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Although the initiating complex of lectin pathway (called M1 in this study) generates C3/C5 convertases similar to those assembled by the initiating complex (C1) of the classical pathway, activation of complement component C5 via the lectin pathway has not been examined. In the present study kinetic analysis of lectin pathway C3/C5 convertases assembled on two surfaces (zymosan and sheep erythrocytes coated with mannan (E(Man))) revealed that the convertases (ZymM1,C4b,C2a and E(Man)M1,C4b,C2a) exhibited a similar but weak affinity for the substrate, C5 indicated by a high K(m) (2.73-6.88 microm). Very high affinity C5 convertases were generated when the low affinity C3/C5 convertases were allowed to deposit C3b by cleaving native C3. These C3b-containing convertases exhibited K(m) (0.0086-0.0075 microm) well below the normal concentration of C5 in blood (0.37 microm). Although kinetic parameters, K(m) and k(cat), of the lectin pathway C3/C5 convertases were similar to those reported for classical pathway C3/C5 convertases, studies on the ability of C4b to bind C2 indicated that every C4b deposited on zymosan or E(Man) was capable of forming a convertase. These findings differ from those reported for the classical pathway C3/C5 convertase, where only one of four C4b molecules deposited formed a convertase. The potential for four times more amplification via the lectin pathway than the classical pathway in the generation of C3/C5 convertases and production of pro-inflammatory products, such as C3a, C4a, and C5a, implies that activation of complement via the lectin pathway might be a more prominent contributor to the pathology of inflammatory reactions.

Objectives: The relative positions of the tibial tuberosity and the center of patella are crucially important to determine the Q angle. The aim of this study was to evaluate a geometric method to analyze the positions of the center of patella and the tibial tuberosity, to document and discuss possible sexual differences.Methods: One hundred paired limbs (50 males and 50 females) of healthy adult Indian volunteers were studied. Trigonometric analysis was used to accurately determine the relative positions of the center of patella and the tibial tuberosity with respect to the medial and lateral joint lines. Two ratios R1 and R2 determined mediolateral placement of the center of patella and tibial tuberosity. Gender differences were documented using appropriate statistics.Results: The center of the patella was more medially placed with respect to the joint lines in females. The mean values of R1 were not significantly different in males (1.48±1.02) and females (1.51±0.62). The tibial tuberosity was usually more laterally placed with respect to the joint lines. The mean values of the tibial tuberosity were significantly greater in females (2.09±0.90) as compared to males (1.29±0.67).Conclusion: In supine position, the tibial tuberosity was significantly placed more laterally in females possibly contributing to a greater Q angle in females.

Sanazole/AK-2123/γ-Radiation/DNA damage/Apoptosis/Radiosensitizer. Sanazole (AK-2123) (N-2′-methoxy ethyl)-2-(3″-nitro-1″-triazolyl)acetamide, which has completed phase III clinical trials as a radiosensitizer, enhanced γ-radiation induced apoptosis in murine fibrosarcoma upon i.p. administration at 40 mg/kg body weight one hour prior to irradiation. A microscopic examination of Giemsa-May-Grunwald stained cells has shown a higher frequency of condensed nuclei and fragmented nuclei in the tumor cells. The administration of sanazole to tumor-bearing animals enhanced the radiation-induced internucleosomal fragmentation in the nuclear genome of tumor cells. Higher levels of caspase-3 activity were also observed in the cell extracts of tumours from AK-2123 administered mice. Exposure to γ-radiation of AK2123-treated mouse further enhanced the caspase-3 activity, indicating the induction of apoptosis. The radiation sensitization property of sanazole was discernible by comparing the relative tumor diameter following irradiation after i.p. administration of AK-2123 and irradiation alone; it was higher during the first few days followed by the treatment.

The studies reported in this communication had two major objectives: first to validate the in-house developed SCGE-Pro: a software developed for automated image analysis and data processing for Comet assay using human peripheral blood leucocytes exposed to radiation doses, viz. 2, 4 and 8 Gy, which are known to produce DNA/chromosome damage using alkaline Comet assay. The second objective was to investigate the effect of gamma radiation on DNA damage in mouse peripheral blood leucocytes using identical doses and experimental conditions, e.g. lyses, electrophoretic conditions and duration of electrophoresis which are known to affect tail moment (TM) and tail length (TL) of comets. Human and mouse whole blood samples were irradiated with different doses of gamma rays, e.g. 2, 4 and 8 Gy at a dose rate of 0.668Gy/min between 0 and 4 degrees C in air. After lyses, cells were electrophorased under alkaline conditions at pH 13, washed and stained with propidium iodide. Images of the cells were acquired and analyzed using in-house developed imaging software, SCGE-Pro, for Comet assay. For each comet, total fluorescence, tail fluorescence and tail length were measured. Increase in TM and TL was considered as the criteria of DNA damage. Analysis of data revealed heterogeneity in the response of leucocytes to gamma ray induced DNA damage both in human as well as in mouse. A wide variation in TM and TL was observed in control and irradiated groups of all the three donors. Data were analyzed for statistical significance using one-way ANOVA. Though a small variation in basal level of TM and TL was observed amongst human and mouse controls, the differences were not statistically significant. A dose-dependent increase in TM (P0.001) and TL (P0.001) was obtained at all the radiation doses (2-8 Gy) both in human and mouse leucocytes. However, there was a difference in the nature of dose response curves for human and mouse leucocytes. In human leucocytes, a linear increase in TM and TL was observed up to the highest radiation dose of 8 Gy. However, in case of mouse leucocytes, a sharp increase in TM and TL was observed only up to 4 Gy, and there after saturation ensued. In human samples, the dose response of both TM and TL showed best fits with linear model (r(TM)=0.999 and r(TL)=0.999), where as in mouse, the best fit was obtained with Sigmoid (Boltzman) model. From the present data on leucocytes with increase in TM and TL as the criteria of DNA damage, it appears that mouse is relatively more sensitive to radiation damage than humans.

Mannan-binding lectin (MBL) mediates innate immune responses, such as activation of the complement lectin pathway and phagocytosis, to help fight infections. In the present study, employing recombinant forms of human MBL (rMBL), the role of wild type MBL (rMBL/A) and its structural variant rMBL/C in mediating THP-1 phagocytosis of fluorescent-labeled zymosan was examined and compared to MBL purified from human plasma (pMBL/A). Flow cytometric analyses revealed that opsonization of zymosan with rMBL/A and pMBL/A (0.5-30microg/ml) resulted in a 1.9- and 2.7-fold enhancement in its uptake by THP-1 cells in the presence of serum that was depleted of both MBL and the classical pathway component, C1q (MBL/C1q Dpl serum). In contrast, no enhancement in phagocytosis was observed when zymosan was opsonized with rMBL/C. Addition of MBL monoclonal antibody, EDTA, or mannan to the opsonization reaction mixture inhibited THP-1 phagocytosis of pMBL/A opsonized zymosan. Heat inactivation of MBL/C1q Dpl serum abolished the 2-fold increase in phagocytosis and in the absence of serum the direct opsonic activity of MBL did not contribute significantly to the uptake of zymosan into THP-1 cells. Activation products of complement components C3 and C4 were deposited on zymosan opsonized with pMBL/A and rMBL/A but not rMBL/C indicating that MBL-mediated phagocytosis of zymosan requires activation of the complement lectin pathway. The findings imply that impaired MBL-mediated phagocytosis may put individuals homozygous for the mutant allele MBL/C but not wild type MBL/A at increased risk to infections such as yeast.

The complement lectin pathway, an essential component of the innate immune system, is geared for rapid recognition of infections as each C4b deposited via this pathway is capable of forming a C3/C5 convertase. In the present study, role of C4b-binding protein (C4BP) in regulating the lectin pathway C3/C5 convertase assembled on zymosan and sheep erythrocytes coated with mannan (E(Man)) was examined. While the C4BP concentration for inhibiting 50% (IC(50)) formation of surface-bound C3 convertase on the two surfaces was similar to that obtained for the soluble C3 convertase (1.05nM), approximately 3- and 41-fold more was required to inhibit assembly of the C5 convertase on zymosan (2.81nM) and E(Man) (42.66nM). No difference in binding interactions between C4BP and surface-bound C4b alone or in complex with C3b was observed. Increasing the C4b density on zymosan (14,000-431,000 C4b/Zym) increased the number of C4b bound per C4BP from 2.87 to 8.23 indicating that at high C4b density all seven alpha-chains of C4BP are engaged in C4b-binding. In contrast, the number of C4b bound per C4BP remained constant (3.79+/-0.60) when the C4b density on E(Man) was increased. The data also show that C4BP regulates assembly and decay of the lectin pathway C3/C5 convertase more stringently than the classical pathway C3/C5 convertase because of a approximately 7- to 13-fold greater affinity for C4b deposited via the lectin pathway than the classical pathway. C4BP thus regulates efficiently the four times greater potential of the lectin pathway than the classical pathway in generating the C3/C5 convertase and hence production of pro-inflammatory products, which are required to fight infections but occasionally cause pathological inflammatory reactions.

Udgivelsesdato: 2009-Apr-27 Inefficient activation of complement lectin pathway in individuals with variant mannan-binding lectin (MBL) genotypes has been attributed to poor formation of higher order oligomers by MBL. But recent studies have shown the presence of large oligomers of MBL (approximately 450 kDa) in serum of individuals with variant MBL alleles. The recombinant forms of MBL (rMBL) variants except MBL/B that assemble into higher order oligomers have not yet been reported. In the present study, structural/functional properties of recombinant forms of wild type MBL (rMBL/A) and its three structural variants, rMBL/B, C, and D generated in insect cells were examined. Western blot analysis indicated covalently linked monomers to hexamers while gel filtration chromatography exhibited non-covalently linked higher order oligomers in addition to prevalent low oligomeric forms. Mannan binding determined by ELISA showed rMBL/A but not the structural variants bind to mannan. Apparent avidity of monoclonal antibody used was found to be about 18- to 52-fold weaker for rMBL structural variants than rMBL/A. Complement activation varied with maximum impairment apparent in rMBL/C followed by rMBL/B, but rMBL/D was functional to the same extent as rMBL/A. Comparison of rMBL/A to MBL purified from plasma (pMBL/A) indicated 8- and 24-fold weaker binding to mannan by BIAcore analysis and ELISA and about 5-fold lesser efficiency in activating complement. The findings provide new insights on the structural/functional properties of rMBL variants and imply that lectin pathway activation may be impaired in individuals, homozygous for the mutant alleles, MBL/C and to a lesser extent MBL/B but not MBL/D.

Although the initiating complex of lectin pathway (called M1 in this study) generates C3/C5 convertases similar to those assembled by the initiating complex (C1) of the classical pathway, activation of complement component C5 via the lectin pathway has not been examined. In the present study kinetic analysis of lectin pathway C3/C5 convertases assembled on two surfaces (zymosan and sheep erythrocytes coated with mannan (E(Man))) revealed that the convertases (ZymM1,C4b,C2a and E(Man)M1,C4b,C2a) exhibited a similar but weak affinity for the substrate, C5 indicated by a high K(m) (2.73-6.88 microm). Very high affinity C5 convertases were generated when the low affinity C3/C5 convertases were allowed to deposit C3b by cleaving native C3. These C3b-containing convertases exhibited K(m) (0.0086-0.0075 microm) well below the normal concentration of C5 in blood (0.37 microm). Although kinetic parameters, K(m) and k(cat), of the lectin pathway C3/C5 convertases were similar to those reported for classical pathway C3/C5 convertases, studies on the ability of C4b to bind C2 indicated that every C4b deposited on zymosan or E(Man) was capable of forming a convertase. These findings differ from those reported for the classical pathway C3/C5 convertase, where only one of four C4b molecules deposited formed a convertase. The potential for four times more amplification via the lectin pathway than the classical pathway in the generation of C3/C5 convertases and production of pro-inflammatory products, such as C3a, C4a, and C5a, implies that activation of complement via the lectin pathway might be a more prominent contributor to the pathology of inflammatory reactions.

Among the several strains of halobacteria grown heterotrophically, ribulose bisphosphate carboxylase activity was detected in those which accumulate poly (β-hydroxybutyrate), viz. Haloferax mediterranei, Haloferax volcanii and Halobacterium marismortui. In H. mediterranei, the activity was present in cell extracts prepared after growth on a variety of carbohydrates. The ribulose bisphosphate carboxylase activity in H. mediterranei was inhibited by carboxyarabinitol bisphosphate, and the enzyme cross-reacted with antibodies raised against the spinach enzyme. CO2 fixation by cell extract was stimulated by the addition of ATP and NADH. Preliminary data suggested that hydrogen could be a possible reductant.

The effect of vinblastine sulfate on gamma-radiation-induced DNA strand breaks in different tissues of tumour bearing mice, was studied by single-cell gel electrophoresis. Intraperitonial administration of different doses (0.25-2.0mg/kg body weight) of vinblastine sulfate 30 min prior to 4 Gy gamma-radiation exposure showed a dose-dependent decrease in the yield of DNA strand breaks in murine fibrosarcoma, blood leukocytes and bone marrow cells. The dose-dependent protection of cellular DNA against radiation-induced strand breaks as evidenced from comet tail length, tail moment and percent DNA in the tail, was more pronounced in bone marrow cells than in the cells of the tumor fibrosarcoma. In fibrosarcoma cells, the decrease in comet tail length, tail moment and percent DNA in the tail was detected at lower doses of vinblastine sulfate administration and these parameters were not significantly altered at higher doses, from that of the control irradiated. From this study, it appears that in addition to anticancer activity, vinblastine sulfate could offer protection to the normal tissues against gamma-radiation-induced DNA strand breaks.

DNA strandbreaks / Thymine glycol / Radiation protection / Tocopherol Monoglucoside Alpha-tocopherol monoglucoside (TMG), a water-soluble derivative of α -tocopherol, has been examined for its ability to protect DNA against radiation-induced strand breaks. Gamma radiation, up to a dose of 6 Gy (dose rate, 0.7 Gy/ minute), induced a dose-dependent increase in single strand breaks (SSBs) in plasmid pBR322 DNA. TMG inhibited the formation of γ radiation induced DNA single strand breaks (SSBs) in a concentration-dependent manner; 500 µ M of TMG protected the single strand breaks completely. It also protected thymine glycol formation induced by γ -radiation in a dose-dependent manner, based on an estimation of thymine glycol by HPLC.

The CO2-fixing enzyme of Calvin cycle ribulose-1,5-bisphosphate-carboxylase/oxygenase has been isolated from a halophilic bacterium, Haloferax mediterranei grown heterotrophically. A homogeneous preparation was obtained from sonicated extract of the cells by three steps, resulting in a specific activity of 52 nmol.min-1.mg protein-1. The physicochemical and catalytic properties of the enzyme were studied. The halobacterial ribulose-bisphosphate carboxylase is an oligomer of 54-kDa and 14-kDa subunits as detected by SDS/PAGE. By sucrose-density-gradient centrifugation, the molecular mass of the enzyme was estimated as approximately 500 kDa indicating a hexadecameric nature. No evidence for an additional form of the enzyme devoid of small subunits was obtained. The enzyme required Mg2+ for activity, KCl for activity and stability, and an optimal pH of 7.8. In contrast to many halophilic proteins, ribulose-bisphosphate carboxylase from H. mediterranei is not an acidic protein. From the comparison of amino acid composition of halobacterial enzyme with its counterparts from a few eukaryotic and eubacterial sources, the S delta Q values showed that these proteins share some compositional similarities.

Mannan-binding lectin (MBL), a pattern-recognition molecule binds to sugars present on the cell surface of microorganisms and activates the lectin pathway of complement. Three naturally occurring point mutations that occur in exon 1 of MBL 2 gene give rise to the structural variants of MBL and are correlated with impaired immune response. Wild type human MBL A and its variant, MBL B (Gly to Asp) were expressed in the yeast, Pichia pastoris. The yeast system has several advantages such as easy to manipulate, less expensive and generally gives high expression levels. Western blot analysis under reduced conditions of rMBL A and rMBL B expressed in the yeast culture medium exhibited a molecular size similar to that of native MBL (32 kDa) purified from human plasma (hMBL). Analysis under nonreduced conditions showed rMBL A and rMBL B to form higher order oligomers (monomers to hexamers) as observed for hMBL. Functional analysis of the rMBL molecules ability to bind sugars measured by ELISA, upon extensive dialysis of the culture medium, indicated that both rMBL A and rMBL B bind to mannan. Functional studies related to complement activation are currently being investigated. The estimated expression level of rMBL A and rMBL B (~0.3mg/L) indicates that the yeast system may be a better expression system of rMBL than the insect cell system (~0.2mg/L). Research supported by NIH grant HL073804

Activation of C5 via the lectin pathway of complement was analyzed by examining C5 cleaving properties of lectin pathway C3/C5 convertases. C4b-dependent C3/C5 convertase, ZymM1,C4b,C2a and EManM1C4b,C2a (M1 represents MBL-MASPs) assembled on zymosan or sheep erythrocytes coated with mannan (EMan) exhibited a weak affinity for C5 (Km = 2.7–5.3 μM). Very high affinity C5 convertases (ZymM1,C3bC4b,C2a and EManM1,C3bC4b,C2a) were generated when low affinity C4b-dependent C3/C5 convertases were allowed to deposit C3b by cleaving native C3. ZymM1,C3bC4b,C2a and EManM1,C3bC4b,C2a exhibited Km (4.9–8.3 nM) well below the normal concentration of C5 in blood (0.37 μM). Although, the Km, Vmax, and kcat of C3/C5 convertases of lectin pathway were similar to those reported for classical pathway, the ability of C4b to bind C2 indicated that every C4b deposited on zymosan/EMan was capable of forming a convertase. The findings differ from those reported for classical pathway C3/C5 convertase where one out of four C4b molecules deposited formed an enzyme. The four times greater potential of the lectin pathway than the classical pathway in generating C3/C5 convertases and producing proinflammatory products, such as C3a, C4a, and C5a implies that activation of complement via the lectin pathway might be a more prominent contributor to the pathology of inflammatory reactions. Research supported by NIH grant HL073804

Radioprotective compounds are of importance in clinical radiation therapy, because normal tissues should be protected against radiation injury while using higher doses of radiation to obtain better cancer control. We investigated the radioprotection of cellular DNA in cancer and in various cells and tissues, in a murine system following exposure to gamma-radiation and tocopherol monoglucoside (TMG) administration. We used single-cell gel electrophoresis (comet assay) and studied the progression of murine fibrosarcoma following radiation exposure and administration of TMG. The administration of TMG to tumor-bearing mice protected the cellular DNA against radiation-induced strand breaks as shown by the decrease in comet tail length, tail moment, and percentage of DNA in the tails of the cells of normal tissues. The same parameters were not altered in the cells of fibrosarcoma. Our results showed that the administration of TMG immediately after exposure to gamma-radiation can protect normal tissues against radiation damages in tumor-bearing mice. Local gamma-radiation exposure (5 Gy) of the tumor retarded the tumor growth. Administration of TMG did not protect cancer cells from radiation damage because the growth curves of cancer cells treated with radiation alone and those treated with TMG after irradiation were not significantly different.

The effect of rifampicin on the biological properties of bovine brain tubulin was investigated. Assembly of microtubules was almost completely blocked by rifampicin, whereas the depolymerization was not affected. The drug was found to inhibit both colchicine and GTP binding to tubulin. Association of rifampicin with tubulin was confirmed by spectrophotometric method. Binding of rifampicin was found to be dependent both on temperature and time. At 0 degrees for 1 hr 0.84 moles of rifampicin were bound per mole of tubulin.