Antihyaluronidase and Alkaline Phosphatase (ALP) Activities of Medicinal Plants to Combat Echis carinatus Venom-Induced Toxicities.

Snakebite is one of the most neglected diseases of developing countries. Deaths due to snakebite envenoming are quite high in Pakistan, and many deaths are caused by Echis carinatus envenomation. Traditional use of medicinal plants against snakebites is a common practice in Pakistan due to countless benefits. The current study was performed with the objective to evaluate eighteen Pakistani medicinal plants inhibitory potential against hyaluronidase and alkaline phosphatase enzymes of Pakistani Echis carinatus venom. Hyaluronidase activity (0.2-1.6 mg/0.1 mL) and alkaline phosphatase activity (0.1-0.8 mg/0.1 mL) were measured in dose-dependent manner. Crude methanolic extracts of medicinal plants were used for in vitro investigation of their inhibitory activity against toxic enzymes. All active plants were fractioned using different solvents and were again analyzed for inhibitory activity of same enzymes. Results indicated all plants were able to neutralize hyaluronidase that Swertia chirayita (Roxb. ex Flem.) Karst., Terminalia arjuna Wight and Arn, Rubia cordifolia Thumb., and Matthiola incana (L.) R.Br. inhibited maximum hyaluronidase activity equivalent to standard reference (p > 0.5). Pakistani medicinal plants are dense with natural neutralizing metabolites and other active phytochemicals which could inhibit hyaluronidase activity of Pakistani Echis carinatus venom. Further advanced studies at molecular level could lead us to an alternative for envenoming of Pakistani Echis carinatus venom.

Anti-inflammatory and anti-nociceptive property of Capparis tomentosa Lam. root extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Capparis tomentosa Lam. root is one of the commonly used traditional medicines for the treatment of pain and inflammatory conditions by the Kunama ethnic group of Ethiopia. Yet, its ethnomedicinal claim has not been scientifically investigated. AIM OF THE STUDY: This study aims to evaluate the anti-inflammatory and anti-nociception activities of root extracts of Capparis tomentosa Lam. MATERIALS AND METHODS: The anti-inflammatory activity of the 70% ethanolic extracts and solvent fractions was determined using invitro hyaluronidase inhibition assay and in vivo carrageenan-induced paw edema in Swiss albino mice model. The anti-nociception effect was determined using the acetic acid-induced writhing model and tail immersion model in Swiss albino mice. RESULTS: The root extract of Capparis tomentosa has exhibited significant hyaluronidase inhibition activity which was comparable with the reference drug, indomethacin. Significant inhibition of carrageenan-induced paw edema was observed in all the test sample treated mice compared to the negative control with the highest activity observed from butanol fraction which was equivalent to the effect of acetylsalicylic acid. All the root extract and solvent fractions have possessed a significant and dose-dependent abdominal writhing inhibition with the highest effect observed in the aqueous fraction. Additionally, the crude extract was found better in increasing the reaction latency of the mice to the hot water stimulus than the solvent fractions which were significant after 60-120 min compared to the distilled water treated group. The root extract and solvent fractions were also found safe at a single oral dose of 2 mg/g in Swiss albino mice. CONCLUSION: The findings of the present study may partially support the folkloric use of Capparis tomentosa root for the treatment of pain and inflammation.

Metabolite profiling and mechanisms of bioactivity of snake autolysate - A traditional Uzbek medicine.

ETHNOPHARMACOLOGICAL RELEVANCE: Aqueous autolysate from the snake Eryx miliaris (SNA) has been used in traditional medicine of Uzbekistan as anti-inflammatory, hepatoprotective and immunomodulatory agent. However, little is known about the chemical composition and its mechanisms of activity. AIM OF THE STUDY: This is our first attempt to analyse the composition of snake autolysate using gas chromatography with mass spectrometry (GC-MS) and to investigate the mechanisms of anti-inflammatory and hyaluronidase activity of fingerprinted E. miliaris autolysate to support their use in the traditional Uzbek medicine. MATERIALS AND METHODS: Aqueous autolysate was evaporated and derivatised for GC-MS analysis of metabolites. For quantification, lipids were extracted from autolysate by solvent extraction and derivatised by esterification and silylation. Biological activity was evaluated with lipid peroxidation, cyclooxygenase (COX) inhibition and antihyaluronidase activity tests. RESULTS: GC-MS analysis of SNA enabled the identification of 27 compounds. Short chain fatty acids (SCFA, 21%), amino acid/derivatives 39% (incl. 2-piperidinone 19%), phenyl (7%), and OH-Phenyl (10%) derivatives covered 77%. Other derivatives (9%) included succinic acid and 3-indole acetic acid). Long chain fatty acids (C16-C18) accounted for 3%. The lipid concentration of SNA was 1.2 mg/mL (0.12%). Three concentration levels (1.0-20.0 µg/mL) did not inhibit COX-1 and COX-2 in vitro and malondialdehyde level was not decreased by SNA in lipid peroxidation model. However, SNA was a potent inhibitor of the hyaluronidase enzyme activity in a dose dependent manner with IC50 = 0.086 mL/mL. CONCLUSION: The results from GC-MS analyses of SNA lead us to the identification of a wide range of major chemical structures of the metabolites and their derivatives with several categories. Pharmacological studies support the traditional use of SNA and show one of its possible mechanisms of activity via inhibition of hyaluronidase.

Effect of phenolic compounds from the rind of Punica granatum on the activity of three metabolism-related enzymes.

Enzyme activity modulation by synthetic compounds provide strategies combining the inhibitory and therapeutic mode of action of the confirmed inhibitors. However, natural modulators could offer a valuable alternative for synthetic ones for the treatment of different chronic diseases (diabetes, hypertension, cancer) due to the numerous side effects of the latter. In vitro screening assays were conducted for Punica granatum rind methanolic extract against three metabolism-related enzymes: α-amylase, tyrosinase, and hyaluronidase. The obtained results showed that the examined extract retained high multitarget inhibition with inhibition percentages 31.5 ± 1.3%, 75.9 ± 4.7%, and 68.5 ± 5.3% against α-amylase, tyrosinase, and hyaluronidase, respectively. Bioguided fractionation of P. granatum rind extract revealed that quercetin is the major active compound with inhibitory activities: 54.3 ± 2.7%, 94.2 ± 3.5%, and 90.9 ± 2.7% against α-amylase, tyrosinase, and hyaluronidase, respectively. Kinetic studies of enzymes showed that quercetin inhibition was noncompetitive, uncompetitive, and competitive for α-amylase, tyrosinase, and hyaluronidase, respectively. The molecular docking of quercetin with α-amylase and hyaluronidase showed high binding energy with different bonds stabilizing the ligand-protein complex. Compiling all obtained results led to conclude that both P. granatum rind extract and quercetin have multitarget activities with potential therapeutic applications in many metabolic disorders.

Influence of the extracellular matrix on water mobility in subcortical gray matter.

PURPOSE: Water mobility in tissues is related to the microstructure that modulates diffusion and spin relaxation. Previous work has shown that the extracellular matrix (ECM) impacts water diffusion in cartilage. To investigate if similar contributions to image contrast exist for brain, which is characterized by a substantially lower ECM content, diffusion and relaxation were studied in fixed samples from goat and human thalamus before and after enzymatic digestion of ECM compounds. Selected experiments in human corpus callosum were included for comparing subcortical gray matter and white matter. METHODS: Digestion of matrix components was achieved by treatment with hyaluronidase. Nonlocalized pulsed field gradient measurements were performed with b values between 0.6 and 18,000 s/mm2 at 3T and temperatures between 0°C and 20°C, in addition to T1 and T2 relaxation measurements. The data were fitted to multiexponential models to account for different water compartments. After the measurements, the samples were sliced and stained for ECM-sensitive markers to verify efficient digestion. RESULTS: Microstructural alterations associated with hyaluronan digestion did not lead to measurable effects on water diffusion or T 2 . However, T1 of the main relaxographic component, attributed to intra-/extracellular water, decreased by 7%. CONCLUSION: Investigations with very strong gradients did not reveal a detectable effect on water diffusion or T 2 after hyaluronan removal, indicating that the brain ECM content is too low to produce a detectable effect. The subtle alteration of T1 upon hyaluronidase treatment might reflect a modulation of intercompartmental water exchange properties.

In vitro pro-inflammatory enzyme inhibition and anti-oxidant potential of selected Sri Lankan medicinal plants.

BACKGROUND: The extracts of the ten selected Sri Lankan medicinal plants have been traditionally used in the treatment of inflammatory mediated diseases. The extracts were investigated for anti-inflammatory and anti-oxidant potential in vitro to identify bio-active extracts for further chemical characterization. METHODS: In vitro anti-inflammatory activities of total ethanol extracts were investigated measuring the inhibitory activities of four pro-inflammatory enzymes, arachidonate-5- lipoxygenase (A5-LOX), hyaluronidase (HYL), xanthine oxidase (XO) and inducible nitric oxide (iNO) synthase. Cytotoxicity of extracts were determined by MTT assay. Oxidative burst inhibition (OBI) on human whole blood (WB) and isolated polymorphoneutrophils (PMNs) was carried out for a selected bio-active extract. Anti- oxidant activities of the extracts were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging, ferric reducing antioxidant power (FRAP), ferrous ion chelation (FIC) and oxygen radical absorbance capacity (ORAC) assays. Total polyphenol and total Flavonoid contents of the extracts were also determined. The most active plant extract was analysed using Gas chromatography-Mass spectrometry (GC-MS) and High Performance Liquid Chromatography (HPLC). RESULTS: The ethanol bark extract of Flacourtia indica showed the highest A5-LOX (IC50: 22.75 ± 1.94 g/mL), XO (70.46 ± 0.18%; 250 µg/mL) and iNOs inhibitory activities on LPS- activated raw 264.7 macrophage cells (38.07 ± 0.93%; 500 µg/mL) with promising OBI both on WB (IC50: 47.64 2.32 µg/mL) and PMNs (IC50: 5.02 0.38 µg/mL). The highest HYL inhibitory activity was showed by the leaf extracts of Barathranthus nodiflorus (42.31 ± 2.00%; 500 µg/mL) and Diospyros ebenum (41.60 ± 1.18%; 500 µg/mL). The bark and leaf extracts of Callophyllum innophyllum (IC50: 6.99 ± 0.02 µg/mL) and Symplocus cochinchinesis (IC50: 9.85 ± 0.28 µg/mL) showed promising DPPH free radical scavenging activities. The GC-MS analysis of ethanol bark extract of F. indica showed the presence of two major bio-active compounds linoleic acid ethyl ester and hexadecanoic acid, ethyl ester (> 2% peak area). The HPLC analysis showed the presence polyphenolic compounds. CONCLUSION: The ethanol bark extract of F. indica can be identified as a potential candidate for the development of anti-inflammatory agents, which deserves further investigations. The bio-active plant extracts may be effectively used in the applications of cosmetic and health care industry.

Polyphenol-Rich Extracts from Cotoneaster Leaves Inhibit Pro-Inflammatory Enzymes and Protect Human Plasma Components against Oxidative Stress In Vitro.

The present study investigated the phenolic profile and biological activity of dry extracts from leaves of C. bullatus, C. zabelii and C. integerrimus-traditional medicinal and dietary plants-and evaluated their potential in adjunctive therapy of cardiovascular diseases. Complementary UHPLC-PDA-ESI-MS³, HPLC-PDA-fingerprint, Folin-Ciocalteu, and n-butanol/HCl assays of the extracts derived by fractionated extraction confirmed that they are rich in structurally diverse polyphenols (47 analytes, content up to 650.8 mg GAE/g dw) with proanthocyanidins (83.3⁻358.2 mg CYE/g) dominating in C. bullatus and C. zabelii, and flavonoids (53.4⁻147.8 mg/g) in C. integerrimus. In chemical in vitro tests of pro-inflammatory enzymes (lipoxygenase, hyaluronidase) inhibition and antioxidant activity (DPPH, FRAP), the extracts effects were dose-, phenolic- and extraction solvent-dependent. The most promising polyphenolic extracts were demonstrated to be effective antioxidants in a biological model of human blood plasma-at in vivo-relevant levels (1⁻5 µg/mL) they normalized/enhanced the non-enzymatic antioxidant capacity of plasma and effectively prevented peroxynitrite-induced oxidative/nitrative damage of plasma proteins and lipids. As demonstrated in cytotoxicity tests, the extracts were safe-they did not affect viability of human peripheral blood mononuclear cells. In conclusion, Cotoneaster leaves may be useful in development of natural-based products, supporting the treatment of oxidative stress/inflammation-related chronic diseases, including cardiovascular disorders.

Discovery of hyaluronidase inhibitors from natural products and their mechanistic characterization under DMSO-perturbed assay conditions.

The present study discovered four novel hyaluronan-degrading enzyme (hyaluronidase) inhibitors including chikusetsusaponins and catechins through the activity-guided separation of Panax japonicus and Prunus salicina, respectively. Although the discovery resulted in identification of usual frequent hitters, subsequent mechanistic characterizations under our DMSO-perturbed assay conditions and related protocols revealed that chikusetusaponin IV would serve as an aggregating and non-specific binding inhibitor, while (-)-epicatechin would interact specifically with enzyme at the catalytic site or more likely at a kind of catechin-binding site with a relatively week inhibitory activity. The latter description might provide a possible explanation for the well-known fact that a series of catechin have been described as frequent hitters in biological assays with a moderate activity. Thus, the present study demonstrated a practical and robust methodology to characterize initial screening hits mechanistically molecule-by-molecule in the early stage of natural product-based drug discovery.

Functional Elucidation of Nemopilema nomurai and Cyanea nozakii Nematocyst Venoms' Lytic Activity Using Mass Spectrometry and Zymography.

BACKGROUND: Medusozoans utilize explosively discharging penetrant nematocysts to inject venom into prey. These venoms are composed of highly complex proteins and peptides with extensive bioactivities, as observed in vitro. Diverse enzymatic toxins have been putatively identified in the venom of jellyfish, Nemopilema nomurai and Cyanea nozakii, through examination of their proteomes and transcriptomes. However, functional examination of putative enzymatic components identified in proteomic approaches to elucidate potential bioactivities is critically needed. METHODS: In this study, enzymatic toxins were functionally identified using a combined approach consisting of in gel zymography and liquid chromatography tandem mass spectrometry (LC-MS/MS). The potential roles of metalloproteinases and lipases in hemolytic activity were explored using specific inhibitors. RESULTS: Zymography indicated that nematocyst venom possessed protease-, lipase- and hyaluronidase-class activities. Further, proteomic approaches using LC-MS/MS indicated sequence homology of proteolytic bands observed in zymography to extant zinc metalloproteinase-disintegrins and astacin metalloproteinases. Moreover, pre-incubation of the metalloproteinase inhibitor batimastat with N. nomurai nematocyst venom resulted in an approximate 62% reduction of hemolysis compared to venom exposed sheep erythrocytes, suggesting that metalloproteinases contribute to hemolytic activity. Additionally, species within the molecular mass range of 14-18 kDa exhibited both egg yolk and erythrocyte lytic activities in gel overlay assays. CONCLUSION: For the first time, our findings demonstrate the contribution of jellyfish venom metalloproteinase and suggest the involvement of lipase species to hemolytic activity. Investigations of this relationship will facilitate a better understanding of the constituents and toxicity of jellyfish venom.

Hyaluronidase Inhibitory Activity of Pentacylic Triterpenoids from Prismatomeris tetrandra (Roxb.) K. Schum: Isolation, Synthesis and QSAR Study.

The mammalian hyaluronidase degrades hyaluronic acid by the cleavage of the ß-1,4-glycosidic bond furnishing a tetrasaccharide molecule as the main product which is a highly angiogenic and potent inducer of inflammatory cytokines. Ursolic acid 1, isolated from Prismatomeris tetrandra, was identified as having the potential to develop inhibitors of hyaluronidase. A series of ursolic acid analogues were either synthesized via structure modification of ursolic acid 1 or commercially obtained. The evaluation of the inhibitory activity of these compounds on the hyaluronidase enzyme was conducted. Several structural, topological and quantum chemical descriptors for these compounds were calculated using semi empirical quantum chemical methods. A quantitative structure activity relationship study (QSAR) was performed to correlate these descriptors with the hyaluronidase inhibitory activity. The statistical characteristics provided by the best multi linear model (BML) (R² = 0.9717, R²cv = 0.9506) indicated satisfactory stability and predictive ability of the developed model. The in silico molecular docking study which was used to determine the binding interactions revealed that the ursolic acid analog 22 had a strong affinity towards human hyaluronidase.

Hyaluronan degrading silica nanoparticles for skin cancer therapy.

We report the first nanoformulation of Hyaluronidase (Hyal) and its enhanced adjuvant effect over the free enzyme. Hyaluronic acid (HA) degrading enzyme Hyal was immobilized on 250 nm silica nanoparticles (SiNP) maintaining specific activity of the enzyme via the layer-by-layer self-assembly technique. This process was characterized by dynamic light scattering (DLS), zeta potential, infrared and UV-Vis spectroscopy, transmission electron microscopy (TEM) and enzymatic activity measurements. The nanoparticles were tested in vivo as adjuvants of carboplatin (CP), peritumorally injected in A375 human melanoma bearing mice and compared with the non-immobilized enzyme, on the basis of equal enzymatic activity. Alcian Blue staining of A375 tumors indicated large overexpression of hyaluronan. At the end of the experiment, tumor volume reduction with SiNP-immobilized Hyal was significantly enhanced compared to non-immobilized Hyal. Field emission scanning electron microscopy (FE-SEM) images together with energy dispersive X-ray spectroscopy (EDS) spectra confirmed the presence of SiNP on the tumor. We mean a proof of concept: this extracellular matrix (ECM) degrading enzyme, immobilized on SiNP, is a more effective local adjuvant of cancer drugs than the non-immobilized enzyme. This could prove useful in future therapies using other or a combination of ECM degrading enzymes.

Inhibition of hyaluronidase by N-acetyl cysteine and glutathione: role of thiol group in hyaluronan protection.

Hyaluronidase inhibitors have immense applications in pathophysiological conditions associated with hyaluronan-hyaluronidase system. The present study demonstrates the inhibitory efficacy of clinically accepted antioxidant N-acetyl cysteine (NAC) against hyaluronidase of serum, testis, and snake and bee venoms. The experimental and molecular dynamic simulation data suggest the non-competitive inhibition and involvement of thiol groups of both NAC and glutathione in exertion of inhibition. The bioavailability, less-toxic and antioxidant nature of NAC and glutathione could become valuable in the management of pathologies triggered by extracellular matrix degradation and to increase the endurance of hyaluronan based biomaterials/supplements, which are highly exciting aspects.

Experimental evaluation of hyaluronidase activity in combination with specific drugs applied in clinical techniques of interventional pain management and local anaesthesia.

BACKGROUND: Hyaluronidase is an enzyme additive used in local anaesthesia and interventional pain reducing procedures such as adhesiolysis of epidural scar tissue after spinal surgery. Only a limited number of studies describe the influence of drugs on hyaluronidase activity. Postulated effects and effectiveness of hyaluronidase are only based on clinical observations. OBJECTIVE: The aim of this study is to investigate the influence of the combined drugs on the activity of hyaluronidase under standardized conditions and to verify the effectiveness of the enzyme. DESIGN: An ELISA-based microtiter-technique is used to evaluate the activity of hyaluronidase in combination with local anaesthetics, corticosteroids, NaCl 10%, and iodinated contrast media. METHODS: Microtiter plates were coated with biotinylated hyaluronate and incubated with hyaluronidase in combination with the above-mentioned drugs. The activity of hyaluronidase was determined by an avidin-peroxidase-based procedure using an ELISA reader. Incubations were carried out at room temperature as well as at 37 degrees C. RESULTS: The data show that drugs affect the activity of hyaluronidase in different ways. Iodinated contrast media, NaCl (10%), and the absence of corticosteroids reduce hyaluronidase activity. In contrast, higher activities were detected at a lower NaCl concentration (0.9%). We cannot attribute a significant influence to local anaesthetics. CONCLUSIONS: Hyaluronidase is effective in all combinations with drugs. To get the maximum effect calculated use of accompanying drugs is necessary.

New potent indole derivatives as hyaluronidase inhibitors.

Because of the physiologic importance of hyaluronidases, the identification of potent and selective inhibitors of hyaluronidases has become increasingly important. A variety of assay methods have been used for such a purpose, i.e. classical turbidimetric, viscometric and colorimetric. In this study, a modified enzymatic assay has been used to obtain a microtiter plate-based sensitive activity screening. All inhibitors were tested in a stains-all assay at pH 7 and in a Morgan-Elson assay at pH 3.5. Among the tested compounds, 1, 2, 3, 6, 7, 8, 16, 17 and 18 showed good inhibition of more than 50%, so the IC(50) values of these derivatives were determined in the range of 25-41 microm. The IC(50) value of the most active hyaluronidase inhibitor Vcpal (6-palmitoyl-L-ascorbic acid) was measured as 8.36 microm. All inhibitors including Vcpal showed twofold less activity at pH 3.5 in a Morgan-Elson assay. Examination of substituent effects on the activity showed that para-positions of benzamide needs to be chlorinated or fluorinated to obtain good inhibitory effect. It was found that the introduction of a p-fluoro benzyl ring in the indole nitrogen has a positive effect for the inhibitory effects of both indole-2- and 3-carboxamide derivatives.

Mast cell stabilization, lipoxygenase inhibition, hyaluronidase inhibition, antihistaminic and antispasmodic activities of Aller-7, a novel botanical formulation for allergic rhinitis.

Allergic rhinitis, also known as hay fever, rose fever or summer catarrh, is a major challenge to health professionals. A large number of the world's population, including approximately 40 million Americans, suffers from allergic rhinitis. A novel, botanical formulation (Aller-7) has been developed for the treatment of allergic rhinitis using a combination of extracts from seven medicinal plants, including Phyllanthus emblica, Terminalia chebula, T. bellerica, Albizia lebbeck, Piper nigrum, Zingiber officinale and P. longum, which have a proven history of efficacy and health benefits. The clinical manifestations of allergy are due to a number of mediators that are released from mast cells. The effect of Aller-7 on rat mesenteric mast cell degranulation was studied by incubating different concentrations of Aller-7 and challenging them with a degranulating agent, compound 48/80. The inhibitory activity of Aller-7 was determined against lipoxygenase and hyaluronidase, the key enzymes involved in the initiation and maintenance of inflammatory responses. Furthermore, most of these manifestations are due to histamine, which causes vasodilatation, increasing capillary permeability and leading to bronchoconstriction. Hence, the antihistaminic activity of Aller-7 was determined is isolated guinea pig ileum substrate using cetirizine as a positive control. The antispasmodic effect of Aller-7 on contractions of guinea pig tracheal chain was determined using papaverine and cetirizine as controls. Aller-7 exhibited potent activity in all these in vitro models tested, thus demonstrating the novel anti-allergic potential of Aller-7.

Xenopus kidney hyaluronidase-1 (XKH1), a novel type of membrane-bound hyaluronidase solely degrades hyaluronan at neutral pH.

In search for Xenopus laevis hyaluronidase genes, a cDNA encoding a putative PH-20-like enzyme was isolated. In the adult frog, this mRNA was only found to be expressed in the kidney and therefore named XKH1. When expressed by means of cRNA injection into frog oocytes, XKH1 solely exhibited at physiologic ionic strength hyaluronidase activity at neutral pH and in weakly acidic solutions. The enzyme was inactive below pH 5.4. In addition to hyaluronic acid hydrolysis, chondroitin sulfate also was degraded at low yield as assessed by fluorophore-assisted carbohydrate electrophoresis analysis of the degradation products. The enzyme is sorted to the outer surface of the cell membrane of XKH1 expressing oocytes. From there, it could not be removed by phospholipase C nor was secreted hyaluronidase activity detectable. We conclude that XKH1 represents a membrane-bound hyaluronan-degrading enzyme exclusively expressed in cells of the adult frog kidney where it either may be involved in the reorganization of the extracellular architecture or in supporting physiological demands for proper renal functions.

Eudesmane derivatives from Tessaria integrifolia.

Five eudesmane-type sesquiterpenoids were isolated from the methanol extract of the aerial part of Tessaria integrifolia Ruiz. et Pavon (Compositae). Their structures were elucidated on the basis of spectroscopic analysis is well as chemical evidence.

pH-adjusted periocular anaesthesia for primary vitreoretinal surgery.

PURPOSE: To evaluate the efficacy of pH-adjusted bupivacaine in conjunction with medial orbital periconal block (periocular anaesthesia). METHODS: Sixty consecutive patients undergoing primary vitreoretinal surgery were enrolled prospectively. RESULTS: Adequate anaesthesia and akinesia with no intraoperative supplementation was achieved in 53 eyes (88.3%). Factors influencing intraoperative supplementation were combined vitrectomy with scleral buckling (p = 0.005) and duration of surgery of more than 2 hours (p = 0.001). No ocular or systemic complication resulted. CONCLUSION: pH-adjusted periocular anaesthesia is safe and effective in patients undergoing primary vitreoretinal surgery.

Structural relationship of sperm soluble hyaluronidase to the sperm membrane protein PH-20.

The sperm plasma membrane protein PH-20 has a hyaluronidase activity that enables acrosome-intact sperm to pass through the cumulus cell layer of the egg. In this study we analyzed the relationship of guinea pig PH-20 and the "classical" soluble hyaluronidase released at the time of the acrosome reaction of guinea pig sperm. PH-20 is a membrane protein, anchored in the plasma and inner acrosomal membranes by a glycosyl phosphatidyl inositol anchor. Several types of experiments indicate a structural relationship of PH-20 and the soluble hyaluronidase released during the acrosome reaction. First, an antiserum raised against purified PH-20 is positive in an immunoblot of the soluble protein fraction released during the acrosome reaction. In the released, soluble protein fraction, the anti-PH-20 antiserum recognizes a protein of approximately 64 kDa, i.e., identical in molecular mass to PH-20 (approximately 64 kDa). Second, the enzymatic activity of the released hyaluronidase is completely inhibited (100%) by the anti-PH-20 antiserum. Third, almost all (97%) of the soluble hyaluronidase is removed from the released protein fraction by a single pass through an affinity column made with an anti-PH-20 monoclonal antibody. These findings suggest that the released, soluble hyaluronidase is a soluble form of PH-20 (sPH-20). During the acrosome reaction, PH-20 undergoes endoproteolytic cleavage into two disulfide-linked fragments whereas the released sPH-20 is not cleaved, suggesting the possible activity of a membrane-bound endoprotease on PH-20. We searched for a cDNA encoding sPH-20 but none was found. This result suggests that sPH-20 may arise from the enzymatic release of PH-20 from its membrane anchor, possibly at the time of acrosome reaction.

Preparation of pH-adjusted local anesthetics.

Characteristics of alkalinized local anesthetics are poorly described with regard to stability and precipitation. Current fixed-volume methods of alkalinization often result in unstable, precipitated solutions of unpredictable pH. We determined a stable, nonprecipitating, anesthetic mixture and used it in a randomized double-blind crossover study of 21 patients to evaluate alkalinized anesthetic solutions in reducing pain on injection. Our solution significantly reduced pain associated with retrobulbar injection (analysis of variance, P = .0362) but not that associated with Nadbath injection. Fixed-volume alkalinization is not recommended because complications can result with the use of precipitated anesthetics.