activity.

BACKGROUND: The characterization of an angiotensin-converting enzyme (ACE) in human pericardial fluid is relevant, considering its role in the angiotensin II release and thus, the role of the pericardium in cardiovascular homeostasis. OBJECTIVE: To isolate and characterize an ACE from human pericardial fluid and to compare the angiotensin I converting activities of the pericardial fluid with that of the serum in patients submitted to cardiovascular surgery. METHODS: The enzyme from human pericardial fluid was purified through chromatographic steps and characterized by polyacrylamide gel electrophoresis (SDS-PAGE), hydrolysis of angiotensin I, bradykinin, Hip-His-Leu and synthetic substrates with internal fluorescence suppression. Lisinopril was used as inhibitor. The ACE activity was measured in blood and pericardial fluid samples of 23 patients submitted to cardiovascular surgery. RESULTS: The purified ACE (MM = 140 kDa), releases angiotensin II, hydrolyses bradykinin and the Hip-His-Leu substrate. The kinetic parameters k cat,(s-1) and k cat/Km (microM-1. s-1) were, respectively: Hip-His-Leu (1.14 and 7 x 10 -4) ; Abz-YRK(Dnp)P-OH (2.60 and 0.77), Abz-LFK(Dnp)-OH (2.77 and 0.36) and Abz-SDK(Dnp)P-OH (1.92 and 0.19). The angiotensin I converting activities (mean +/- SD) in the pericardial fluid and in blood, were, respectively: 3.16 +/- 0.90 mU x mg -1x min-1 and 0.33 +/- 0.11 mU x mg -1x min-1. The difference was significant between the two fluids. CONCLUSION: An ACE that bears great similarity with the somatic enzyme was isolated from human pericardial fluid. The angiotensin I converting activity is higher in the pericardial fluid when compared to the serum activity. These data are important evidence of the role of the pericardial fluid in the metabolism of active peptides.

Enzima conversora de angiotensina no líquido pericárdico: estudo comparativo com a atividade sérica / Angiotensin-converting enzyme in pericardial fluid: comparative study with serum activity

FUNDAMENTO: A caracterização de uma enzima conversora de angiotensina (ECA) no líquido pericárdico humano é relevante diante do seu papel na liberação de angiotensina II e, portanto, do papel do pericárdio na homeostase cardivascular. OBJETIVO: Isolar e caracterizar uma ECA do líquido pericárdico humano. Comparar as atividades conversoras de angiotensina I do fluido pericárdico e do soro de pacientes submetidos à cirurgia cardiovascular. MÉTODOS: A enzima do líquido pericárdico humano foi purificada por meio de etapas cromatográficas e caracterizada por eletroforese em gel de poliacrilamida (SDS-PAGE), hidrólise de angiotensina I, bradicinina, Hip-His-Leu e substratos sintéticos com supressão interna de fluorescência. Lisinopril foi usado como inibidor. A atividade de ECA foi determinada em amostras de sangue e líquido pericárdico de 23 pacientes submetidos à cirurgia cardiovascular. RESULTADOS: A ECA purificada (MM = 140 kDa) libera angiotensina II, hidrolisa a bradicinina e o substrato Hip-His-Leu. Os parâmetros cinéticos k cat,(s-1) e k cat/Km (µM-1. s-1) foram respectivamente: Hip-His-Leu (1,14 e 7 x 10 -4), Abz-YRK(Dnp)P-OH (2,60 e 0,77), Abz-LFK(Dnp)-OH (2,77 e 0,36) e Abz-SDK(Dnp)P-OH (1,92 e 0,19). As atividades conversoras de angiotensina I (média ± DP) do líquido pericárdico e no soro foram, respectivamente, 3,16 ± 0,90 mU x mg -1x min-1 e 0,33 ± 0,11 mU x mg -1x min-1 . A diferença foi significativa entre os dois fluidos. CONCLUSÃO: Uma ECA com grande similaridade com a enzima somática foi isolada do fluido pericárdico humano. A atividade conversora de angiotensina I é maior no líquido pericárdico quando comparada com a atividade do soro. Esses dados constituem importante evidência do papel do líquido pericárdico no metabolismo de peptídeos ativos.

BACKGROUND: The characterization of an angiotensin-converting enzyme (ACE) in human pericardial fluid is relevant, considering its role in the angiotensin II release and thus, the role of the pericardium in cardiovascular homeostasis. OBJECTIVE: To isolate and characterize an ACE from human pericardial fluid and to compare the angiotensin I converting activities of the pericardial fluid with that of the serum in patients submitted to cardiovascular surgery. METHODS: The enzyme from human pericardial fluid was purified through chromatographic steps and characterized by polyacrylamide gel electrophoresis (SDS-PAGE), hydrolysis of angiotensin I, bradykinin, Hip-His-Leu and synthetic substrates with internal fluorescence suppression. Lisinopril was used as inhibitor. The ACE activity was measured in blood and pericardial fluid samples of 23 patients submitted to cardiovascular surgery. RESULTS: The purified ACE (MM = 140 kDa), releases angiotensin II, hydrolyses bradykinin and the Hip-His-Leu substrate. The kinetic parameters k cat,(s-1) and k cat/Km (µM-1. s-1) were, respectively: Hip-His-Leu (1.14 and 7 x 10 -4) ; Abz-YRK(Dnp)P-OH (2.60 and 0.77), Abz-LFK(Dnp)-OH (2.77 and 0.36) and Abz-SDK(Dnp)P-OH (1.92 and 0.19). The angiotensin I converting activities (mean ± SD) in the pericardial fluid and in blood, were, respectively: 3.16 ± 0.90 mU x mg -1x min-1 and 0.33 ± 0.11 mU x mg -1x min-1. The difference was significant between the two fluids. CONCLUSION: An ACE that bears great similarity with the somatic enzyme was isolated from human pericardial fluid. The angiotensin I converting activity is higher in the pericardial fluid when compared to the serum activity. These data are important evidence of the role of the pericardial fluid in the metabolism of active peptides.

Analogues containing the paramagnetic amino acid TOAC as substrates for angiotensin I-converting enzyme.

The angiotensin I-converting enzyme (ACE) converts the decapeptide angiotensin I (Ang I) into angiotensin II by releasing the C-terminal dipeptide. A novel approach combining enzymatic and electron paramagnetic resonance (EPR) studies was developed to determine the enzyme effect on Ang I containing the paramagnetic 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) at positions 1, 3, 8, and 9. Biological assays indicated that TOAC(1)-Ang I maintained partly the Ang I activity, and that only this derivative and the TOAC(3)-Ang I were cleaved by ACE. Quenching of Tyr(4) fluorescence by TOAC decreased with increasing distance between both residues, suggesting an overall partially extended structure. However, the local bend known to be imposed by the substituted diglycine TOAC is probably responsible for steric hindrance, not allowing the analogues containing TOAC at positions 8 and 9 to act as substrates. In some cases, although substrates and products differ by only two residues, the difference between their EPR spectral lineshapes allows monitoring the enzymatic reaction as a function of time.

Estudo da especificidade dos sítios catalíticos da enzima conversora de angiotensina I e desenvolvimento de substratos seletivos para o domínio C / Specificirty study of the catalytic sites of angiotensin I converting enzyme and development of selective substrates for C-domain

No presente trabalho, usamos bibliotecas combinatórias de peptídeos com supressão intramolecular de fluorescência para fazer uni estudo detalhado da especificidade dos subsítios S3 a S1’ da enzima conversora de angiotensina 1 (ECA). Estes estudos permitiram o desenvolvimento de substratos seletivos para o domínio C da enzima. Para definir a especificidade do subsítio S1, ensaiamos uma primeira biblioteca com estrutura geral Abz-GXXZXK(Dnp)-0H, onde Z representa um dos 19 aminoácidos naturais (cisteína foi omitida para evitar dimerização) e X uma mistura destes aminoácidos incorporados aleatoriamente. Nossos resultados mostraram que os peptídeos contendo Arg e Leu em P1, apresentaram maior seletividade para o domínio C da enzima. As especificidades dos subsítios S1', S2 e S3 foram estudadas com as bibliotecas Abz-GXXRZK(Dnp)-OH, Abz-GXZRXK(Dnp)-OH e Abz-GZXRXK(Dnp)-OH, respectivamente. Nestas bibliotecas, um resíduo de Arg foi fixado na posição P1 para facilitar a solubilização dos peptídeos. Os nossos estudos com as bibliotecas permitiram demonstrar que o domínio C tem preferência por Phe em P1', Ile e Val em P3. Com base nestes resultados foram sintetizados os peptídeos Abz-GVILFK(Dnp)-OH e Abz-GVIRFK(Dnp)-OH, que contém os resíduos mais favoráveis em cada posição para o domínio C (P3-P1'). A redução gradativa da cadeia peptídica levou a um aumento de especificidade para este domínio. O substrato Abz-LFK(Dnp)-0H foi hidrolisado com eficiência catalítica 70 vezes maior pela ECA recombinante C-domínio (kcat/KM=36.7 M-1.s-1) do que pela ECA recombinante N-domínio (kcat/Km=0.51 M-1.s-1). Esta série de compostos foi testada também com a ECA purificada de testículo humano. Na segunda etapa do nosso trabalho estudamos uma isoforma de ECA, de aproximadamente 140 kDa, isolada de fluído de pericárdio humano. A enzima hidrolisou os substratos Abz-YRK(Dnp)P-0H (kcat=2,50s-1;Km=3,4 M; kcat/Km= 770 mM-1.s-1), Abz-LFK(Dnp)OH (kcat =2,77 s-1; Km= 7,7 M; kcat/Km= 358 mM-1.s-1) e Hip-His-Leu (kcat=1,14 s-1; Km,=1654 M; kcat/Km=0,69 mM-1.s-1)...