''Secondary'' isozymes derived from the three PGM loci

1. The isozymes derived from the human phosphoglucomutases - PGM₁, PGM₂, PGM₃ have been examined in three tissues (lymphocytoid cells, placentae and red blood cells) in which the average age of the constituent proteins may be expected to differ. The appearance of one or more more negatively charged isozymes would appear to be correlated with increasing overall protein age. It is suggested that these are ‘secondry’ isozymes formed in vivo from the least negatively chared form which presumed to be the primary post translational product of the particular gene. 2. Changes in the PGM₃, isozyme pattern have been observed on storage of the placental extracts. Both the primary and the secondary in vivo isozymes were similarly affected. The storage effects observed are possibly due to reaction with red cell oxidized glutathione contaminating the extract. They may lead to confusion in typing unless controlled. Similar changes were not observed with the PGM₁, and PGM₂, isozymes. 3. The effects of a number of thiol reagents on the three PGMs have been examined and various changes in isozyme pattern have been produced artificially. Both the primary and the secondary in vivo isozymes derived from each allele were similarly affected by any particular treatment. PGM₃, isozymes were more reactive with thiol reagents than PGM₁, or PGM₂, isozymes. These findings suggest that the primary and secondary isozymes derived from each of the three PGM loci each contain at least one reactive sulphydryl group. However, the in vivo changes by which secondary isozymes are generated do not appear to be due to such thiol effects.     

Viral proteins and adenosine triphosphate phosphohydrolase activity of fish lymphocystis disease virus

Fish lymphocystis disease virus (FLDV) was isolated from papilloma-like lesions of various flatfish species, flounder, dab, and plaice. FLDV particles purified by density gradient centrifugation were denatured and analyzed by polyacrylamide gel electrophoresis under denaturing conditions. At least 33 FLDV polypeptides ranging in molecular weight from 220 to 14 K were detectable after staining with Coomassie blue. The patterns of structural polypeptides from different fish species were similar although some slight and distinct differences were found. In addition, polypeptide patterns of FLDV-infected flatfish cells were analyzed and compared with those of uninfected fish cells. A drastic change in the pattern of host cell proteins is observed and new, virus-induced proteins are synthesized as a result of an in vivo infection by FLDV. A nucleoside triphosphate phosphohydrolase activity is associated with FLDV. The enzymatic activity hydrolyzes the γ-phosphate residue of ATP and GTP with a high preference for ATP. The requirements for the ATPase activity and the reaction products are described.     

Aging of the erythrocyte. IX. Fluorescence studies on changes in membrane properties

Fluorescence studies of membranes prepared from density-separated red blood cells demonstrated an increase in lipid viscosity (judging from fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene and 1-aminonaphthalane-8-sulfonate and changes in the physical state of proteins (judging from tryptophan fluorescence intensity and polarization). These alterations may be partly due to lipid peroxidation. In vivo accumulation of products of this process in the membranes was confirmed. Changes were also found in the distribution pattern of cell populations of different mean age in a cell sorter, probably due to alterations in rheological properties of the erythrocytes.     

Aging of the erythrocyte. VII. on the possible causes of inactivation of red cell enzymes

The sequence of inactivation of five bovine erythrocyte enzymes (glucose-6-phosphate dehydrogenase, pyruvate kinase, lactate dehydrogenase, aspartate aminotransferase and aldolase) was compared during in vivo aging of the red blood cell and during treatment of the cells with heat, gamma-radiation and an enzymatic source of the superoxide radical anion. Amounts of oxygen free radicals comparable to those formed physiologically in the cells were able to induce considerable inactivation of some enzymes; in no case was the physiological sequence of inactivation reproduced. These results seem to argue for a multifactorial inactivation of red cell enzymes during intravascular aging of the erythrocyte.     

Generation of T-cell function in organ culture of foetal mouse thymus. I. Mitogen responsiveness

An in vitro model for the study of T-cell ontogeny is presented. The generation of mitogen responsive thymocytes in embryonic mouse thymus using an organ culture system is described. The pattern of development of mitogen responsiveness in vitro is similar to that seen in vivo except that responses were greater and appeared later in culture than in thymus of a temporally equivalent age in vivo. This suggests that organ culture selectively enriches T-cell activity and thus offers a novel approach to the analysis of T-cell reactivity as well as to the understanding of defects in T-cell development.     

Protective effect of stilbenes containing extract-fraction from Cajanus cajan L. on Aβ25–35-induced cognitive deficits in mice

Cajanus cajan (L.) is a traditional Chinese herb medicine which contains a lot of potential active components. In the present study, we identified the effects of the stilbenes containing extract-fraction from C. cajan L. (sECC) on Aβ_25–35-induced cognitive deficits, oxidative stress and cholinergic dysfunction in mice. Mice were treated with sECC (100 and 200 mg/kg/d) for 1-week, and then received a single intracerebroventricular (i.c.v.) injection of Aβ_25–35 (5 μg/mice). Behavioral changes and neuron apoptosis in mice were evaluated using Morris water maze and TUNEL tests. Furthermore, superoxide dismutase (SOD), choline acetyl transferase (ChAT) and acetylcholine esterase (AchE) activity in hippocampus and cortex were analyzed by spectrophotometric method. The data showed that consumption of sECC (200 mg/kg) significantly ameliorated the cognitive deficits and neuron apoptosis caused by i.c.v. injection of Aβ_25–35. At the same time, the decreased SOD and ChAT activity in hippocampus and cortex were markedly increased by sECC (200 mg/kg). sECC has no effect on AchE activity in hippocampus and cortex. These findings suggest that sECC may be a potential candidate for the development of therapeutic agents to manage cognitive impairment associated with Alzheimer's disease (AD) through increasing the activity of ChAT and anti-oxidative mechanism.     

Effects of the type of dietary fat at two levels of vitamine in wistar male rats during development and aging. II. Biochemical and morphometric parameters of the brain

The object of this study was to explore the possible influence of the type of dietary fat at two extreme levels of vitamin E on several biochemically determined changes in the whole brain as well as on a number of quantitatively analyzed ultrastructural variations with age in neurons of the dorso-lateral parietal cortex and in the cerebellar Purkinje cells. For this purpose, six groups of weaning Wistar male rats were fed ad libitum isoenergetic diets containing similar amounts (15 g per 100 g of diet) of saturated fat (coconut oil), unsaturated fat (safflower oil) or a combination of both at two levels of dl-α-tocopherol (2 or 200 mg per 100 g of diet). The determinations were carried out in rats killed at 3, 6, 12, 18 and 24 months. Although in relation to age and irrespective of the type of diet several of the biochemical parameters significantly fluctuated with time, comparisons of the results between the youngest and oldest rats showed no changes in the concentration of RNA, triglycerides, phospholipids, cholesterol and collagen, a significant increase in DNA and vitamin E, and a significant decrease in total protein. While in vitro lipoperoxidation of whole brain homogenates (malonaldehyde production) increased from 3 to 18 months, there was a precipitous decline at 24 months. None of these biochemical changes were consistently influenced by the type of diet. In vivo peroxidation in the total lipids of the whole brain (conjugated dienes) was never observed in rats fed the saturated fat diets and was only detected at 6, 12 and 18 months in some of the rats fed unsaturated or combined fat diets. At 24 months, however, none of the rats from the various dietary groups showed in vivo lipoperoxidation. It would appear that the dietary levels of vitamin E had no effect on the extent of in vivo lipoperoxidation in rats fed the unsaturated fat diets but may have had some preventive effect in rats fed the combined fat diets for 18 months. The possible nosologic implications, if any, of the eventual occurrence of in vivo lipoperoxidation remained uncertain since no correlation could be found between this phenomenon and the other biochemical and morphometric parameters. In the cerebral cortex, the numerical density of neurons, astrocytes and dark oligodendrocytes did not change with age, but microglial cells increased and light oligodendrocytes significantly decreased. No major quantitative variations were found in mitochondria and rough endoplasmic reticulum of cortical neurons with age. The numerical density of Purkinje cells decreased with age and, while in these cells the numerical density of mitochondria decreased, the fractional volume of these organelles as well as the density of rough endoplasmic reticulum remained unchanged with age. The fractional cytoplasmic volume of lipofuscin significantly increased almost linearly with time in cortical neurons and Purkinje cells. While no correlation was found between lipofuscin accumulation in the cerebral cortical neurons and the variations in the other morphometric parameters of these cells, a highly significant negative correlation was detected between the increment of lipofuscin and the decrement of Purkinje cell number (r = ?0.96) as well as with the decrement in the number of mitochondria in these cells (r = ?0.90), suggesting a possible deleterious effect of this pigment. None of the morphometric parameters, including lipofuscin, were clearly influenced by either the type of dietary fat or by the levels of vitamin E.     

Effects of donor's age on growth kinetics of rabbit articular chondrocytes in culture

The _vitro proliferative capacity of articular chondrocytes derived from young and old rabbits was investigated to examine if the modifications incurred can be related to the _{in vivo} aging. Determinations were made of the cartilage cell density, cell volume, cell number at confluency, plating efficiency, growth curve and DNA content distributions.The old donor cells were characterized by a decline in all the parameters of cartilage growth studied: cell number at confluency, cell replication rate (from 20 h to 45 h) as well as an increase in cell volume. The mean cycle time in vitro increased from 17.5 h compared to 27 h during in vitro aging, essentially because of an elongation of the G₁ phase.Chondrocytes derived from young and old donors may be an appropriate model system for studying the in vitro effects of drugs on rheumatoid diseases as a function of in vivo aging.     

Hydrogen peroxide is produced by E. coli challenged haemocytes and regulates phagocytosis, in the medfly Ceratitis capitata. The active role of superoxide dismutase

Hydrogen peroxide (H₂O₂) participates as a second messenger in cell signaling. In this paper, the role of H₂O₂ was investigated, in Escherichia coli phagocytosis by the haemocytes of the medfly Ceratitis capitata. Block of H₂O₂ synthesis by specific enzymic inhibitors, namely N-ethylmaleimide (NEM) for NADPH oxidase and diethyldithiocarbamate (DDC) for SOD, resulted in the increase of E. coli phagocytosis. Immunoblot analysis, flow cytometry and confocal microscopy, revealed the constitutive expression of SOD, in the medfly haemocytes. Phagocytosis increased by small interfering RNA (siRNA) for SOD, revealing the active involvement of SOD and H₂O₂. Immunoblot analysis showed an increase of the ERK1/2 phosphorylation, in the presence of the above H₂O₂ synthesis enzymic inhibitors. In addition, confocal microscopy showed no co-localization of SOD with β integrin subunit. It appears that SOD participates in the regulation of bacterial phagocytosis, due to involvement of the produced H₂O₂ in the differential phosphorylation of MAP kinases.     

Myelination in rat brain aggregating cell cultures.

Aggregates of fetal rat brain were maintained in rotating culture for 30–40 days and were analyzed morphologically and biochemically. At 4 days in culture all cells were undifferentiated. At 26 days in vitro over 90% of all cells within the aggregates could be identified as neurons, astrocytes or oligodendrocytes. Myelinated axons and morphologically mature synapses were present at 26 days. Myelination started between 18 and 19 days in culture as determined biochemically. Myelin basic protein sulphatide synthesis and 2′,3′-cyclic nucleotide 3′-phosphohydrolase activity increased with in vitro age. The amount of myelin observed within the aggregates was much lower than observed at the corresponding age in vivo. Neurons and neuronal processes were undergoing severe degeneration in the 40-day aggregates and synaptic contacts were not maintained. There were no normal myelinated axons at 40 days although multilammellar membranes were found intra- and extracellularly. The ganglioside pattern of the aggregates were qualitatively similar to rat whole brain. Quantitatively the G_M3ganglioside was elevated in comparison to whole rat brain.Our results indicate that aggregating rat brain cultures provide a useful in vitro system for the biochemical and morphological analysis of myelin formation.     

Stimulation of proliferation in stationary primary cultures of monkey and rabbit aortic smooth muscle cells. I. Effects of lipoprotein fractions of hyperlipemic serum and lymph.

Medial explants of thoracic aorta from rhesus monkey or rabbit show outgrowth within 5–10 days when cultured in 90% basal Eagle's medium and 10% normal serum. After 5–6 weeks of rapid growth, cell colonies around the explant reach a stationary phase with low mitotic activity. Homologous hypercholesterolemic serum and lymph stimulate these stationary cultures into another proliferative phase, when 5% of the normal serum is replaced in the culture medium. Measurement of increase in colony size and evaluation of [³H]thymidine incorporation by autoradiography serve to indicate increased proliferation. Low density lipoproteins (LDL) from hyperlipemic serum and lymph have the greatest stimulatory effect, while high density lipoproteins (HDL) and the lipid-free bottom fraction, as well as LDL from normal serum and lymph, have no effect. These stationary cultures are valuable for the study of agents suspected of having a stimulatory effect on cell proliferation of aortic lesions in vivo.     

Mechanism of inactivation of a Fasciola proteolytic enzyme by peptide aldehydes and alkylating agents

A proteolytic enzyme of the liver fluke Fasciola sp. was purified as described previously by ammonium sulfate fractionation, gel filtration on Sephadex G-200 column and l-phenylalanine-agarose chromatography. Leupeptin, a peptide aldehyde of microbial origin, competitively inhibited the enzyme activity with respect to the substrate $\text{α-N-}\text{benzoyl-}\text{l}\text{-argininamide}$; the apparent K_i value for leupeptin is 45 000-fold less than the apparent K_m for the substrate. Incubation of the enzyme with leupeptin resulted in time-dependent inactivation of the globinolytic activity, with an inactivation constant (K_inact) of 0.4 μM giving the half-maximum inactivation velocity, and with a minimum inactivation half-time (T) of 2.7 min at infinite concentration of this compound. The inactivated enzyme was not reactivated by extensive dialysis. These results imply that leupeptin yields an affinity labelling of an active site of the enzyme. The activity of the Fasciola proteolytic enzyme was also inactivated by other peptide aldehydes and alkylating agents and inactivation constants observed were 0.5 μM for chymostatin, 13 μM for antipain, 2 μM for $\text{p-}\text{toluenesulfonyl-}\text{l}\text{-lysine}$ chloromethyl ketone, 140 μM for $\text{p-}\text{toluenesulfonyl-}\text{l}\text{-phenylalanine}$ chloromethyl ketone and 40 μM for iodoacetate under the conditions used.     

An immunological basis for the anaemia of acute Salmonella gallinarum infection of chicken. I. Haematological changes and their association with in vivo modification of the erythrocytes

The haematological changes occurring during the course of acute Salmonella gallinarum infection of chicken have been investigated. A severe, acute anaemia, together with reticulocytosis and hepato-splenomegaly, were regularly observed. The findings indicated that these changes were due to increased extravascular destruction of erythrocytes rather than by inhibition of haemopoietic activity.

Coincidentally with the development of the anaemia, erythrocytes became regularly modified serologically in vivo, suggesting a possible relationship between these two phenomena. In contrast, in chickens which died of the per-acute form, where normal haematologic values were found, in vivo erythrocyte modification was not detected.

As the anaemia increased in severity, a change in the type and in vivo erythrocyte modification was also frequently noted.

It is considered that in vivo erythrocyte modification initiates the severe haematological changes observed and it is postulated that the underlying mechanism of increased erythrocyte destruction is immunological.

     

Molecular association in aqueous solutions of high molecular weight poly(N-vinyl-2-pyrrolidone)

A viscosimetric method was used to investigate the molecular association in aqueous solutions of poly(N-vinyl-2-pyrrolidone), (PVP), of molecular weight 700 000. Limiting viscosity numbers [η] and Huggins constants k_H of the polymer solutions were observed to decrease upon addition of denaturing agents such as thiourea and guanidinium sulfate. Guanidinium sulfate was found to be even more effective in its denaturing action as compared to urea and thiourea. The decrease in [η] and k_H values increased with increasing concentrations of the denaturing agents. This behaviour was explained on the basis of the rupture of bridges formed by water molecules among different PVP chains, hydrogen bonding being responsible for these intermolecular associations. While no change in the [η] values was observed for PVP in 2 M thiourea solutions upon increasing the temperature from 25°C to 35 and 45°C, for the same increase in temperature the [η] values of aqueous PVP solutions showed substantial decreases. This is also attributed to the breaking of hydrogen bonds existing between PVP molecules.     

Antioxidant Activity In Vitro and Hepatoprotective Effect of Phlomis Maximowiczii In Vivo

Background

A number of medicinal plants and there compounds played a major role in the treatment of hepatic disorders. They were widely used for the treatment of these disorders, and oxidant stress injury was one of the liver injury mechanisms. The present study evaluated the antioxidant activity and the hepatoprotective effect of each extracts of Phlomis maximowiczii.

Materials and Methods

The antioxidant activity was assayed by the methods of ABTS, FRAP and DPPH in vitro. Hepatoprotective effect of P. maximowiczii extracts was examined using carbon tetrachloride-induced acute liver injury in mice.

Results

P. maximowiczii n-butanol (PMBU) extract, ABTS (IC₅₀=18.96 µg/mL), DPPH (IC₅₀=25.15 µg/mL), and FRAP (RACT₅₀=2775.6±144.18 µmol/g), showed higher scavenging capacity than that of P. maximowiczii ethyl acetate (PMEA). The n-butanol extract could significantly reduce the level of GPT, GOT and MDA (P<0.05, P<0.001 and P<0.001, respectively) and increase the level of SOD (P<0.001), respectively.

Conclusion

The antioxidant activity of n-butanol extract in vitro was related with the level of MDA and SOD in vivo, and hepatoprotective effect of n-butanol extract also had relationship with its antioxidant activity in vivo.     

Staphylococcal catalase protects intracellularly survived bacteria by destroying H2O2 produced by the murine peritoneal macrophages

To determine the interrelationship between the hydrogen peroxide (H₂O₂) mediated killing and the potential role of bacterial catalase and SOD in the evasion of host defense, we examined three clinical isolates of Staphylococcus aureus and evaluated their intracellular survival mechanism within murine peritoneal macrophages. Fluorescent microscopy and bacterial colony-forming unit (cfu) count revealed that phagocytic capacity of murine peritoneal macrophages was highest after 2 h of in vitro infection with S. aureus. To understand whether catalase and SOD contributing in the intracellular survival, were of bacterial origin or not, 3 amino 1,2,4 triazole (ATZ) and Diethyldithiocarbamic acid (DDC) were used to inhibit specifically macrophage derived catalase and SOD respectively. Catalase activity from the whole staphylococcal cell in presence of ATZ suggested that the released catalase were of extracellular origin. Scanning electron microscopy revealed the degraded host cell membrane integrity during prolonged infection. Purified bacterial catalase from the intracellularly survived S. aureus recovered after 5 h of infection and its inhibition by ATZ in the zymography strengthened the scope of involvement of these anti-oxidants in the intracellular survival of S. aureus.     

Quantitative studies on antigenic recognition. 3. Nonspecific inhibition of the recognition process

Attempts were undertaken to evaluate the inhibitory effect of various agents on the process of recognition of transplantation antigens by lymphoid cells from normal mice, rats and Syrian hamsters. These effects were measured by estimating quantitatively a product of antigenic recognition (PAR) present in supernatants of mixed spleen cell cultures in which either the recognizing (parental strain aggressor) cell partner or the recognized ( F₁ hybrid target) cell partner was treated prior to cocultivation. The inhibitory agents used were heat, trypsin, ALS and rabbit anti-PAR antisera. Heat inactivation of cells resulted in inhibition of the process and affected both partners of cell mixtures. All other agents failed t o harm antigens on F₁ target cells but neutralized the recognizing potential of aggressor cells. In this respect, trypsin inhibited both mouse and rat aggressor cells to about equal extents. Anti-lymphocyte serum failed to show strain and even species specificity. Sera prepared against PAR also displayed no strain specificity but revealed species specificity.     

Passive anaphylaxis in mice with γG antibodies: V. Competitive effects of different immunoglobulins and inhibition of reactions with antiglobulin sera

Mouse antisera to DNP conjugates were fractionated on agar block electrophoresis, and each fraction was tested for ability to give anaphylactic reactions in vivo in mice and guinea-pigs, and in vitro on mouse mast cells. Concentrations of IgG₁ and IgG₂ globulin were measured for each fraction, and the results indicate that mouse IgG₁ globulin mediates both the in vivo PCA reaction in the mouse and the in vitro mast cell sensitization.

PCA reactions in guinea-pigs with mouse antisera are mediated only by electrophoretic fractions containing IgG₂ globulin. PCA reactions in guinea-pigs could be blocked by the addition of IgG_2a myeloma containing sera to the sensitizing anti-DNP preparation, but not by addition of IgG_2b or IgG₁ myeloma sera.

From six different IgG₁ myeloma sera tested on their capacity to block PCA reactions in mice, five showed strong inhibitory activity whereas the sixth was inactive. However, also IgG_2a and IgG_2b myelomas were found capable of blocking PCA reactions in mice. The passive in vitro sensitization of mouse mast cells by purified mouse anti-DNP antibodies could also be blocked by the addition of specific anti-IgG₂ sera. Possible interpretations of these findings are discussed.

     

Tracking transplanted cells in live animal using upconversion fluorescent nanoparticles

With the emergence of cell transplant as an attractive treatment modality for various diseases, there is a parallel need to track the fate of these cells to assess their therapeutic effectiveness. Here, we report the use of upconversion fluorescent nanoparticles, silica/NaYF₄:Yb,Er, to dynamically track live myoblast cells in vitro and in a living mouse model of cryoinjured hind limb. Nanoparticles loaded into cells were confirmed for its intracellular uptake by confocal imaging, spectrophotometry and inductively coupled plasma analysis. Loaded nanoparticles demonstrated absolute resistance to photobleaching and were applied for dynamic imaging to real time track in vitro cell migratory activity for a continuous 5 h duration using a time-lapse confocal microscope. Direct observation on the direction, speed and cell–cell interaction of migrating cells was clearly visualized. In vivo confocal imaging of nanoparticle-loaded cells intravenously injected into a mouse tail vein showed them flowing in the ear blood vessels. Nanoparticle-loaded cells were also unambiguously identified with superior contrast against a negligible background at least 1300 μm deep in a fully vascularized living tissue upon intramuscular injection. Spatiotemporal migratory activity of the transplanted cells within the three-dimensional living tissue was captured for at least 7 days post-delivery. Direct in vivo visualization of cell dynamics in the native tissue was unobtrusively followed over a 4 h time course and revealed subtle migratory activity of the transplanted cells. With these unique optical properties, we present silica/NaYF₄:Yb,Er nanoparticles as a new fluorescent live cell tracker probe for superior in vitro and in vivo dynamic imaging.     

Antibodies to guinea-pig lymphokines: V. Suppression of normal lymphocyte transfer, immune lymphocyte transfer and direct reactions in the guinea-pig by anti-lymphokine globulin

An antiserum (anti-lymphokine globulin, ALkG) directed against highly-purified products of activated lymphocytes, inhibits normal lymphocyte transfer (NLT), immune lymphocyte transfer (ILT) and direct reactions (DR) elicited by lymph node cells from strain 2 guinea-pigs in the skin of strain Bio B recipients. When injected i.d. around the site of injection of donor cells or i.v. into Bio B guinea-pigs, ALkG partially suppressed the initial inflammatory phase associated with NLT reactions and totally abolished the flare-up phase.

ALkG is directed against three newly synthesized lymphocyte products (one of them being migration inhibitory factor, MIF) which are involved in the mediation of delayed hypersensitivity reactions in vivo. Since ALkG does not contain cytotoxic antibodies against guinea-pig lymphocyte cell antigens and since its inhibitory activity is not absorbed by lymphoid cells, it is probable that the inhibition of NLT reactions is not mediated by the lysis of donor or host cells but rather by the inactivation of a soluble factor elaborated in vivo as a result of the allogeneic interaction.

The injection of ALkG 2 days after the initiation of the NLT reaction only produces a partial suppression of the inflammatory response associated with the flare-up phase indicating that ALkG must be present during the first 24–48 h in vivo in order effectively to inhibit the development of the NLT reaction, possibly by blocking the action of a humoral substance mediating the flare-up reaction. These findings suggest that ALkG is capable of inhibiting some product(s) of allogeneic cell interactions not only in vitro (ALkG has previously been shown to inhibit the guinea-pig mixed leucocyte reaction by reacting with a blastogenic factor produced during the MLC reaction) but also in vivo suggesting that similar lymphocyte products may be responsible for both in vivo (NLT) an in vitro (MLC) reactivity.