Purification and characterization of lymphocytic clonal growth factor (LCGF) derived from human-human hybridoma SH-76 cells

Human-human hybridoma SH-76 cells were found to produce a factor that supported the growth of lymphocytic cells at low densities. The factor was purified from serum-free conditioned medium of the hybridoma cells by a successive application of ammonium sulfate precipitation, DEAE-Toyopearl, TSK G3000 SW and DEAE-5PW column chromatograph. The purified factor was a 72K single protein. The factor showed marked growth stimulating effect on lymphocytic cell lines, but had no effect on the growth of human adhesive cancer cell lines. Thus, the factor is a lymphocytic clonal growth factor (LCGF), as found previously in human plasma (Miyata, 1988). The LCGF of SH-76 cells could be produced in growth factor-free RPMI medium and purified easily from the conditioned medium. The factor is inactivated by heating at over 80°C, but is much more stable than the LCGF in human plasma.     

Ammonia regulation of phosphate-activated glutaminase displays regional variation and impairment in the brain of aged rats

The regulation of PAG by ammonia in whole brain (Sprague-Dawley) and regional (Fischer-344) synaptosomal preparations from adult and aged animals was assessed. Whole brain synaptosomal preparations from both age groups displayed a significant decrease in PAG activity with increasing ammonium chloride concentrations, however, the aged rats exhibited a significant attenuation in ammonia-induced PAG inhibition. PAG activity measured in synaptosomes prepared from the striatum (STR), temporal cortex (TCX) and hippocampus (HIPP) was also inhibited by ammonium chloride. The STR showed the greatest degree of ammonia-induced PAG inhibition (55%) followed by the HIPP (30–35%) and the TCX (25–30%). This reduction in PAG activity was significantly attenuated in STR from aged rats at ammonium chloride concentrations greater than 50 M and in the TCX, PAG activity was significantly attenuated in the aged rats at ammonia concentrations of 0.5 and 1.0 mM. Ammonia regulation of PAG activity in the HIPP appeared to be unaffected by age. Ammonium chloride concentrations up to 5 mM had no effect on GLU release from cortical slices, although GLN efflux was significantly enhanced. These findings suggest that isozymes of PAG may exist in different brain regions based on their differential sensitivity to ammonia. The attenuation of ammonia-induced PAG inhibition seen in aged rats may have deleterious effects in the aged brain.Abbreviations PAG phosphate-activated glutaminase: L-glutamine amidohydrolase; EC 3.5.1.2 - STR striatum - TCX temporal cortex - HIPP hippocampus     

Methyl mercury uptake by free and immobilized cyanobacterium

Methyl mercury uptake in free cells and different immobilizates of the cyanobacteriumNostoc calcicola has been examined. The general growth of the immobilized cyanobacterial cells could be negatively correlated with methyl mercury uptake. Alginate spheres proved most efficient in terms of uptake rate (0.48 nmol mg protein^–1 min^–1, 10 min) and total bioaccumulation (10.71 nmol mg protein^–1, 1 h) with a bioconcentration factor of 3.3×10³. Alginate biofilms showed a faster methyl mercury accumulation rate (0.83 nmol mg protein^–1 min^–1, 10 min) with a saturation of 10.28 nmol mg protein^–1 reached within only 30 min (bioconcentration factor, 3.1×10³). Foam preparations with a slow initial uptake approximated biofilms but were characterized by a lower bioconcentration factor (2.8×10³). Free cells, in comparison, maintained the initial slow rate of uptake (0.62 nmol mg protein^–1 min^–1, 10 min), saturating at 30 min (8.81 nmol mg protein^–1), and the resultant lowest bioconcentration factor (2.7×10³). Cell ageing (30 days) brought a drastic reduction (3-fold) in organomercury uptake by free cells while alginate spheres maintained the same potential. Foam preparations of the same age showed a significant improvement in methyl mercury uptake followed by only a marginal decline in alginate biofilms. Data are discussed in the light of the physiological efficiency and longevity of immobilized cells.     

Ca2+ transport activities of inside-out vesicles prepared from density-separated erythrocytes from rat and human

The plasma membrane Ca²⁺ ATPase in erythrocytes is vital for the maintenance of intracellular Ca²⁺ levels. Since the cytoplasmic Ca²⁺ concentration is elevated in older erythrocytes, the properties of the Ca²⁺ transport ATPase were examined during cell aging using inside-out vesicles (IOVs) prepared from density-separated, young (less dense, Ey) and old (more dense, Eo) rat and human erythrocytes. The transport of Ca²⁺ and the coupled hydrolysis of ATP were measured using radiolabeled substrates. The calmodulin-independent Ca²⁺ transport activity (Ey, 38.8 vs. Eo, 23.3 nmols/min/mg IOV protein) and the Ca²⁺ dependent ATP phosphohydrolase activity (Ey, 53.5 vs. Eo, 48.8 nmols/min/mg protein) were greater in IOVs prepared from younger (less dense) rat erythrocytes. The calmodulin-independent Ca²⁺ transport activity in IOVs from human erythrocytes was 12.9 nmols/min/mg IOV protein for Ey and 10.7 nmols/min/mg IOV protein for Eo. Inside-out vesicles from older (more dense) cells exhibited a lower pumping efficiency as determined by the calculated stoichiometry, molecule of Ca²⁺ transported per molecule of ATP hydrolyzed (rat: Ey, 0.74 vs. Eo, 0.49; human: Ey, 1.22 vs. Eo, 0.77). The enzymatic activity of rat and human Ey IOVs was labile. The Ca²⁺ transport activity in Ey but not Eo IOVs rapidly declined during cold storage (4°C). The decrease in Ca²⁺ transport activity during aging may accentuate the age-related decline in several erythrocytic properties.Abbreviations IOV Inside-Out Vesicles - Ey Erythrocytes enriched with young (less dense) cells - Eo Erythrocytes enriched with old (more dense) cells - ACEase Acetylcholinesterase     

Sperm aging in the male after sexual rest: Contribution to chromosome anomalies

The chromosome complement was studied in first-cleavage metaphases of mouse zygotes resulting from sperm aged in the male physiologically, after sexual rest. Females were inseminated by control males mating at 3-day intervals while experimentals mated to males that had had a sexual rest of 14 or more days. A total of 1954 eggs were collected 33–35 h post-HCG from 101 superovulated females mated to 42 controls and 43 experimental males. The fertilization rate was similar in both groups, being 84% and 85%, respectively. G-banded or Q-banded chromosomes were analyzed in 301 (68.3%) controls and 392 (49%) experimental first-cleavage metaphases. The overall rate of chromosome anomalies in controls was 4.45% as compared to 10.94% in experimentals, a highly significant difference. In the experimental group compared to controls, the frequency of trisomy, triploidy, structural rearrangements, and tetraploidy increased from 3.9% to 6.9%, 0% to 1.6%, 0.8% to 2.8%, and 0% to 1.3%, respectively. The genomic source of origin of the abnormalities was determined on the basis of differential condensation of the genomes. In the experimentals, grossly unbalanced sperm (diploids, disomics, double disomics, and those with large fragments) fertilized significantly more oocytes compared to controls. Our results implicate an advantage either in numbers or fertilizing capability for chromosomally abnormal sperm in a physiologically aged population.     

MORPHOLOGICAL VARIABILITY IN POPULATIONS OF CHRYSOPHYCEAN RESTING CYSTS. I. GENETIC (INTERCLONAL) AND ENCYSTMENT TEMPERATURE EFFECTS ON MORPHOLOGY1

Many chrysophycean species produce resting cysts (statospores) with purportedly species-specific morphology. I investigated variation in the cyst morphology of a single species that may result from genetic differences among the vegetative clones involved and from variation in the temperature of the environment during cyst development. Populations of Dinobryon cylindricum Imhof cysts were produced under defined conditions in vitro and then sampled for morphological analysis based on SEM micrographs. Morphological data is presented and then used in a multivariate discriminant analysis to determine the utility of each morphological character in distinguishing the six populations studied. Results suggest that some features of cyst morphology (i.e. cyst diameter) are invariant among the populations, while other features show distinctive variation. The density of spines covering the cyst body as well as the morphology of those spines appear correlated to the specific clones involved, and thus may represent useful phenotypic genetic markers. The length and definition of both the spines and the cyst collar, on the other hand, are markedly influenced by encystment temperature. The implications of these findings for paleoecological studies is discussed.     

Segregation products of male mice doubly heterozygous for the RB(6.16) and RB(16.17) translocations: Influence of sperm karyotype on fertilizing competence under varying mating frequencies

The meiotic segregants of male mice heterozygous for Rb(6.16)24Lub and Rb(16.17)7Bnr were viewed, for the first time, at first cleavage metaphase. Chromosomes were analyzed after G-banding, C-banding, and karyotyping. To study sperm aging effects, chromosomes of 202 one-cell zygotes derived from males mating at intervals of approximately 3,14, and 21 days were examined. At least 89.6% of sperm-derived complements were products of 2:2 segregation; at most, a possible 6.4% were 3:1 segregants. The six expected types of 2:2 segregants, both balanced and unbalanced, were equifrequent in the total zygote population derived from sperm of all ages. When the data were analyzed according to mating frequency, the 3-day sperm population considered most likely to be fresh showed a deficiency of the segregant nullisomic for chromosome 6 and disomic for chromosome 17, when compared with the reciprocal segregant (P < 0.025) as well as to all other 2:2 segregants (P < 0.05). However, these sperm fertilized in greater numbers (P < 0.01) than their reciprocal segregant (disomic for 6 and nullisomic for 17) in the 14-day sperm population. While sperm with chromosomal abnormalities are capable of fertilization, the competence of segregants nullisomic for 6 and disomic for 17 apparently depends on the prior storage period in the male. Further, the results suggest that the effect of aneuploidy on sperm function is dependent on the specific chromosome(s) involved.     

Age-dependent modifications of mitochondrial proteins in cerebral cortex and striatum of rat brain

The protein composition of free mitochondria purified from cerebral cortex and striatum during aging was analyzed by gel electrophoresis. Mitochondria were isolated from cerebral cortex and striatum of 4-, 12-, and 24-month-old rat brain. The percent amount of mitochondrial proteins after gel-electrophoretic separation was determined densitometrically. A significant decrease in the amount of two polypeptides (with molecular weights of 20 and 16 kDa, respectively) in both brain regions during aging was found. The decrease was higher in the striatum indicating a greater vulnerability of this brain area to the aging process. The age-dependent modifications of mitochondrial proteins observed may play an important role in several mitochondrial functions, such as energy transduction and transport processes as well as in structural changes occurring with age, causing altered membrane permeability and fluidity.     

Increased choline acetyltransferase activity by Chinese herbal medicine Sho-saiko-to-go-keishi-ka-shakuyaku-to in aged rat brain

The effect of a Chinese herbal medicine Sho-saiko-to-go-keishi-ka-shakuyaku-to (TJ-960) on the brain choline acetyltransferase (CAT) activity was studied in adult (3.5 months of age) and aged (24 months of age) rats. After oral administration of 5% TJ-960 solution for 3 months, CAT activity in the hippocampus, pons-medulla oblongata and striatum of aged rats was significantly lower than that of adult rats. CAT activity in the cerebellum, however, was significantly higher in the aged rats, as compared to the adult rats. TJ-960 significantly increased CAT activity in the hippocampus and striatum of aged rats, but did not affect the activity of the enzyme in the adult rat brain.     

Age-related effect induced by oxidative stress on the cerebral glutathione system

In the forebrain from male Wistar rats aged 5, 15 and 25 months, age-related putative alterations in the glutathione system (reduced and oxidized glutathione; redox index) were chronically induced by the administration in drinking water of free radical generators (hydrogen peroxide, ferrous chloride) or of inhibitors of endogenous free radical defenses (diethyl-dithio-carbamate, an inhibitor of superoxide dismutase activity). In hydrogen peroxide administered rats, both reduced glutathione and the cerebral glutathione redox index markedly declined as a function of aging, whereas oxidized glutathione consistently increased. In contrast, chronic iron intake failed to modify the reduced glutathione in forebrain from the rats of the different ages tested, whereas the oxidized glutathione was increased in the older brains. The chronic intake of diethyl-dithio-carbamate enhanced the concentrations of reduced glutathione in the forebrains from the rats of the different ages tested, the oxidized glutathione being unchanged. In 15-month-old rats submitted to chronic oxidative stress, ergot alkaloids (and particularly dihydroergocriptine) interfered with cerebral glutathione system, while papaverine was always ineffective. The comprehensive analysis of the data indicates that: (a) both the type of oxidative stress and the age of the animals modulate the cerebral responsiveness to the putative modifiers in the level of tissue free radicals; (b) aging magnifies the cerebral alterations induced by oxidative stress; the (c) cerebral glutathione system may be modified by metabolic rather than by circulatory interferences; (d) a balance between the various cerebral antioxidant defenses is present, the perturbation of an antioxidant system resulting in the compensatory modified activity of component(s) of another system.