Evaluation of secretory activity in carp pancreatocytes (Cyprinus carpio L. ) when using lactobacillus-based chelate and probiotic supplemental feed complexes / Avaliação da atividade secretora em pancreatócitos de carpa (Cyprinus carpio L. ) ao usar quelato à base de lactobacilos e complexos suplementares de alimentação probiótica

The experimental research was carried out on the juvenile carp (Cyprinus carpio L.). The impact from supplemental feeds consisting of variable concentrations of chelate compounds, biogenic trace elements and probiotic lactobacillus-based product Bacillus subtilis VKPM B-2335 was evaluated. Optical and qualitative parameters of the lactobacillus base were studied in order to identify the major group of substances potentially able to influence the end result. The purpose of this research was to identify changes in the structure of the zymogen granules and their dimensions at which supplemental feeds produce a stimulating effect on the synthesis of zymogens in exogenous cells of the secretory part of pancreas. At the outcome of the study, for the first time, it was possible to prove that the integrated action of chelates and lactobacillus-based probiotics complemented each other. Metal chelate compounds contributed to enlargement of the zymogen granules, if compared to the control values. The bacterial products accelerated production of the zymogen granules in acinar cells diffusely located in carp hepatopancreas.

A pesquisa experimental foi realizada em carpas juvenis (Cyprinus carpio L.). O impacto de suplementos alimentares consistindo em concentrações variáveis de compostos quelatos, oligoelementos biogênicos e produto probiótico baseado em lactobacilos Bacillus subtilis VKPM B-2335 foi avaliado. Parâmetros ópticos e qualitativos da base de lactobacilos foram estudados a fim de identificar o maior grupo de substâncias potencialmente capazes de influenciar o resultado final. O objetivo dessa pesquisa foi identificar mudanças na estrutura dos grânulos de zimogênio e suas dimensões nas quais alimentos suplementares produzem um efeito estimulante na síntese de zimogênios em células exógenas da parte secretora do pâncreas. No resultado do estudo, pela primeira vez, foi possível comprovar que a ação integrada dos quelatos e dos probióticos à base de lactobacilos se complementava. Compostos quelatos metálicos contribuíram para o aumento dos grânulos de zimogênio, se comparados aos valores de controle. Os produtos bacterianos aceleraram a produção dos grânulos de zimogênio nas células acinares localizadas difusamente no hepatopâncreas da carpa.

Partial characterization of digestive proteases in sheepshead, Archosargus probatocephalus (Spariformes: Sparidae)

Digestive proteases were partially characterized in sheepshead juveniles, using biochemical and electrophoretic techniques. Results showed higher activity level of the stomach proteases (2.39 ± 0.02 U mg protein-1) compared to the intestinal proteases (1.6 ± 0.1 U mg protein-1). The activity of trypsin, chymotrypsin, leucine aminopeptidase and carboxypeptidase A was also recorded. The optimum temperature of the stomach proteases was recorded at 45 °C, while for intestinal proteases was recorded at 55 °C. Stomach proteases showed less stability to temperature changes than intestinal proteases. An optimum pH of 2 was recorded for stomach proteases with high stability under acidic conditions, while an optimum pH of 9 was recorded for intestinal proteases showing high stability under alkaline conditions. Stomach proteases were inhibited around 78% with Pepstatin A, indicating the presence of pepsin as the main protease. The stomach proteases zymogam revealed one active band with Rf of 0.49, this enzyme was completely inhibited by Pepstatin A. The intestinal proteases zymogram revealed four active proteases (51.3, 34.9, 27.8 and 21.2 kDa) that were inhibited by TLCK, which mainly represent a trypsin-like serine proteases. It can be conclude that digestion in sheepshead can be considered as a carnivorous species with an omnivorous tendency.(AU)

Se caracterizaron parcialmente las proteasas digestivas de juveniles del sargo, utilizando técnicas bioquímicas y electroforéticas. Los resultados muestran mayores niveles de actividad en las proteasas estomacales (2.39 ± 0.02 U mg proteina-1) comparados con los de las proteasas intestinales (1.6 ± 0.1 U mg protein-1), también se registró la actividad de tripsina, quimotripsina, leucina aminopeptidasa y carboxipeptidasa A. La temperatura óptima de las proteasas estomacales fue de 45 °C, mientras que la de las proteasas intestinales fue de 55 °C. El pH óptimo fue de 2 para las proteasas estomacales con alta estabilidad a condiciones ácidas, mientras que el pH óptimo para las proteasas intestinales fue de 9, mostrando una alta estabilidad en condiciones alcalinas. Las actividades de las proteasas estomacales fue inhibida en un 78% con Pepstatina A, lo que indica la presencia de pepsina, como principal proteasa. El zimograma de proteasas estomacales reveló una sola banda con actividad proteasa, con Rf de 0.49, completamente inhibida por Pepstatina A. El zimograma de proteasas intestinales reveló cuatro bandas (51.3, 34.9, 27.8 y 21.2 kDa). Todas las bandas se inhibieron con TLCK, lo que muestra la presencia principalmente de serina proteasas tipo tripsina. Se concluye que la digestión del sargo puede ser considerada como la de una especie carnívora con tendencia al omnivorismo.(AU)

Estratégias para o enfrentamento do absenteísmo em consultas odontológicas nas Unidades de Saúde da Família de um município de grande porte: uma pesquisa-ação / Strategies for tackling absenteeism in dental appointments in the Family Health Units of a large municipality: action research

O objetivo deste trabalho foi analisar os motivos das faltas às consultas odontológicas em Unidades de Saúde da Família (USF) e implementar estratégias para sua redução por meio da pesquisa-ação. O estudo foi realizado em 12 USF de Piracicaba/SP, de 01 de janeiro a 31 de dezembro de 2010. A amostra se consistiu de 385 usuários, entrevistados por telefone, sobre os motivos das faltas, além de 12 cirurgiões-dentistas e 12 enfermeiras. Realizaram-se duas oficinas com os profissionais: uma para problematização dos dados coletados nas entrevistas e elaboração de estratégias; e outra após 4 meses, para avaliação. O maior motivo de faltas foi a coincidência do horário de funcionamento das unidades com o de trabalho dos usuários. Dentre as estratégias ressaltou-se a realização de palestras sobre saúde bucal, educação permanente nas reuniões de equipe, capacitação dos Agentes Comunitários de Saúde, participação em grupos terapêuticos e parcerias entre Equipe de Saúde Bucal e equipamentos sociais da comunidade. A adoção de prontuário único foi a estratégia desafiadora encontrada pelos profissionais. Concluiu-se que as estratégias implementadas levaram à diminuição das faltas em 66,6% e o caráter motivador das oficinas possibilitou a reflexão crítica para o redirecionamento da prática em saúde.

The aim of this study was to analyze the reasons for missed appointments in dental Family Health Units (FHU) and implement strategies to reduce same through action research. This is a study conducted in 12 FHUs in Piracicaba in the State of São Paulo from January, 1 to December, 31 2010. The sample was composed of 385 users of these health units who were interviewed over the phone and asked about the reasons for missing dental appointments, as well as 12 dentists and 12 nurses. Two workshops were staged with professionals: the first to assess the data collected in interviews and develop strategy, and the second for evaluation after 4 months. The primary cause for missed appointments was the opening hours of the units coinciding with the work schedule of the users. Among the strategies suggested were lectures on oral health, ongoing education in team meetings, training of Community Health Agents, participation in therapeutic groups and partnerships between Oral Health Teams and the social infrastructure of the community. The adoption of the single medical record was the strategy proposed by professionals. The strategies implemented led to a 66.6% reduction in missed appointments by the units and the motivating nature of the workshops elicited critical reflection to redirect health practices.

Pro-prostate-specific antigen and its related indexes in the diagnosis of prostate cancer / 中华男科学杂志

Pro-prostate-specific antigen (proPSA) is the precursor of PSA and a form of free PSA (fPSA). In recent years, a lot of studies have been done on proPSA, the roles of its related indexes in the diagnosis of prostate cancer, and the value of its clinical application. The correlated indexes of proPSA include proPSA, % pPSA, p2PSA, % p2PSA and prostate health index (PHI). They are more effective than total PSA (tPSA) and fPSA in the diagnosis of prostate cancer, especially % p2PSA and PHI, which may significantly increase our ability to detect and identify PCa and lower the rate of unnecessary biopsies. This article presents an overview on the advances in the studies of proPSA and the application of its related indexes in the diagnosis of prostate cancer.

Listeriosis en México: importancia clínica y epidemiológica / Listeriosis in Mexico: Clinical and epidemiological importance

La listeriosis es una enfermedad transmitida por alimentos (ETA) y ocasionada por Listeria monocytogenes. La importancia de ésta se debe a su impacto clínico, la alta tasa de mortalidad y el efecto económico derivado de los brotes asociados con el consumo de alimentos. En México, las fallas en los sistemas de vigilancia epidemiológicos son causa de información imprecisa sobre la incidencia de la listeriosis y sobre su caracterización como ETA. En este trabajo se presentan datos referentes a la presencia de la bacteria en alimentos, reportes de casos de la enfermedad y patologías relacionadas con infección por L. monocytogenes. La falta de datos exactos sobre la importancia de esta bacteria plantea la necesidad de concientizar a las instancias correspondientes para definir estrategias de búsqueda intencionada de L. monocytogenes en alimentos y de la recopilación de información clínica precisa que permita conocer la importancia clínica y epidemiológica de la listeriosis en México.

Listeriosis is caused by Listeria monocytogenes, an important food-borne disease due to its clinical forms, high mortality rate, and the economic impact in both clinical and food production industries. In Mexico, the lack of epidemiological surveillance systems leads to the need of accurate data on the incidence of listeriosis and its association with food-borne disease. In this paper, we present data about the presence of this bacterium in food, reports related to clinical cases of listeriosis, and information of diseases in which L. monocytogenes may be involved. However, in most of these cases the etiology was not established. Given this, there's a need to inform and warn the appropriate entities, to define strategies for the mandatory search of L. monocytogenes through the whole food production chain and clinical suspects, for the epidemiological importance and control of listeriosis in Mexico.

Expression of limulus Factor C in silkworm larvae by Bac-to-Bac/BmNPV baculovirus expression system / 生物工程学报

Limulus Factor C, a serine protease zymogen from the amoebocytes of the limulus, has high affinity for endotoxin. When Factor C is activated by endotoxin, it hydrolyses artificial tripeptide substrate and measurable products are released, so it can be used as an alternative reagent for endotoxin analysis. Factor C gene of Tachypleus tridentatus was obtained through RT-PCR and the recombinant protein was expressed by Bac-to-Bac/BmNPV baculovirus expression system in silkworm larvae. The activity of Factor C was detected with diluted serum of silkworm larvae, and the sensitivity of endotoxin detected was 0.2 EU/mL when the serum was diluted at 1:500. The silkworm larvae expressed limulus Factor C could be used to develop a new low-cost endotoxin test reagent.

Role of (pro)rennin receptor in cardiomyocytes of heart failure rat model / 华中科技大学学报（医学）（英德文版）

The role of (pro)rennin receptor (PRR) in cardiomyocytes of a heart failure (HF) rat model was studied. Spontaneously hypertensive rats (SHR) with HF (SHR-HF) or not were identified by two-dimensional (2-D) ultrasound. Age-matched Wistar Kyoto normotensive (WKY) rats were used as controls. PRR short hair RNA (sh-RNA) was injected into the heart of SHR-HF. Simultaneously SHR and controls received the same shRNA injection into the heart. Scramble shRNA was injected into the heart as controls. The expression of PRR mRNA and protein in cardiomyocytes was detected by using real-time PCR and Western blotting respectively. The heart function was evaluated by 2-D ultrasound, including eject fraction (EF%), fractional shortening (FS%), left ventricle thickness (LV), and inter-ventricular septal thickness (IVS). The number of apoptotic cardiomyocytes was counted by using flow cytometry. The results showed that the mRNA and protein expression levels of PRR were significantly higher in cardiomyocytes of SHR-HF group than in those of SHR group or control group. The apoptosis of myocytes in SHR-HF group was increased as compared with SHR group or control group. After knock-down of PRR with shRNA in SHR-HF group, the apoptosis of myocytes was reduced, resulting in the improved heart function. It was suggested that down-regulation of PRR might protect the heart from development of HF in SHR-HF by inhibiting the apoptosis of cardiomyocytes.

Propeptide processing and proteolytic activity of proenzymes of the Staphylococcal and Enterococcal GluV8-family protease.

Proenzymes with various lengths of propeptides have been observed in GluV8 from Staphylococcus aureus and GluSE from S. epidermidis. However, the production mechanism of these proenzymes and roles of truncated propeptides have yet to be elucidated. Here we demonstrate that shortening of propeptide commonly occurs in an auto-catalytic manner in GluV8-family members, including those from coagulase negative Staphylococci and Enterococcus faecalis. Accompanied with propeptide shortening, the pro-mature junction (Asn/Ser-1-Val1) becomes more susceptible towards the hetero-catalytic maturation enzymes. The auto-catalytic propeptide truncation is not observed in Ser169Ala inert molecules of GluV8-family members. A faint proteolytic activity of proenzymes from Staphylococcus caprae and E. faecalis is detected. In addition, proteolytic activity of proenzyme of GluV8 carrying Arg-3AlaAsn-1 is demonstrated with synthetic peptide substrates LLE/Q-MCA. These results suggest that GluV8-family proenzymes with shortened propeptides intrinsically possess proteolytic activity and are involved in the propeptide shortening that facilitates the final hetero-catalytic maturation.

Codon optimization, expression and enzymatic comparison of Rhizopus oryzae lipases pro-ROL and m-ROL in Pichia pastoris / 生物工程学报

Rhizopus oryzae lipase (ROL) is not only a biocatalyst used in a broad range of biotechnological fields, but also a model to investigate the function of intramolecular chaperone in the post-translational processing of lipase. In this study, we cloned and expressed the mature lipase gene (m-ROL) containing the pre-sequence (pro-ROL) of R. oryzae HU3005 in Pichia pastoris GS115 and characterized their enzymatic activities. m-ROL exhibited higher hydrolysis activity towards middle-chain substrates (C10 and C12) at pH 9.0, whereas pro-ROL preferred short-chain substrates (C4) and displayed maximal activity at pH 8.0. Moreover, pro-ROL possessed better thermal stability than m-ROL. This enzymatic discrepancy between m-ROL and p-ROL may be due to the pre-sequence that affects the folding and conformation of the mature lipase domain. To improve the expression level of m-ROL in P. pastoris, overlap extension PCR was conducted to substitute eight less-frequently used codons of m-ROL with frequently used codons. After methanol-induced expression for 72 h, the activity and protein content of the codon optimized m-ROL reached 132.7 U/mL and 50.4 mg/L, while the activity of the parental m-ROL and pro-ROL are 28.7 U/mL and 14.4 mg/L, 29.6 U/mL and 14.1 mg/L, respectively.

Distribution and relative activity of matrix metalloproteinase-2 in human coronal dentin / 国际口腔科学杂志·英文版

The presence of matrix metalloproteinase-2 (MMP-2) in dentin has been reported, but its distribution and activity level in mature human coronal dentin are not well understood. The purpose of this study was to determine the MMP-2 distribution and relative activity in demineralized dentin. Crowns of twenty eight human molars were sectioned into inner (ID), middle (MD), and outer dentin (OD) regions and demineralized. MMP-2 was extracted with 0.33 mol x L(-1) EDTA/2 mol xL(-1) guanidine-HCl, pH 7.4, and MMP-2 concentration was estimated with enzyme-linked immunoabsorbant assay (ELISA). Further characterization was accomplished by Western blotting analysis and gelatin zymography. The mean concentrations of MMP-2 per mg dentin protein in the dentin regions were significantly different (P = 0.043): 0.9 ng (ID), 0.4 ng (MD), and 2.2 ng (OD), respectively. The pattern of MMP-2 concentration was OD > ID > MD. Western blotting analysis detected -.66 and -72 kDa immunopositive proteins corresponding to pro- and mature MMP-2, respectively, in the ID and MD, and a -66 kDa protein in the OD. Gelatinolytic activity consistent with MMP-2 was detected in all regions. Interestingly, the pattern of levels of Western blot immunodetection and gelatinolytic activity was MD > ID > OD. The concentration of MMP-2 in human coronal dentin was highest in the region of dentin that contains the dentinoenamel junction and least in the middle region of dentin. However, levels of Western blot immunodetection and gelatinolytic activity did not correlate with the estimated regional concentrations of MMP-2, potentially indicating region specific protein interactions.

Gene synthesis of the bovine prochymosin gene and high-level expression in Kluyvermyces lactis / 生物工程学报

Chymosin is an important industrial enzyme widely used in cheese manufacture. To improve expression efficiency of recombinant bovine chymosin in Kluyveromyces lactis strain GG799, we designed and synthesized a DNA sequence encoding bovine prochymosin gene (GenBank Accession No. AA30448) by using optimized codons. The synthesized prochymosin gene was amplified by two-step PCR method, and then cloned into the expression vector pKLAC1, resulting in pKLAC1-Prochy. pKLAC1-Prochy was linearized and transformed into K. lactis GG799 by electrotransformation. Positive clones were screened by YEPD plates containing 1% casein. A recombinant strain chyl with highest activities and multi-copy integration which was detected by using specifical integration primers was chosen and fermented in flask. Prochymosin was expressed in K. lactis successfully. SDS-PAGE analysis revealed that the purified recombinant bovine prochymosin had a molecular mass of 41 kDa. After acid treatment, molecular weight of chymosin is about 36 kDa, the same as native bovine chymosin. Activity tests showed that the chymosin activity of the culture supernatant was 99.67 SU/mL after 96 h cultivation. The activities of chymosin were not prominent increased when galactose was used as carbon source instead of glucose, which proved that the fermentation of recombinant strain does not need galactose inducing. The recombinant K. lactis strain obtained in this study could be further used to produce recombinant chymosin for cheese making.

New approaches toward anti-Rhipicephalus (Boophilus) microplus tick vaccine / Novas estratégias para o desenvolvimento de uma vacina contra o carrapato Rhipicephalus (Boophilus) microplus

The tick Rhipicephalus (Boophilus) microplus (formerly Boophilus microplus) is the major ectoparasite affecting livestock in America, Asia, Africa, and Oceania. Conventional tick control is based on the use of acaricides but immunization of bovines with tick gut proteins induces only a partial protective immune response. Based on this information, distinct research groups have explored the possibility of protecting the animals by inducing an immune response against other tick proteins. However, the antigens so far described do not induce the necessary protection for suppressing the use of acaricides. Currently, several groups are engaged in identifying new tick proteins to be used as targets for the development of new vaccines. This approach focuses on the enhancement of the immunogenicity of antigens already tested by incorporating new adjuvants or formulations and by searching for new antigens. This paper reviews the work done by Brazilian researchers to develop a vaccine against this tick.

O carrapato Rhipicephalus (Boophilus) microplus (anteriormente Boophilus microplus) é o principal ectoparasita que afeta bovinos na América, Ásia, África e Oceania e o seu controle é tradicionalmente realizado através do uso de acaricidas. Experimentos de imunização com proteínas do carrapato mostram que a resposta imune desenvolvida pelos bovinos vacinados protege, em parte, os animais do parasitismo. Baseado nessas observações, vários grupos de pesquisa exploram a possibilidade de proteger os animais pela indução de uma resposta imune contra proteínas do carrapato. Entretanto, os antígenos já caracterizados não asseguram o grau de proteção necessário para suprimir o uso de acaricidas. Portanto, esses grupos de pesquisa estão engajados na tentativa de identificar novas proteínas que possam ser utilizadas para o desenvolvimento de novas vacinas, as quais possam induzir maior imunogenicidade de que os antígenos já testados, através do uso de novas formulações e/ou pela incorporação de adjuvantes. O presente artigo apresenta uma revisão da literatura sobre os resultados obtidos por pesquisadores brasileiros no desenvolvimento de vacinas contra o carrapato.

Recombinant expression of bovine chymosin in Pichia pastoris / 生物工程学报

To express bovine chymosin in yeast, we amplified the prochymosin gene from the plasmid pMD18T-Prochy by PCR, and then cloned the gene into the expression vector pPICZaA, resulting in pPICZaA-Prochy. Pichia pastoris GS115 was used as host cells. Integration of the prochymosin cDNA into the Pichia pastoris genome was confirmed by PCR and sequencing analysis. Chymosin was expressed in Pichia pastoris successfully, and a strong band at about 37 kD was shown by SDS-PAGE. Activity tests showed that the chymosin activity of the culture supernatant was 12.2 SU/mL. This is the first report of successful expression of chymosin in Pichia pastoris. The recombinant Pichia pastoris strain obtained in this study could be further used to produce recombinant chymosin for cheese making.

Nucleotide sequence of cDNA encoding for preprochymosin in native goat [Capra hircus] from Iran

Prochymosin is one of the most important aspartic proteinases used as a milk-clotting enzyme in cheese production. In the present investigation we report sequence of cDNA encoding goat [Capra hircus] preprochymosin and compare its nucleotide and deduced amino acid sequences with sequences of other ruminants preprochymosin. As bovine prochymosin, the caprine prochymosin cDNA encodes 365 amino acids with a prosegment of 42 amino acids and the mature goat chymosin begins with glycine. The preprochymosin nucleotide sequence reported in this study differs from other reported goat sequence [AY389343] in three nucleotides, two of which alter the amino acids at positions 19p and 139

Improvement on the activity of microbial transglutaminase with Streptomyces hygroscopicus by the addition of surfactant CTAB / 生物工程学报

Effect of CTAB addition on the accumulation of microbial transglutaminase (MTG) with Streptomyces hygroscopicus was investigated. The results showed that the addition of CTAB enhanced MTG accumulation, and the optimal addition time and concentration of CTAB were 32 h and 1%. The maximum MTG activity in the culture broth was 5.04 u/mL and increased by 21.8% compared with the control. With the addition of CTAB, pro-MTG was activated to become MTG. CTAB could enhance the production of pro-MTG by relieving the product inhibition, and the accumulation of MTG was improved.

Investigation of the degradation and the mechanisms of artificial basement membrane co-cultured with oral carcinoma-associated fibroblasts / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the degradation of artificial basement membrane (matrigel) co-cultured with oral carcinoma-associated fibroblasts (CAFs) and its possible mechanism.</p><p><b>METHODS</b>CAFs and normal fibroblasts (NFs) were incubated on matrigel for 24, 48, 72 h. Equivalent amounts of conditioned medium were collected and assayed for total protein, hydroxyproline and matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) activity by gelatin zymography.</p><p><b>RESULTS</b>Oral CAFs were superior to oral NFs in total protein and hydroxyproline density, CAFs present more pro-MMP-2 and activated MMP-2.</p><p><b>CONCLUSION</b>CAFs were superior to NFs in degradation of matrigel. CAFs might play a key role in the reconstitution of extracellular matrix and the progression of tumor.</p>

Relationship of the disturbed balance between matrix metalloproteinases and their tissue inhibitors and the invasion of malignant salivary gland tumours / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To detect protein expression of MMPs and TIMPs in various salivary gland neoplasms and to investigate their roles in invasion and metastasis of the malignant salivary gland tumors.</p><p><b>METHODS</b>Immunohistochemistry and Gelatin zymography analyses for MMP-2, MMP-9, MT1-MMP, TIMP-1 and TIMP-2 were performed in 26 malignant and 28 benign salivary gland tumors.</p><p><b>RESULTS</b>The expression of MMP-2 and MMP-9 was significantly higher in carcinomas than in adenomas (P < 0.05). The MMP-2/TIMP-1 and MMP-2/TIMP-2 was also significantly higher in carcinomas than in adenomas (P < 0.05). There was a cooperated effect among MMP-2, MT1-MMP and TIMP-2. The expression of active MMP-2, proMMP-9 and active MMP-9 was significantly higher in malignant tumors than in benign tumors (P < 0.05).</p><p><b>CONCLUSION</b>MMP-2 and MMP-9 may play important roles in invasion of malignant salivary gland tumors. A disturbed balance between MMP-2, MMP-9, TIMP-1 and TIMP-2 in malignant salivary gland tumors was detected. It was the absolute increase of MMP-2 and MMP-9 to induce the unbalance.</p>

Effects of interaction between vascular endothelial cells and monocytes on expression of matrix metalloproteinase-2 and of tissue inhibitor of metalloproteinases 2 and regulation of pravastatin / 中华病理学杂志

<p><b>OBJECTIVE</b>To analyze the effects of interaction between vascular endothelial cells and monocytes on the expression of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2), as well as the regulation of pravastatin.</p><p><b>METHODS</b>A co-cultured system of monocytes and endothelial cells was established through addition of THP-1 to human umbilical vein endothelial cells (HUVECs) in various rates. After 24 hours, the changes in activity and expression of MMP-2 and TIMP-2 in the co-culture system were studied by zymography and reverse zymography. The 1:1 co-culture system was selected and one control group (no pravastatin added) and experimental groups (with concentration of pravastatin being 0.1, 0.5 and 1.0 micromol/ml respectively) were studied. All groups were cultured for another 24 hours and analyzed in the same way.</p><p><b>RESULTS</b>Compared to the single cultured HUVECs, the activity of proMMP-2 in the co-cultured system increased by 2.09, 2.46 and 2.07 folds respectively (number = 8, P < 0.01). There was also activated MMP-2 secretion in the co-culture system. The secretion of proMMP-2 and active MMP-2 in the 1:1 co-cultured system was most obvious. After pravastatin treatment, the activity of proMMP-2 and MMP-2, decreased significantly (number = 8, P < 0.01). MMP-2 secretion was completely suppressed after 1.0 micromol/ml pravastatin treatment. Reverse zymography revealed that, compared to the single culture HUVECs or THP-1, the secretion of TIMP-2 decreased in the co-cultured system, regardless of the ratio of mixture. However, pravastatin had no obvious effect on TIMP-2.</p><p><b>CONCLUSIONS</b>Interaction between vascular endothelial cells and monocytes may contribute to the secretion and activation of MMP-2 and suppress secretion of TIMP-2. Pravastatin may inhibit the secretion and activation of MMP-2.</p>

Delay of Photoreceptor Cell Degeneration in rd Mice by Systemically Administered Phenyl-N-tert-butylnitrone

PURPOSE: To study the effect of systemic administration of phenyl-N-tert-butylnitrone (PBN) on the degeneration of photoreceptor cells in rd mice. METHODS: PBN was injected intraperitoneally into FVB/rd mice on postnatal days (P) 5 to 14 (group A), and P10 to 18 (group B). At days P14, 16, 18, 20 and 27, morphological changes and apoptosis were analyzed by staining with hematoxylin and eosin or DAPI. The effect of PBN on apoptosis was analyzed in retinal pigment epithelial (RPE) cells by the measurement of caspase-3 activity. RESULTS: In control and group B mice, the outer nuclear layer (ONL) of the retina was composed of 8-10 rows at P12, and rapidly decreased to one row at P18. In group A mice, the ONL was preserved with 5-7 rows at P18, and decreased to one row at P22. PBN inhibited caspase-3 activity in cultured RPE cells. CONCLUSIONS: PBN delayed, but did not block, the degeneration of photoreceptor cells in rd mice. PBN may exert its inhibitory effect during the early phase of photoreceptor cell degeneration.