Reviving the use of inhibitors of matrix metalloproteases in spinal cord injury:a case for specificity

At present, there are no restorative therapies in the clinic for spinal cord injury, with current treatments offering only palliative treatment options. The role of matrix metalloproteases is well established in spinal cord injury, however, translation into the clinical space was plagued by early designs of matrix metalloprotease inhibitors that lacked specificity and fears of musculoskeletal syndrome prevented their further development. Newer, much more specific matrix metalloprotease inhibitors have revived the possibility of using these inhibitors in the clinic since they are much more specific to their target matrix metalloproteases. Here, the evidence for use of matrix metalloproteases after spinal cord injury is reviewed and researchers are urged to overcome their old fears regarding matrix metalloprotease inhibition and possible side effects for the field to progress. Recently published work by us shows that inhibition of specific matrix metalloproteases after spinal cord injury holds promise since four key consequences of spinal cord injury could be alleviated by specific, next-generation matrix metalloprotease inhibitors. For example, specific inhibition of matrix metalloprotease-9 and matrix metalloprotease-12 within 24 hours after injury and for 3 days, alleviates spinal cord injury-induced edema, blood-spinal cord barrier breakdown, neuropathic pain and restores sensory and locomotor function. Attempts are now underway to translate this therapy into the clinic

Advances in tenascin-C biology

Tenascin-C is an extracellular matrix glycoprotein that is specifically and transiently expressed upon tissue injury. Upon tissue damage, tenascin-C plays a multitude of different roles that mediate both inflammatory and fibrotic processes to enable effective tissue repair. In the last decade, emerging evidence has demonstrated a vital role for tenascin-C in cardiac and arterial injury, tumor angiogenesis and metastasis, as well as in modulating stem cell behavior. Here we highlight the molecular mechanisms by which tenascin-C mediates these effects and discuss the implications of mis-regulated tenascin-C expression in driving disease pathology

The Effects of Natural Epigenetic Therapies in 3D Ovarian Cancer and Patient-Derived Tumor Explants: New Avenues in Regulating the Cancer Secretome

High mortality rates in ovarian cancer have been linked to recurrence, metastasis, and chemoresistant disease, which are known to involve not only genetic changes but also epigenetic aberrations. In ovarian cancer, adipose-derived stem cells from the omentum (O-ASCs) play a crucial role in supporting the tumor and its tumorigenic microenvironment, further propagating epigenetic abnormalities and dissemination of the disease. Epigallocatechin gallate (EGCG), a DNA methyltransferase inhibitor derived from green tea, and Indole-3-carbinol (I3C), a histone deacetylase inhibitor from cruciferous vegetables, carry promising effects in reprograming aberrant epigenetic modifications in cancer. Therefore, we demonstrate the action of these diet-derived compounds in suppressing the growth of 3D ovarian cancer spheroids or organoids as well as post-treatment cancer recovery through proliferation, migration, invasion, and colony formation assays when compared to the synthetic epigenetic compound Panobinostat with or without standard chemotherapy. Finally, given the regulatory role of the secretome in growth, metastasis, chemoresistance, and relapse of disease, we demonstrate that natural epigenetic compounds can regulate the secretion of protumorigenic growth factors, cytokines, extracellular matrix components, and immunoregulatory markers in human ovarian cancer specimens. While further studies are needed, our results suggest that these treatments could be considered in the future as adjuncts to standard chemotherapy, improving efficiency and patient outcomes

The Effects of Natural Epigenetic Therapies in 3D Ovarian Cancer and Patient-Derived Tumor Explants: New Avenues in Regulating the Cancer Secretome.

High mortality rates in ovarian cancer have been linked to recurrence, metastasis, and chemoresistant disease, which are known to involve not only genetic changes but also epigenetic aberrations. In ovarian cancer, adipose-derived stem cells from the omentum (O-ASCs) play a crucial role in supporting the tumor and its tumorigenic microenvironment, further propagating epigenetic abnormalities and dissemination of the disease. Epigallocatechin gallate (EGCG), a DNA methyltransferase inhibitor derived from green tea, and Indole-3-carbinol (I3C), a histone deacetylase inhibitor from cruciferous vegetables, carry promising effects in reprograming aberrant epigenetic modifications in cancer. Therefore, we demonstrate the action of these diet-derived compounds in suppressing the growth of 3D ovarian cancer spheroids or organoids as well as post-treatment cancer recovery through proliferation, migration, invasion, and colony formation assays when compared to the synthetic epigenetic compound Panobinostat with or without standard chemotherapy. Finally, given the regulatory role of the secretome in growth, metastasis, chemoresistance, and relapse of disease, we demonstrate that natural epigenetic compounds can regulate the secretion of protumorigenic growth factors, cytokines, extracellular matrix components, and immunoregulatory markers in human ovarian cancer specimens. While further studies are needed, our results suggest that these treatments could be considered in the future as adjuncts to standard chemotherapy, improving efficiency and patient outcomes

Effect of Dietary Treatment with Stearidonic Acid and Eicosapentaenoic Acid on Membrane Phospholipid Fatty Acid Composition and Glycogen-Elicited Peritoneal Neutrophil Survival in Absence or Presence of Bacterial Endotoxin

We previously found that enteral nutrition with diets containing elevated levels of eicosapentaenoic acid (EPA; 20:5 n-3) reduced the number of neutrophils in bronchoalveolar lavage fluid and improved clinical outcome of patients with or at risk for adult respiratory distress syndrome (ARDS). We have also shown that the treatment of the neutrophil-like cell line, HL-60, with EPA can induce apoptosis. It is not known, however, if the addition of EPA to the diet would result in similar occurrences in neutrophils. It has been suggested that the use of diets enriched with stearidonic acid (SDA; 18:4 n-3) may provide a novel source of n-3 fatty acids while offering similar beneficial effects on health as diets containing EPA. Therefore, we examined whether dietary SDA can be used as a precursor to increase EPA content in liver tissue. It is well known that endotoxin increases neutrophil lifespan and that this may be important in maintaining innate immune response. The second part of this study incorporated the use of glycogen-elicited peritoneal neutrophils from the rat to examine the effect of dietary intervention with n-3 fatty acids (either EPA or SDA) on constitutive and endotoxin induced neutrophil survival. Male Long-Evans rats (n=18) were randomly divided into 3 dietary groups and fed a modified US-17 diet providing 200kcal/kg/day and containing 2% oleic acid (OA; 18:1 n-9) (n=6), 1% EPA/1% OA (n=6), or 1% SDA/1% OA (n=6) for 2 weeks. Liver phospholipids were analyzed using gas chromatography and neutrophil lifespan was evaluated using flow cytometry. Dietary supplementation with EPA significantly increased the concentration of EPA and docosapentaenoic acid (DPA, 22:5n3), but not docosahexaenoic acid (DHA, 22:6n3) in liver tissue as compared with animals fed OA. Dietary supplementation with SDA also increased tissue contents of EPA and DPA, but not DHA as compared with OA fed controls. The increase in tissue EPA was about one-half as effective in animals fed a diet containing SDA as those fed EPA. SDA was about two-thirds as effective as EPA at increasing tissue DPA content. These results indicate that, when fed at current dietary levels, SDA can increase the incorporation of EPA and DPA into the membrane phospholipids of liver tissue in a similar manner to dietary EPA. This provides evidence that SDA may provide an alternative to EPA for increasing tissue levels of EPA and DPA, but not DHA. Flow cytometry analysis of neutrophils following 20h incubation in either presence or absence of endotoxin indicated that diet did not significantly affect constitutive or endotoxin-mediated neutrophil survival when fed at current levels. There was a non-significant trend towards an increase in both constitutive viability and endotoxin mediated cell survival (p=0.069) in both SDA and EPA fed animals as compared with control animals that were fed OA. While these results do not fully explain the role that EPA or SDA play in reducing neutrophil influx during ARDS, they do provide evidence that it is not through an increase of neutrophil apoptosis or an attenuation of the neutrophil response to endotoxin. These findings may indicate the importance of dietary EPA for maintaining host immune response, while providing beneficial effects in regards to inflammation. Because of similar action between dietary EPA and SDA these results might also provide further proof as to the potential for dietary SDA to be used as an alternative for dietary EPA

Matrix metalloproteinase activity and inhibition in articular cartilage : effects on composition and biophysical properties and relevance to osteoarthritis

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 1995.Includes bibliographical references (leaves 172-183).by Lawrence Justin Bonassar.Ph.D

Studies on Herbal Constituents Active against Snakes Venom by In Vivo and In Vitro Methods

In the obtainable investigation selected medicinal plant was found to have neutralization of snake venom activities. The aerial parts of M.quadrifolia had been extracted successively by using soxhlet extraction with petroleum ether, chloroform, acetone, ethyl acetate, methanol. In preliminary phytochemical appraisal alkaloids, flavonoids, glycosides, terpenoids, phenolic compounds, tannins, steroids, saponins were found in the methanol extract of M.Quadrifolia. Total phenol content and flavanoids content were estimated. Presence of phenol and flavanoid might be answerable for their potential neutralization of snake venom. Thus, methanol extract were focused for further investigation.2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-2,3-dihydro-4H-chromen-4-one was isolated from methanol extract of M.Quadrifolia. Acute toxicity study established that the plants M.Quadrifolia dose not shown any sign of abnormalities and mortality even at maximum dose in experimental animals for a period of 48hrs initially and then during a week observation. Invitro antivenom studies promosing neutralization action of the plant extract, several invitro studies done in methanol extract of M.Quadrifolia have shown venom PLA2 inhibition activity, fibrinolysis induced protease inhibition and inhibition of venom induced procoagulant activity as major antivenom. The in vivo studies on M.Quadrifolia methanol extract and isolated compounds shown promising venom neutralizing activities against N. kaouthia and R. viper venom. The PLA2 was highly present in the Elapidae and Viperidae family snakes, protease and zinc metalloprotease is highly present in the Viperidae family snakes. The plant compound 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-2,3-dihydro-4H-chromen-4-one isolated from methanol extract of M.Quadrifolia shown potentially neutralize the enzymes caused by snake envenomation in experimental animals. Snakebite cases are very frequent in the rural areas and the fatalities die to lack of suitable treatment and high cost. Herbal antidotes if administered in proper dose levels and occurrence might prove effective in saving a valuable life. The present investigation was an exploit to identify the M.Quadrifolia, having antivenom activity. The whole study was carried out in animal models and then requires further investigation. The study also suggests the use of the herbal in kind ship with snake venom antiserum, to give greater protection against venom induced lethal action in animal models. Therefore it can be suggested that, the plant extract may be precious in those tropical areas where antiserum is not available, potentially the antiserum action to afford a higher grade of protection. The present study continual to explore aerial parts of M.Quadrifolia as it was whispered that there are more compounds present in the extract having greater snake venom neutralizing capacity. Therefore an endeavor to validate once again, the claim of traditional literature of the usefulness of this plant in snakebite cases

Regulatory role of ambient pH in the expression of pathogenicity determinant gene products of Beauveria bassiana and Metarhizium anisopliae

Entomopathogenic fungi (EPF) are the one of the potential cause of the morbidity and mortality of insects. In agro-forestry uses, they are applied mainly in the form of conidial preparations in dry, aqueous or oil formulations. This approach, while practical, works in a hit and miss fashion leading to a frustrating dilemma of why successes and failure perpetuate. The fundamental solution is to bridge gaps in our knowledge about conidia of EPF in varied environments where they confront a diversity of insect hosts to start their pathogenesis.This thesis was undertaken to examine the effects of hydration and the regulatory role of ambient pH on proteases which are the primary pathogenicity determinants in Beauveria bassiana and Metarhizium anisopliae. The approaches used were those of biochemical, proteomics and functional proteomics. Novel aspects of pH regulation/homeostasis during the soaking of conidia in water, (type II water, which had a maximum electrical conductivity of 1ìS/ cm at 298K/ 25° C) were identified. Hydrated conidia showed swelling in type II water as assessed by (Multisizer IIITM (Coulter CounterTM). Release of proteases, metabolic activity through liberation of ammonia and citrate and synthesis of protein, RNA and DNA was established. It was deduced that conidial enzymes are either attached by loose hydrogen bonding or were associated to the spore membranes. Water soaked or hydrated conidia can secrete citrate and ammonia to modify the ambient pH and maximize the activity of secreted proteases. Pr1- and- Pr2-like proteases were liberated by washing conidia in tween (Tw), water (Ww) and buffer. The washing of conidia in buffers (pH 4-10) affected the release/activity of Pr1 and Pr2. The thesis shows a newly designed native IPG strip zymography to identify the release of 4 and 8 isoforms of proteases, respectively from conidia. The 2-DE zymography (copolymerized gelatin) of protease from Tw of B. bassiana and M. anisopliae indicated one band (Mr 70 kDa; pI 6.3) and six isozymes (Mr 115-129 kDa; pI 3.7-9.0), respectively, which were identified using mass spectrometry (MALDI-TOF) as a serine-like protease. Six metalloprotease isozymes from M. anisopliae but only one from B. bassiana was documented by 1-DE native zymography combined with 2-D spot densitometry scans. Cationic PAGE native zymography separated two basic protease isozymes from Tw extract of M. anisopliae depending upon the pH of the incubation buffer. However, one activity band was identified from B. bassiana. Furthermore, only one activity band was apparent during 1st and 2nd Ww up to day 2 for both EPF. SDS PAGE (non-dissociating) zymogram of secreted protease isozymes from Tw of B. bassiana revealed three bands of Mr100, 60, and 36.3 kDa. The isozymes observed at day 2 and 3 had a Mrs of 35.4 and 25 kDa, and 24.7 and 20.3 kDa at day 4. The SDS PAGE zymograms for M. anisopliae indicated two isozymes of Mr 103 and 12 kDa, respectively. During the 1st Ww and incubation of spores at day 2 and 3, a 12 kDa band was observed. These results confirm the presence of diversity of proteases and their isozymes with unique molecular sizes.This thesis research discovered and characterized a diversity of proteins/enzymes not previously reported from any other fungi. A newly designed enzyme overlay membrane (EOM) technique revealed three isoforms of Pr1-like subtilisin from Tw of M. anisopliae (pI 8.1-9.7) and B. bassiana (pI 8.4-9.7). Conversely, only one isoform of Pr2-like trypsin was identified from M. anisopliae and no Pr2-like activity was observed from B. bassiana. Use of metalloprotease (MEP) inhibitors in conjunction with EOM analysis revealed their release during treatment in Tw. In M. anisopliae four activities (pI 4.4-7.5) of thermolysin-like MEP were observed. However, Tw of B. bassiana showed one activity band (pI 5.5). In addition, an isozyme of neutral MEP containing Zinc from M. anisopliae (pI 6.1) and one from B. bassiana (pI 6.5-7.6), respectively, was identified. MALDI-TOF and Q-TOF analysis revealed the presence of proteins similar to ROD 1, Ü- and â-glucanases, elastase, lipase 5 and galectin 7, which are important during the initial phase of germination and pathogenesis. In addition subtilisin (Pr1-like), trypsin (Pr2-like) and NAGase synthesis from the germinating conidia and mycelia under the supply of different carbon and nitrogen (C/ N) sources was studied. The regulation of the synthesis of cuticle-degrading enzymes (CDE) from germinating conidia and mycelia was hypothesized to be controlled through regulatory derepression and nutritional starvation. Pr1 and Pr2 are regulated in a different manner in conidia and mycelia. Both enzymes are regulated through a multiple control mode. It was concluded that C/ N repression occurs only when it is necessary for infective structures to establish a nutritional relationship with the host cuticular structures. In addition, C/ N sources have a significant effect upon pH modulation, ammonia production and protease secretion. Furthermore, the synthesis of Pr1 and Pr2 from germinating conidia was affected by the (inducer pH) pHi of the growth media. Growing mycelia of B. bassiana under acidic (4.0), neutral (7.0) and basic (11.0) pH conditions produce ammonia which modifies the pH thereby creating environments suitable for protease. Growth, morphology, radial extension rate and conidiation at different pHi revealed that both EPF modify the pH of growth medium effectively as opposed to the saprophytic fungus, Aspergillus nidulans. The presence of MEPs and Pr2-like trypsin suggests that these enzymes can act as a back up system for Pr1 to breach the cuticle and facilitate penetration before appressoria formation. The diversity of isozymes released from conidia suggests that the EPF are pre-adapted to pathogenic mode of life style, further contributing complexity to their interaction with host insects. Such isozymes can circumvent protease inhibitors present in the insect cuticle and the hemolymph. In addition, these isozymes may offer selective advantages in exploring new habitats (substrates) either as pathogen or saprophyte

Rational anti-malarial drug design

Malaria is a mosquito-borne disease caused by several species of plcismodium protozoa. It is one o f the major killer diseases o f the world, causing 2 million deaths annually. The most dangerous form is caused by Plasmodium falciparum with infections often ending terminally. Attempts to eradicate malaria have failed due to the emergence of drug resistant strains and thus there is a need for novel anti-malarial drugs. Recently an identical aminopeptidase has been isolated from the both the human malarial parasite P. falciparum and the rodent malarial parasites P. berghi and P. chabaudi. It is significantly different in both size and structure to mammalian aminopeptidases and thus provides a prime target for chemotherapeutic intervention. Investigations were directed towards the design and synthesis of novel selective inhibitors of this enzyme. Initially over forty amino acid and dipeptide derivatives with N- and C-terminal modifications were prepared. These included 7V-cinnamoyl, A^S-dinitrobenzoyl, Ntoluene-/?- sulphonyl, N-2-nitrophthaloyl, vV-phenylureido and 7V-chloroacetyl amino acids, amino esters, amino amides and amino alcohols. The design rationale and synthesis of /V-2-hydroxy-2-phenylacetyl, AL2-hydroxy-3-phenylpropionyl, 7V-3-hydroxy-3 - phenylpropionyl and 2-thio-2-phenylacetyl amino acid derivatives are also presented, along with accompanying structure-activity analysis. The compounds were characterized by I.R. and N.M.R. spectroscopy, as well as high resolution M.S. X-ray crystallography was used to elucidate the crystal structures and hydrogen bonding interactions of two of the compounds. All o f the compounds were tested for inhibitory potency in a novel bioassay based on the liberation of fluorescent 7-amino-4-methyl-coumarin from L-leucine-7- amido-4-methyl-coumarin upon enzymatic hydrolysis of the amide bond. Many of the compounds possessed modest enzyme inhibitory potency in the micromolar range [3- nitrocinnamoyl glycine ethyl ester (3c);IC5o=6.6|jM]. Some of the compounds also acted as potent activators of the aminopeptidase [3-nitrophthaloyl-L-leucine methyl ester (10c); activation at concentration of 10(iM=132.8%], The most effective inhibitor was ./V-2-thio-2-phenylacetyl phenethylamide (30) (IC5o~5.2(_iM)