Calcium and Protein Kinase C Regulate the Actin Cytoskeleton in the Synaptic Terminal of Retinal Bipolar Cells

The organization of filamentous actin (F-actin) in the synaptic pedicle of depolarizing bipolar cells from the goldfish retina was studied using fluorescently labeled phalloidin. The amount of F-actin in the synaptic pedicle relative to the cell body increased from a ratio of 1.6 ± 0.1 in the dark to 2.1 ± 0.1 after exposure to light. Light also caused the retraction of spinules and processes elaborated by the synaptic pedicle in the dark.Isolated bipolar cells were used to characterize the factors affecting the actin cytoskeleton. When the electrical effect of light was mimicked by depolarization in 50 mM K⁺, the actin network in the synaptic pedicle extended up to 2.5 μm from the plasma membrane. Formation of F-actin occurred on the time scale of minutes and required Ca²⁺ influx through L-type Ca²⁺ channels. Phorbol esters that activate protein kinase C (PKC) accelerated growth of F-actin. Agents that inhibit PKC hindered F-actin growth in response to Ca²⁺ influx and accelerated F-actin breakdown on removal of Ca²⁺.To test whether activity-dependent changes in the organization of F-actin might regulate exocytosis or endocytosis, vesicles were labeled with the fluorescent membrane marker FM1-43. Disruption of F-actin with cytochalasin D did not affect the continuous cycle of exocytosis and endocytosis that was stimulated by maintained depolarization, nor the spatial distribution of recycled vesicles within the synaptic terminal. We suggest that the actions of Ca²⁺ and PKC on the organization of F-actin regulate the morphology of the synaptic pedicle under varying light conditions.     

Aluminum effects on the kinetics of calcium uptake into cells of the wheat root apex

The effects of aluminum on the concentration-dependent kinetics of Ca²⁺ uptake were studied in two winter wheat (Triticum aestivum L.) cultivars, Al-tolerant Atlas 66 and Al-sensitive Scout 66. Seedlings were grown in 100 M CaCl₂ solution (pH 4.5) for 3 d. Subsequently, net Ca²⁺ fluxes in intact roots were measured using a highly sensitive technique, employing a vibrating Ca²⁺-selective microelectrode. The kinetics of Ca²⁺ uptake into cells of the root apex, for external Ca²⁺ concentrations from 20 to 300 M, were found to be quite similar for both cultivars in the absence of external Al; Ca²⁺ transport could be described by Michaelis-Menten kinetics. When roots were exposed to solutions containing levels of Al that were toxic to Al-sensitive Scout 66 but not to Atlas 66 (5 to 20 M total Al), a strong correlation was observed between Al toxicity and Al-induced inhibition of Ca²⁺ absorption by root apices. For Scout 66, exposure to Al immediately and dramatically inhibited Ca²⁺ uptake over the entire Ca²⁺ concentration range used for these experiments. Kinetic analyses of the Al-Ca interactions in Scout 66 roots were consistent with competitive inhibition of Ca²⁺ uptake by Al. For example, exposure of Scout 66 roots to increasing Al levels (from 0 to 10 M) caused the K _m for Ca²⁺ uptake to increase with each rise in Al concentration, from approx. 100 M in the absence of Al to approx. 300 M in the presence of 10 M Al, while having no effect on the V _max. The same Al exposures had little effect on the kinetics of Ca²⁺ uptake into roots of Atlas 66. The results of this study indicate that Al disruption of Ca²⁺ transport at the root apex may play an important role in the mechanisms of Al toxicity in Al-sensitive wheat cultivars, and that differential Al tolerance may be associated with the ability of Ca²⁺-transport systems in cells of the root apex to resist disruption by potentially toxic levels of Al in the soil solution.We would like to thank Dr. Lionel F. Jaffe, Director of the National Vibrating Probe Facility, Marine Biological Laboratory, Woods Hole, Mass., USA, for making his calcium-selective vibrating-mi-croelectrode system available for a portion of this work. The research presented here was supported in part by USDA/NRI Competitive Grant number 91-37100-6630 to Leon Kochian. Contribution from the USDA-ARS, U.S. Plant, Soil and Nutrition Laboratory, Cornell University, Ithaca, N.Y. This research was part of the program of the Center for Root-Soil Research, Cornell University, Ithaca, N.Y. Department of Soil, Crop and Atmosphere Science, paper No. 1741.     

Modulation of the antigenic phenotype of human breast carcinoma cells by modifiers of protein kinase C activity and recombinant human interferons

Summary In the present study we have analyzed the effect of a synthetic protein kinase C (PKC) activator 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol (ADMB) and the natural PKC-activating tumor-promoting agents 12-O-tetradecanoylphorbol 13-acetate (TPA) and mezerein on the antigenic phenotype of T47D human breast carcinoma cells. All three agents increased the surface expression of the tumor-associated antigen BCA 225 and various cellular antigens, including HLA class II antigens, intercellular adhesion molecule 1 (ICAM-1) and c-erbB-2. Expression of the same antigens was also upregulated to various extents in T47D cells by recombinant fibroblast (IFN) and immune (IFN) interferon. Shedding of BCA 225 from T47D cells was induced by TPA, mezerein, IFN and IFN, whereas ADMB did not display this activity. The ability of ADMB, TPA and mezerein to modulate the antigenic phenotype of T47D cells appears to involve a PKC-mediated pathway, since the PKC inhibitor, H-7, eliminates antigenic modulation. In contrast, the ability of IFN and IFN to enhance the synthesis, expression and shedding of BCA 225, as well as to enhance HLA class II antigens, c-erbB-2 and ICAM-1 expression, was either unchanged or modestly reduced by simultaneous exposure to H-7. Analysis of steady-state mRNA levels for HLA class I antigens, HLA class II-DR antigen, ICAM-1 and c-erbB-2 indicated that the ability of H-7 to inhibit expression of these antigens in TPA-, mezerein- and ADMB-treated cells was not a consequence of a reduction in the steady-state levels of mRNAs for these antigens. The results of the present investigation indicate that the biochemical pathways mediating enhanced antigenic expression in T47D cells induced by TPA, mezerein and the synthetic PKC activator ADMB are different from those induced by recombinant interferons. Furthermore, up-regulation of antigenic expression in T47D cells can occur by a PKC-dependent or a PKC-independent pathway.     

Demography of spawning grayling (Thymallus arcticus) in the Beaverlodge River,Alberta

Changes in a grayling spawning population are described that occurred in relation to both natural and man-induced events. The spawning population was monitored from 1982 to 1987 with a trap on a fishway on the Beaverlodge River, Alberta. Most of the spawning run in each year was tagged with recaptures noted in subsequent years. Male grayling grew slightly faster than females. Growth rates were variable and appeared to be quite low in years of low water flow. Grayling first spawned at 2 to 6 years of age. The majority of tagged grayling would have returned to spawn a second time after a delay of one to two years were it not for the high adult mortality. It is hypothesized that the variable time to first maturity and the delay in the second spawning is a response to highly variable juvenile recruitment. Annual mortality rates increased with age, suggesting an increased vulnerability of larger fish to angling. The population appears highly sensitive to over-exploitation, because of the high mortality rates and the delayed time to spawning of the adults.Deceased.Deceased.     

Inhibition of flower pigmentation by antisense CHS genes: promoter and minimal sequence requirements for the antisense effect

Introduction of a constitutive antisense full-length chalcone synthase (CHS) cDNA gene in petunia can result in an inhibition of flower pigmentation. We have evaluated some of the factors which may be important for the effectiveness of an antisense CHS gene.Antisense CHS genes encoding half-length or quarter-length RNA complementary to the 3 half of CHS mRNA are able to affect flower pigmentation, while a gene encoding RNA complementary to the 5 half of CHS mRNA did not show phenotypic effects in transgenic petunia plants. We demonstrate that the RNA encoded by the latter gene has a much lower average steady-state level in leaf tissue than the RNAs encoded by the other antisense gene constructs. We have compared the CaMV 35S and endogenous CHS promoter strengths and intrinsic stabilities of sense and antisense CHS RNAs. From the data we conclude that the constitutive antisense CHS genes are not likely to provide an excess of antisense RNA compared to the CHS mRNA derived from the endogenous genes.Effective inhibition of flower pigmentation is also observed when the antisense CHS gene is under control of the homologous CHS promoter. The results indicate that the mechanism of antisense inhibition cannot solely operate via RNA duplex formation between sense and antisense RNA.     

Typhloplanid flatworms (Mesostoma and related genera): Mechanisms of predation and evidence that they structure aquatic invertebrate communities

Mesostoma species and other typhloplanid flatworms are predators found in a wide variety of habitats. Laboratory observations of some Mesostoma spp found in shallow aquatic habitats (referred to as littoral species) have revealed a wide variety of prey killing mechanisms: 1) mucus trapping, 2) sit-and-wait predation, 3) releasing a toxin into the water, and 4) active searching. We review the existing literature on these mechanisms. We also describe for the first time the predatory behavior of a pelagic Mesostoma sp. found in Brazilian lakes.The existing literature is also reviewed to assess the potential impact of Mesostoma species and related genera on the aquatic invertebrate community. Mesostoma has a high potential for population increase and has been found in high densities in some shallow aquatic habitats. Single prey experiments show that a number of Mesostoma species feed heavily on mosquito larvae, some chironomid larvae and some daphnids but considerably less on most copepods and ostracods. Prey preference experiments reflect the same trends. Hence, these predation studies suggest that the flatworms, at high densities, should reduce populations of certain prey speces and, consequently, alter community structure. Field studies support this prediction. Mesostoma species, at high densities, appear to be important predators of mosquito larvae in shallow aquatic habitats even under conditions where high densities of planktivorous fishes had little impact. They also appear to alter zooplankton community structure. However, much of the field evidence is correlational and not experimental. No studies have assessed the impact of pelagic typhloplanids on zooplankton community structure.It is concluded that many more manipulative field experiments are needed to assess the impact of typhloplanids on aquatic invertebrate communities.     

A substitution of cysteine for arginine 614 in the ryanodine receptor is potentially causative of human malignant hyperthermia.

Malignant hyperthermia (MH) is a devastating, potentially lethal response to anesthetics that occurs in genetically predisposed individuals. The skeletal muscle ryanodine receptor (RYR1) gene has been linked to porcine and human MH. Furthermore, a Cys for Arg substitution tightly linked to, and potentially causative of, porcine MH has been identified in the ryanodine receptor. Analysis of 35 human families predisposed to malignant hyperthermia has revealed the presence, and cosegregation with phenotype, of the corresponding substitution in a single family. This substitution, by analogy to the findings in pig, may be causal for predisposition to MH in this family.     

Influence of birth weight on differences in infant mortality by social class and legitimacy.

OBJECTIVE--To investigate the influence of birth weight on the pronounced social class differences in infant mortality in Britain. DESIGN--Analysis of routine data on births and infant deaths. SETTING--England and Wales. SUBJECTS--All live births and infant deaths, 1983-5. MAIN OUTCOME MEASURE--Mortality in infants by social class, birth weight, and legitimacy according to birth and death certificates. RESULTS--Neonatal and postneonatal mortality (deaths/1000 births) increased with social class. Neonatal and postneonatal mortality was 4.2/1000 and 2.3/1000 respectively for social class I and 6.8/1000 and 5.6/1000 respectively for social class V. Mortality was lower among births registered within marriage (postneonatal 3.5/1000; neonatal 5.2/1000) than among those jointly registered outside marriage (5.1/1000; 6.4/1000); mortality was highest in those solely registered outside marriage (7.2/1000; 7.0/1000). For neonatal mortality the effect of social class varied with birth weight. Social class had little effect on neonatal mortality in low birthweight babies and increasing effect in heavier babies. For postneonatal mortality the effect of social class was similar for all birth weights and was almost as steep as for all birth weights combined. CONCLUSION--Birth weight mediates little of the effect of social class on postneonatal mortality.     

Toxicity of 4-nitroquinoline 1-oxide for Crithidia fasciculata.

Growth inhibition of Crithidia fasciculata by 4-nitroquinoline 1-oxide (NQO) was observed in defined and complex media at 28 C. Aromatic amino acids, cystein, and nicotinic acid, among several other substances, were ineffective in overcoming NQO toxicity. Dicoumarol and bovine albumin reversed NQO inhibition. While bovine albumin probably acted by the extra-cellular binding of NQO, dicoumarol inhibited the activity of DT-diaphorase, which reduces NQO to 4-hydroxyaminonitroquinoline 1-oxide (HAQO). The DT-diaphorase from C. fasciculata had the same characteristics as the enzyme from rat liver. The specific protection by dicoumarol against NQO inhibition suggests that HAQO is the active toxic substance for C. fasciculata.     

Properties of Two Homologous Alkaline Proteases from Streptomyces rectus

Some physicochemical properties of two thermostable proteases from Streptomyces rectus are described. The enzymes were judged to be identical with respect to molecular weight, inactivation with serine protease inhibitors, and in primary structure by peptide analysis. Amino acid analysis indicated the enzymes had identical compositions except for their amide content. The molecular weights of the enzymes were judged to be 28,000 by sedimentation equilibrium, 26,200 by sedimentation diffusion, and 29,100 from amino acid analysis. Titration of the proteases with diisopropylfluorophosphate and phenylmethane sulfonylfuoride indicate equivalent weights of 28,500 and 32,800 g, respectively, for the proteins. The pentapeptide around the serine residue reacting with diisopropylfluorophosphate was isolated and had the composition: Asx(1), Gly(1), Thr(1), Ser(1), Met(1).