Molecular species of epidermal growth factor carrying immunosuppressive activity

The suppression of antibody formation to sheep red cells in mice by partially purified fractions of mouse submaxillary gland was shown to be caused by epidermal growth factor (EGF). Purification of EGF by the method of Savage and Cohen resolved three components referred to as EGF a, EGF b, and EGF c. All three induced premature eye opening in neonatal mice, but only EGF a (identified as EGF 1-53) had full immunosuppressive activity. EGF c was shown by micropeptide mapping of chymotryptic and thermolytic digests and amino-terminal analysis to differ from EGF a only by the presence of beta-aspartyl instead of an asparaginyl residue. EGF b differed from EGF a in that it lacked the N-terminal asparagine. EGF shortened enzymatically at its carboxy terminal by two or five amino acids did not have any immunosuppressive activity. These findings suggest that, in contrast to some other biological effects of EGF, intact amino and carboxy terminals are required for the expression of immunosuppressive activity.     

Biochemical characterization of propionyl CoA carboxylase deficiency: Heterogeneity within a single genetic complementation group

Liver tissues and fibroblasts from patients with propionic acidemia assigned to the pcc BC genetic complementation group have previously been shown to contain normal or near-normal quantities of structurally altered propionyl CoA carboxylases (PCC). Biochemical comparisons of PCCs from extracts of three livers and one placenta belonging to the pcc BC complementation group revealed that the K _m values for the enzyme's major substrates, propionyl CoA, bicarbonate, and ATP, and its monovalent activator, potassium, were similar to those of normal PCC. PCC in extracts of one of the livers, however, had an altered isoelectric point (pI = 5.4) compared to that of PCC from normal and other PCC-deficient tissues (pK = 4.6–4.7). Thermostability in the presence of sucrose or ATP differed among several of the mutant PCCs, including the PCC with an altered pI, and from that of normal PCC. To confirm these results and to determine whether valid inferences may be derived from comparisons of mutant and normal PCC in crude extracts, PCC was purified from normal liver and from one of the PCC-deficient livers. The biochemical parameters of the purified carboxylases were similar to those observed in liver extracts. These studies further-more confirmed that, whether purified or in extracts, PCC from the pcc BC group reflects structural mutations. Nevertheless, the abnormal enzyme structure appears to have no corresponding effect on the clinical features of the disorder in various affected individuals. Moreover, there is biochemical heterogeneity within the pcc BC complementation group that probably represents different interallelic gene mutations.This work was supported by NIH Research Grants Am 25675 and AM 26127. B. Wolf is the recipient of NIH Research Career Development Award AM 00677 and is aided by Basil O'Connor Starter Research Grant 5-263 from The National Foundation-March of Dimes. This article is No. 131 from the Department of Human Genetics at the Medical College of Virginia.     

Crucial factors determining the spatio-temporal distribution patterns of threeAthalia sawflies feeding on common cruciferous plants

Three Athalia sawflies, A. japonica, A. rosae and A. infumata, feeding on cruciferous plants, coexist in Japan. However, it is not known what ecological strategies they use and what environmental factors are crucial to such strategies. I attempted to explain these questions by examining the relationship between the spatio-temporal distribution patterns of three Athalia sawflies and their habitats in three districts (Lowland, Intermediate and Mountain). The three sawflies have different spatio-temporal distribution patterns, though they usually used common cruciferous plants. A. japonica was abundant in spring and autumn but disappeared during summer in all the districts. In the Lowland, populations of A. rosae and A. infumata, like that of A. japonica, crashed in summer. However A. rosae occurred mainly in summer in the Intermediate and Mountain. Although A. infumata occurred in the same seasons as A. rosae in all districts, population levels of A. infumata were always lower than those of A. rosae. The crucial factors controlling their population patterns were the availability of host plants and temperature. Population crashes of A. rosae and A. infumata were due to food depletion, and those of A. japonica were due to heat stress. On the other hand, their population patterns may be interpreted as phenological synchronization with their original host plants, though they all existed on common cruciferous plants. The three sawflies may have evolved different strategies to escape from unfavorable habitat conditions. Such strategies are speculated to be summer diapause in A. japonica, long distance migration in A. rosae, and local dispersal in A. infumata.     

Recovery of photosynthesis and phtosystem II fluorescence in Chlamydomonas reinhardtii after exposure to three levels of high light

Recovery from 60 min of photoinhibitory treatment at photosynthetic photon flux densities of 500, 1400 and 2200 μMmol m^?2 s^? was followed in cells of the green alga Chlamydomonas reinhardtii grown at 125 μMmol m^?2 s^?1. These light treatments represent photoregulation, moderate photoinhibition and strong photoinhibition, respectively. Treatment in photoregulatory light resulted in an increased maximal rate of oxygen evolution (P_max) and an increased quantum yield (Φ), but a 15% decrease in F_v/F_M. Treatment at moderately photoinhibitory light resulted in a 30% decrease in F_v/F_M and an approximately equal decrease in Φ. Recovery in dim light restored F_v/F_M within 15 and 45 min after high light treatment at 500 and 1400 μMmol m^?2 s^?1, respectively. Convexity (Θ), a measure of the extent of co-limitation between PS II turnover and whole-chain electron transport, and Φ approached, but did not reach the control level during recovery after exposure to 1400 μMmol m^?2 s^?1, whereas P_max increased above the control. Treatment at 2200 μMmol m^?2 s^?1 resulted in a strong reduction of the modeled parameters Φ, Θ and P_max. Subsequent recovery was initially rapid but the rate decreased, and a complete recovery was not reached within 120 min. Based on the results, it is hypothesized that exposure to high light results in two phenomena. The first, expressed at all three light intensities, involves redistribution within the different aspects of PS II heterogeneity rather than a photoinhibitory destruction of PS II reaction centers. The second, most strongly expressed at 2200 μmol m^?2 s^?1, is a physical damage to PS II shown as an almost total loss of PS II_α and PS II Q_B-reducing centers. Thus recovery displayed two phase, the first was rapid and the only visible phase in algae exposed to 500 and 1400 μmol m^?2 s^?1. The second phase was slow and visible only in the later part of recovery in cells exposed to 2200 μmol m^?2 s^?1.     

Analysis of fluorescence induction in thylakoids with the method of moments reveals two different active Photosystem II centres

There is presently a debate concerning the number of phases in fluorescence induction and on the identification of the several possible heterogeneities in PS II centres. However, the usual methods of analysis present numerical problems, including a lack of robustness (robustness being defined as the ability to give the correct answer in the presence of distortions or artefacts). We present here the adaptation of the method of moments, which was developed for robustness, to the analysis of fluorescence induction. We were thus able to identify three phases in the fluorescence induction in the presence of DCMU. The slowest phase was attributed to the centres inactive in plastoquinone reduction by using duroquinone as electron acceptor. In order to compare fluorescence with and without DCMU, we introduced the rate of photochemistry, defined as the product of the area times the rate constant of an exponential. This quantity is invariant for a given centre no matter what the size of the electron acceptor pool is. The two fastest phases in the presence of DCMU were attributed to active centres because their rate of photochemistry was the same as that of the plastoquinone-reducing phases in the absence of DCMU. Because their reduction of plastoquinone showed different kinetics, these two types of active centres were either separated by more than 250 nm or were associated with discrete plastoquinone pools having restricted diffusion domains.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMBQ 2,5-dimethyl-p-benzoquinone - MOPS 3-[N-Morpholino]propanesulphonic acid - PpBQ Phenyl-p-benzoquinone     

Estrogen-stimulation of postconfluent cell accumulation and foci formation of human MCF-7 breast cancer cells

Foci, nodules of cellular overgrowth, that appear after confluence are an in vitro characteristic of malignant transformation. A well-studied in vitro model of estrogen-dependent tumors is the MCF-7 cell line, derived from a pleural metastasis of a human breast adenocarcinoma. We report that cultivation of MCF-7 cells, using routine methods, results in extensive estrogen-stimulated postconfluent cell accumulation characterized by discrete three-dimensional arrays. Side view Nomarski optical sections revealed these to be principally multicellular foci with occasional domes and pseudoacinar vacuoles. This effect on MCF-7 cell growth occurs in media containing fetal bovine serum but not with calf serum or charcoal-dextran-treated fetal bovine serum unless supplemented with estrogens. Foci formation starts 5-6 days after confluence, and the number of foci generated is a function of the concentration of added estrogens. Foci formation is suppressed by the antiestrogens Tamoxifen and LY 156758. Addition of progesterone, testosterone, or dexamethasone had little or no effect, while various estrogens (ethinyl estradiol, diethylstilbestrol, and moxestrol) induced foci development. Clones derived from single cells of the initial MCF-7 population revealed a wide variance in estrogen-induced foci formation, demonstrating heterogeneity of this tumor cell line. The postconfluent cell growth of the estrogen receptor-deficient cell line, MDA-MB-231, contrasted with MCF-7 by developing an extensive multilayer morphology devoid of discrete structures. The tumorigenic potential of the MCF-7 cells used in our experiments was confirmed by their estrogen-dependent growth in immunosuppressed male BDF1 mice. These data suggest an estrogen receptor-based mechanism for the development of multicellular foci during postconfluent growth of MCF-7 cells. After confluence, foci, in contrast to the quiescent surrounding monolayer, retain proliferating cells. Focus formation, therefore, reflects the heterogeneous responsiveness of these cells to estrogens and should provide a model permitting in vitro comparisons between the progenitor cells of multicellular foci and the monolayer population.     

Fibroblast heterogeneity in glucocorticoid regulation of collagen metabolism: Genetic or epigenetic?

Summary Cultured fibroblasts derived from normal human dermis show a consistent 62% inhibition of collagen synthesis by hydrocortisone, whereas cultures derived from keloids average only 30% inhibition and show a much larger strain to strain variation ranging from 75% inhibition to 49% stimulation. Examination of fibroblast clones indicates that this high variation among keloid strains is not due to differences in the proportion of normal and keloid cells in the mass culture populations. Small but significant differences in the effect of hydrocortisone on collagen deposition are also seen among these clonal populations, but are not related to the type of tissue from which cultures were derived. Two to three-fold differences among clones derived from a single individual were observed, possibly suggesting functional heterogeneity of dermal fibroblasts with regard to collagen metabolism under control conditions and in response to hydrocortisone. However, this variation among clones may simply reflect differences in clonal growth, inasmuch as both collagen synthesis and deposition, and the effect of hydrocortisone on these processes, are strongly affected by population density. This work was supported in part by PHS grants, CA-17229 from the National Cancer Institute and AG-02046 from the National Institute on Aging, DHHS; and by Grant RIM 78-17313 from the National Science Foundation.     

Enzyme histochemistry of the mesocoxal muscles of Periplaneta americana

Histochemical techniques have been employed to characterize enzymatic activity in the mesocoxal muscles of the cockroach, Periplaneta americana. Through our studies of the enzymes myosin-ATPase, NADH reductase, succinic dehydrogenase (SDH), and lactic dehydrogenase (LDH), we were able to classify fibers within these muscles according to criteria established for muscle fibers of vertebrates. Many of the mesocoxal muscles possess two different and distinct populations of fibers, whereas the remaining muscles are homogeneous with respect to their constituent fibers. The data presented here indicate biochemical heterogeneity for muscles of differing structural and functional features and possible neurotrophic influences upon oxidative enzymes and myosin-ATPase isozymes.