A study on biochemical changes during cultivation of Rhizopus oryzae in deproteinized whey medium in relation to chitosan production.

UNLABELLED: The kinetics of cultivation of zygomycete filamentous fungus Rhizopus oryzae in deproteinized whey medium in relation to chitosan production was studied here to optimize chitosan production from R. oryzae as well as utilize whey, a by-product of sweetmeat industry as a cheap source of sugar in the cultivation process. Chitosan content of R. oryzae biomass was found to be increased with time during cultivation and reached maximum (13·6%) after 72 h and then declined steadily. Maximum 1·13 g of chitosan was obtained from one litre of deproteinized whey medium. Concentration of lactose in the medium was observed to be reduced from 45·0 to 11·7 g l(-1) during cultivation resulting in decrease in biochemical oxygen demand (BOD) of whey by approx. 60%, and this was important from environmental point of view before discharging whey into any water body. However, no significant change in pH or titratable acidity was noted during the entire course of cultivation, probably due to good buffering capacity of the medium. Molecular weight of chitosan varied from 130 to 230 kDa depending on the time of cultivation, but no significant change in degree of deacetylation of chitosan (approx. 87%) was found during cultivation. SIGNIFICANCE AND IMPACT OF THE STUDY: Whey is the largest by-product of dairy industries, and its disposal is a big environmental issue because of its high biological oxygen demand (BOD) value. This study will help to lower BOD value of whey by using it as a cultivation medium for fungus R. oryzae that contains chitosan, a very commercially important material on its cell wall. Moreover, the study on biochemical changes in whey during cultivation process with R. oryzae will help to understand the exact changes occurring in the medium and optimize cultivation process to isolate chitosan in larger extent with better and uniform physicochemical properties.

Optimization of fermentation conditions for green pigment production from Bacillus cereus M¹ 16 (MTCC 5521) and its pharmacological application.

UNLABELLED: Optimal culture conditions for the production of green pigment was investigated. The optimal culture condition for the production of an extracellular green pigment by growing Bacillus cereus M(1) 16 (MTCC 5521) in a complex medium containing (g l(-1) ) Peptone-4.0, Beef Extract-9.0, NaCl-7.0, MgSO4 .7H2 O-1.0 and KH2 PO4 -5.0 was as follows pH-7.0 at 30°C for 72 h in a 5 l fermenter. Aeration rate and agitator speed had no effect on the pigment production. Thin layer chromatogram of the pigment extracted from the fermented broth with chloroform on silica gel GF254 using ethyl acetate and hexane (1 : 1) as solvent showed three fractions. The major fraction (C3 ) was separated out and identified as 9-methyl-1, 4, 5, 8-tetra-azaphenanthrene. Acute toxicity test revealed the nontoxic nature upto a dose of 2000 mg kg(-1) , b.wt., of mice. MTT assay showed the cytotoxic nature in HL60 cells having an IC50 of 2.47 mmol. So, this biopigment may have application in food, textile colorant and pharmaceutical industry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the optimum production of a biopigment (9-methyl-1, 4, 5, 8-tetra-azaphenanthrene) by fermentation of a complex medium with Bacillus cereus M(1) 16 (MTCC 5521) in submerged fermentation. This is the first investigation of toxicity and cytotoxicity activities of this biopigment. The study showed that the purified pigment had no toxicity to healthy albino mice but a high cytotoxicity activity in HL60 cancer cell line in vitro. The biopigment had further displayed dyeing capability to both solidified agar and cotton cloth. Therefore, it may represent a nontoxic and natural alternative to chemical dyes and pigments.

Biosorption of cadmium and nickel by functionalized husk of Lathyrus sativus.

The husk of Lathyrus sativus (HLS) has been functionalized by introducing thio groups with the help of carbon disulphide treatment in alkaline environment. Elemental analysis indicates that sulphur content of the functionalized biomass increases to 3.7% from 0.36% of the pristine biomass suggesting the incorporation of thio group on HLS. A conspicuous change in the surface morphology of the biomass due to functionalization is depicted by SEM images. EDX data support the introduction of sulphur group on the HLS. The involvement of the hydroxyl groups mainly in the functionalization process is demonstrated by Fourier transform infrared (FTIR) spectroscopic study. The adsorption capacity of the functionalized biomass with respect to cadmium and nickel is observed to increase by about 50% compared to that of pristine one. Similar to the case of unmodified HLS the adsorption process involving the functionalized one obeys Langmuir isotherm model and attains equilibrium in 10 min compared with 60 min in the case of unmodified biomass.

Adsorption of nickel on husk of Lathyrus sativus: behavior and binding mechanism.

Husk of Lathyrus sativus (HLS) has been found to be a good sorbent for the removal of nickel(II) from its aqueous solution. The adsorption process depends on pH of the solution with an optimum at 5.0, and follows Langmuir isotherm model (correlation coefficient 0.998). Initial adsorption rate is very fast and reaches equilibrium following pseudo-second order kinetics within 60 min. Amino, carboxyl, hydroxyl and phosphate groups of the biomass are involved in chemical interaction with nickel ions as revealed from SEM-EDX and FTIR studies. Chemical modifications of the functional groups of the biosorbent show that amino groups contribute largely (approximately 57%) for the binding of nickel ions and probably undergo chelation through dative bond formation. HLS biomass has been found to adsorb both nickel and cadmium equally from their mixed solution to the extent of approximately 70% indicating the importance of this sorbent in industrial effluent treatment.

Adsorption of cadmium on husk of Lathyrus sativus: physico-chemical study.

Adsorption of cadmium (II) from aqueous solution by low-cost biosorbents was investigated. Husk of Lathyrus sativus (HLS) was found to be the most efficient in this respect and removed approximately 95% of the metal. The influence of pH, temperature, contact time and metal ion concentration on the adsorption process by HLS was studied. Hydrogen ion concentration of the solution greatly influenced the process with an optimum at pH 5.0-6.0, whereas temperature had no significant effect. The process was very fast and more than 90% of the total adsorption took place within the first 5 min and was found to follow pseudo-second order rate kinetics. The adsorption data can better be explained by Langmuir isotherm model and the calculated maximum adsorption capacity was 35 mg/g of HLS at pH 5.0 and 30 degrees C. Scanning electron micrographs showed that cadmium was present as micro precipitate on the surface of the adsorbent. Cadmium replaced calcium of the biomass as revealed from the EDX analysis indicating that the adsorption proceeds through ion exchange mechanism. Cadmium could be desorbed from the loaded biomass by lowering pH approximately 1.0 with mineral acid.

Purification and characterization of an extracellular agglutinin from Tricophyton rubrum with specificity towards sialic acid containing glycoconjugates.

An agglutinin, a monomeric glycoprotein with a molecular mass of about 6.5 kDa and containing 18% sugar has been purified to an apparent homogeneity from a 21 days old culture filtrate of an anthropophilic dermatophyte Tricophyton rubrum. It is a human blood group non-specific agglutinin which also agglutinates animal erythrocytes and Ehrlich ascites carcinoma and Sarcoma-180 cells. It is thermally stable and exhibits maximum activity at pH 8. Amino acid analysis shows a significant amount of glycine, with no cysteine. Glycoproteins inhibited the hemagglutination of the agglutinin, but not the simple sugars, including sialic acid. Fetuin is the most potent inhibitor among the glycoproteins tested. This inhibition gives a hint to binding with Galbeta1-3GalNAc or Galbeta1-4GlcNAc residue containing sialic acid at the terminal position with alpha 2-6 or alpha 2-3 linkage.

Murine hypodense eosinophils induce tumour cell apoptosis by a granzyme B-dependent mechanism.

PURPOSE: Eosinophils have been shown to potentiate anti-tumour cytotoxicity in both clinical and animal studies. The mechanism by which eosinophils induce tumour cell damage, however, has largely been speculative. The purpose of this study was to identify the mechanisms involved in eosinophil-induced tumour cell cytotoxicity. METHODS: To investigate eosinophil cytotoxicity, eosinophils were isolated from the peritoneal cavity of Mesocestoides corti-infected BALB/c mice, and were separated into normodense (ND) and hypodense (HD) populations using discontinuous Percoll density gradient centrifugation. The tumoricidal activity of ND and HD eosinophils was assessed using the [51Cr]-release cytotoxicity assay (a measure of cytolytic activity) and the JAM assay (a measure of apoptotic activity). Investigation of apoptosis-inducing molecules in HD eosinophils was undertaken by RT-PCR. The calcium chelator EGTA, serine protease inhibitor aprotinin and a competitive substrate for granzyme B were used to assess the role of perforin and granzyme B in HD eosinophil killing. RESULTS: Cytotoxic activity induced by HD eosinophils was significantly greater than that of ND eosinophils, and apoptosis was the principal killing mechanism. RT-PCR analysis revealed that HD eosinophils express mRNA for perforin, granzyme B and Fas ligand. Furthermore, HD eosinophil killing was markedly inhibited by EGTA, intracellular aprotinin and the granzyme B competitive substrate. CONCLUSIONS: These data are consistent with a hypothesis that murine HD eosinophils elicit tumoricidal activity via a granzyme B-dependent mechanism.

Declining clinical autopsy rates versus increasing medicolegal autopsy rates in Halifax, Nova Scotia.

The downward trend in the rate of clinical autopsies has been extensively documented in the literature. This decline is of concern when the benefits of the clinical autopsy are considered. In contrast, the rate of medicolegal autopsies has not been studied in such detail. What little reference there is to medicolegal autopsy rates suggests an absence of the same downward trend. A retrospective review of autopsy data over a 13-year period from the Queen Elizabeth II Health Sciences Centre in Halifax, Nova Scotia, and from the Office of the Chief Medical Examiner of Nova Scotia was conducted. This review showed a difference between the rates of clinical and medicolegal autopsies for the metro Halifax area. The clinical autopsy rate was consistently less than 30% and declined to 15% in 1999, while the medicolegal autopsy rate was consistently greater than 40% and rose to 62% in 1999. The literature proposes many reasons for the decline in the clinical autopsy rate, but none for this difference between rates. The explanation proposed here is the changing and currently uncertain purpose of the clinical autopsy versus the clear, and consistent over time, purpose of the medicolegal autopsy.

Fast track thrombolysis in acute myocardial infarction: a quality improvement project.

Thrombolytic therapy for acute myocardial infarction has been proved to be most effective if given very early in the course of evolving infarction. This study was undertaken to optimise such treatment by overcoming the in-hospital delay in the existing set-up of an industrial hospital. A quality improvement project was undertaken to analyse the existing system of thrombolysing 46 consecutive patients of acute myocardial infarction treated in six months. By following the breakthrough sequence and proceeding in steps, the causes for delay in door to needle time were identified and rectified over two months. Impact of such measures in 32 patients of acute myocardial infarction thrombolysed consecutively in the next five months was studied. Door to needle time in the baseline group (n = 46) was in the range of 15-145 minutes and the average was 48.9 minutes. Only 32.6 percent of the patients in this group were thrombolysed within 30 minutes of arrival in the hospital. After the corrective measures were implemented in a study group of 32 patients, 27 with clear indication on admission were thrombolysed on the fast track i.e. with minimum delay. Five patients with doubtful need were put on the slow track and subsequently thrombolysed. Patients with no indication or a contra-indication for thrombolysis were excluded from this study. In the fast group, door to needle time reduced to an average of 22.56 minutes with a range of 7 to 67 minutes and 75 percent of the thrombolysed patients received the infusion within 30 minutes of arrival in the hospital. Differences in door to needle time between the two groups were statistically significant. Streamlining the hospital systems and procedures can help reduce the door to needle time in thrombolysing patients of acute myocardial infarction. This could be achieved within the existing resources by applying the principles of total quality improvement.

Inhibition of bacterial respiration by a low-molecular weight lectin, scyllin, from Scylla serrata crab hemolymph.

Interaction of plant and/or invertebrate lectins with mammalian cells and different microorganisms is well known. In the present study, we have demonstrated that scyllin, a low molecular weight (MW 4000) lectin from the edible crab Scylla serrata hemolymph, purified by GalNAc-Sepharon affinity column followed by Mono-Q ion exchanger in FPLC exhibits antimicrobial activity against Bacillus cereus and Escherichia coli by inhibiting endogenous respiration as well as exogenous glucose oxidation. In both the cases oxygen consumption has been measured in an oxygraph. Scyllin has produced 50% inhibition of endogenous respiration at a concentration of 110 micrograms/ml and 125 micrograms/ml in B. cereus and E. coli respectively. It also reduced the exogenous glucose oxidation by 50% at a concentration of 12 micrograms/ml and 80 micrograms/ml respectively in B. cereus and E. coli. From the above study the mechanism of bacterial growth inhibitory property of scyllin is suggested though the other studies such as inhibition of nucleic acid biosynthesis, cell wall biosynthesis etc. to evaluate its total mode of inhibitory action are not yet obtained.

Typing of Shigella dysenteriae strains of different serogroups by lectins.

Ten different serogroups of Shigella dysenteriae were typed with the aid of lectins of known sugar specificity resulting from their interactions with the carbohydrates on lipopolysaccharides in the outer membrane of bacteria as evidenced by the agglutination-inhibition assay with simple carbohydrates. Lipopolysaccharides of two serogroups of Shigella were precipitated with different lectins and the results were corroborated by those derived from the agglutination assay suggesting that Shigella dysenteriae can be characterized on the species level with the aid of lectins.

Tumor localization of monoclonal antibodies against human renal carcinoma in a xenograft model.

We investigated the localization of intravenously injected DAL K45 and DAL K29, two monoclonal antibodies (MABs) against human renal cell carcinoma (RCC), and their F(ab)2 fragments in nude mice bearing intrarenal transplants of the RCC line Caki-1. More of the MABs or their F(ab)2s specifically localized in the tumor than in any normal tissue with the exception of blood. Compared to parent MABs, F(ab)2s were cleared faster from all tissues. In serum, the MABs and F(ab)2s showed a single radioactive peak retaining partial immunoreactivity. DAL K45-F(ab)2 showed the highest tumor:normal tissue localization ratios and the most distinct gamma-camera image at 24 h.

Monoclonal antibodies against Epstein-Barr virus transformed B lymphocytes from a CLL patient.

Immunization of BALB/c mice with EBV-CLL-1 cells, derived from Epstein-Barr virus transformed B lymphocytes from a chronic lymphocytic leukemia (CLL) patient, yielded 2 monoclonal antibodies (IgG1 Kappa and IgG2a Kappa) against a membrane antigen on a subset of normal B lymphocytes and non-Hodgkin's lymphomas. Immunofluorescence revealed strong reactivity of the antibodies with EBV-CLL-1 cells and with most lymphocytes in tonsil follicles, in the intestinal wall, around splenic arterioles and near Hassall's corpuscles in the neonatal thymus as well as with a small proportion of lymphocytes in some large reactive lymph node follicles, weak reactivity with 1/5 of peripheral blood B lymphocytes (PBL), and no reactivity with platelets, granulocytes and non-lymphoid tissues. PBL from 3 CLL patients showed weak staining of only larger cells. Intense fluorescence was observed in several non-Hodgkin's lymphomas of various histological types and in Burkitt's lymphoma lines but not in the 3 T lymphoblastoid and 12 nonlymphoid tumor lines examined. The antibodies precipitated Mr 22,000 and 33,000 bands from surface labeled RAJI or EBV-CLL-1 cells and cross-competed in a binding inhibition assay. The antibodies had approximately 6 million binding sites per EBV-CLL-1 or RAJI cell but were not cytotoxic. This high antigen-density and limited expression in normal cells may permit their use for immunocytological diagnosis and targeting cytotoxic agents and radionuclides against appropriate lymphoma cells.

Lectin typing of Pseudomonas aeruginosa strains of different serogroups, Habs and Fisher types.

Sixteen Habs and three Fisher types of Pseudomonas aeruginosa were typed with lectins of know specificity resulting from their interaction with bacterial cell surface carbohydrates as evidenced by agglutination-inhibition assay with simple carbohydrates. Lipopolysaccharides of few strains of Pseudomonas are precipitated with different lectins and the results are corroborated by those of agglutination suggesting that Pseudomonas aeruginosa can be characterized intraspecifically by lectins.

Ligational behavior of two biologically active N-S donors toward oxovanadium(IV) ion and potentiation of their antibacterial activities by chelation to metal ions. Part III.

Chelating behavior of two biologically active ligands, pyridine-2-carboxaldehyde thiosemicarbazone (PT) and pyridine-2-carboxaldehyde-(4-phenyl)thiosemicarbazone (PPT), toward oxovanadium(IV) ion has been studied. The ligands are found to react in the thioketo form (pH 2-4), yielding the complexes [VO(PT)X2](X = Cl-, Br-, ClO4-), [VO(PT)(SO4)H2O], [VO(PPT)2X]X (X = Cl-, Br-, ClO4-) and [VO(PPT)2SO4]. Reactions of [VO(PT)(SO4)H2O] and [VO(PPT)2X]X (X = Cl-, Br-, ClO4-) with a monodenate Lewis base (B) like pyridine lead to the formation of [VO(PT)(SO4)Py]H2O and [VO(PPT)2py]X2 respectively. Bonding sites of the donor molecules around the oxometal cation have been located. Nature of the EPR spectra and magnetic moment values point to the monomeric character of the complexes and suggest a distorted octahedral donor environment for the oxovanadium(IV) ion. Status of the metal-oxygen multiple bond in all the complexes has been computed in terms of the V-O(1) stretching force constant. The ligands themselves and most of their oxovanadium(IV) complexes are found to exert powerful in vitro antibacterial activities towards E. coli.

A study of erythrocyte fatty acids, adenosinetriphosphatase and acetylcholinesterase in cystic fibrosis.

1. The proportions of both saturated and polyunsaturated fatty acids were measured in the erythrocytes in Cystic Fibrosis (CF) patients along with two membrane bound enzymes ATPase and acetylcholinesterase. Linoleic acid was found to be significantly decreased and arachidonic acid increased in CF patients. The proportion of saturated fatty acids were not significantly different from the controls. Only adenosinetriphosphatase activity was found to be reduced and not acetylcholinesterase in CF patients.

A study of the salivary glycoprotein in cystic fibrosis patients and controls: fucose incorporation and protein pattern.

The incorporation of fucose to glycoprotein acceptors prepared from the saliva of Cystic Fibrosis (CF) patients was compared with the incorporation into acceptors from controls. The CF acceptor glycoprotein incorporated significantly more fucose in the presence of either patients' or control plasma. The fucosyl transferase activity in the patients' plasma was not significantly different from controls. Fucosidase activity was similar also for both groups. The protein bands of the acceptor glycoproteins from the patients' saliva differed from those of the control in number and electrophoretic mobility. On the basis of these studies of fucose incorporation we propose that glycoprotein in the salivary secretion of CF patients are qualitatively different from normal.