Ca release induced by cyclic adenosine diphosphoribose (cADPr) in sea urchin egg homogenates: mechanisms of release and heterogeneity of the Ca compartments

A rapid superfusion system measuring the amounts, kinetics, and Ca dependencies of released 45Ca, was used to examine the effects of ryanodine (RY), caffeine (CF), and cyclic ADP ribose (cADPr) on sea urchin egg homogenates. The RY-sensitive compartment had more than twice the Ca release capacity of the CF-sensitive or cADPr-sensitive compartment. cADPr-stimulated 45Ca release required calcium with half-maximal activation at approximately 0.2 to 0.6 microM [Ca2+]. K(1/2) for cADPr activation was approximately 100 nM, and in spite of the Ca requirement for cADPr-stimulated release, the cADPr affinity was not affected by [Ca2+]. Peak 45Ca release rate with cADPr (3 microM) was greater than with CF (20 mM), yet the release amounts were similar and both were [Ca2+]-dependent. When activated with CF and cADPr simultaneously, 45Ca release was large and, no longer [Ca2+]-dependent. Mg competitively inhibited the Ca activation site(s), yet did not inhibit the activation with CF-plus-cADPr. Pre-release of 45Ca by cADPr with low (approximately 0.1 microM) [Ca2+] right-shifted the [Ca2+] dependence of the remaining cADPr-response. These data suggest that (a) only a portion of RY-sensitive compartments empty when stimulated with cADPr or CF, (b) Ca and cADPr act on non-interacting sites, and (c) cADPr-sensitive compartments represent a heterogeneous population with different [Ca2+] dependencies.     

Urinary retention and the lunisolar cycle: is it a lunatic phenomenon?

OBJECTIVE--To determine whether a relation between urinary retention and temporal rhythms exists. DESIGN--Retrospective analysis of patients presenting over three years. SETTING--Urology departments in two hospitals. PATIENTS--815 Patients presenting as emergency admissions with urinary retention and requiring immediate decompression of the bladder. MAIN OUTCOME MEASURES--Calendar date of each admission to determine circadian, monthly, and seasonal periodicity. RESULTS--No association was found between urinary retention and circadian, monthly, or seasonal rhythms. A significantly higher (p less than 0.001) incidence of urinary retention was observed during the new moon in comparison with other phases of the lunar cycle. CONCLUSIONS--Urinary retention is periodic in nature. This should be considered when the workload of a specialist urological department is organised.     

Regeneration of native bacteriorhodopsin structure following acetylation of epsilon-amino groups of Lys-30, -40, and -41

The chymotryptic fragment of bacteriorhodopsin, C-2 (residues 1-71), has been acetylated completely at its three lysines (residues 30, 40, and 41) by treatment with acetic anhydride. The triacetylated C-2 fragment is able to reassociate with fragment C-1 (residues 72-248) and the complex binds all-trans-retinal to form a native bacteriorhodopsin-like chromophore, which is essentially identical with that formed from fragments C-2 and C-1. Further, the kinetics and pH dependence of chromophore regeneration and the proton pumping of the reconstituted triacetylated C-2 and C-1 complex are indistinguishable from that of the unmodified C-2 and C-1 complex. However, the extent of regeneration of the chromophore from triacetylated C-2 and C-1 is less than that from fragments C-2 and C-1, suggesting that the acetylated C-2 fragment is less stable than unacetylated C-2 in the reconstitution medium. We conclude that the amino groups in Lys-30, -40, and -41 do not contribute to the stabilization of the folded bacteriorhodopsin structure and are not required for proton translocation.     

Ca2+ binding by Myxicola neurofilament proteins

Titrimetric, 45Ca dialysis, and autoradiographic methods were used to examine how axoplasmic proteins from the giant neuron of the marine annelid Myxicola infundibulum bind calcium. Following the autoradiographic method of Maruyama et al., the 150-160 kD neurofilament subunits were identified as prominent intracellular Ca-binding peptides. Using equilibrium dialysis, extracts of axoplasmic proteins (greater than 50% neurofilament subunits) were examined in 300 mM KCl at different concentrations of free Ca and Mg, and at different pH. Axoplasmic proteins showed a high affinity Ca binding site (K1/2 3-6 microM, capacity 3-7 mumole g-1 protein) at pH 6.8 or pH 7.5. Changing the Mg concentration from 0 to 5 mM had no effect on the Ca binding. Elevating the dialysis pH from 7.0 to 9.0 reduced the apparent number of binding sites for Ca. Using microelectrodes to record the free Ca, microtitrations of axoplasmic proteins were completed by adding small amounts of CaCl2 to 100 microliters volumes of protein solutions. In a medium containing ionic constituents closely resembling those of the Myxicola axon, a Ca binding capacity of 5.0 mumole g-1 protein and a K1/2 of approximately 1 microM were measured.     

Selection against genetic defects in conservation schemes while controlling inbreeding

We studied different genetic models and evaluation systems to select against a genetic disease with additive, recessive or polygenic inheritance in genetic conservation schemes. When using optimum contribution selection with a restriction on the rate of inbreeding (ΔF) to select against a disease allele, selection directly on DNA-genotypes is, as expected, the most efficient strategy. Selection for BLUP or segregation analysis breeding value estimates both need 1–2 generations more to halve the frequency of the disease allele, while these methods do not require knowledge of the disease mutation at the DNA level. BLUP and segregation analysis methods were equally efficient when selecting against a disease with single gene or complex polygene inheritance, i.e. knowledge about the mode of inheritance of the disease was not needed for efficient selection against the disease. Smaller schemes or schemes with a more stringent restriction on ΔF needed more generations to halve the frequency of the disease alleles or the fraction of diseased animals. Optimum contribution selection maintained ΔF at its predefined level, even when selection of females was at random. It is argued that in the investigated small conservation schemes with selection against a genetic defect, control of ΔF is very important.     

In Vivo Neurochemical Evaluation of Striatal Serotonergic Hyperinnervation in Rats Depleted of Dopamine at Infancy

Destruction of nigrostriatal dopamine (DA) neurons with 6-hydroxydopamine (6-OHDA) early in development results in hyperinnervation of striatum by the serotonergic afferents deriving from the dorsal raphe nucleus. We have used in vivo microdialysis to investigate the degree to which serotonergic neurotransmission in striatum is altered by this increase in the density of serotonin (5-HT) terminals. The effects of several manipulations known to influence 5-HT function on extracellular 5-HT and 5-hydroxyindoleacetic acid in striatum were compared in adult rats treated neonatally with 6-OHDA and in intact adult rats. Basal levels of 5-HT in extracellular fluid (ECF) of striatum were similar in neonatally DA-depleted rats and in intact rats. Perfusion with the 5-HT reuptake blocker, fluoxetine (100 microM), increased 5-HT in striatal ECF of neonatally DA-depleted rats to levels that were threefold greater than those achieved in intact rats. Likewise, K(+)-depolarization of the 5-HT terminals (100 mM in perfusate) or systemic administration of the 5-HT releaser, (+/-)-fenfluramine (10 mg/kg i.p.), increased the concentration of 5-HT in striatal ECF of neonatally DA-depleted rats to levels approximately threefold greater than those observed in striatum of intact rats. These findings indicate that the 5-HT hyperinnervation of striatum that takes place in rats depleted of DA at infancy is associated with an increased capacity for neurotransmitter release in this system. Concomitant increased in high-affinity 5-HT uptake may prevent the occurrence of any measurable changes in the resting concentration of 5-HT in striatal ECF.     

Engineered selective plant male sterility through pollen‐specific expression of the EcoRI restriction endonuclease

Unintended gene flow from transgenic plants via pollen, seed and vegetative propagation is a regulatory concern because of potential admixture in food and crop systems, as well as hybridization and introgression to wild and weedy relatives. Bioconfinement of transgenic pollen would help address some of these concerns and enable transgenic plant production for several crops where gene flow is an issue. Here, we demonstrate the expression of the restriction endonuclease EcoRI under the control of the tomato pollen‐specific LAT52 promoter is an effective method for generating selective male sterility in Nicotiana tabacum (tobacco). Of nine transgenic events recovered, four events had very high bioconfinement with tightly controlled EcoRI expression in pollen and negligible‐to‐no expression other plant tissues. Transgenic plants had normal morphology wherein vegetative growth and reproductivity were similar to nontransgenic controls. In glasshouse experiments, transgenic lines were hand‐crossed to both male‐sterile and emasculated nontransgenic tobacco varieties. Progeny analysis of 16 000–40 000 seeds per transgenic line demonstrated five lines approached (>99.7%) or attained 100% bioconfinement for one or more generations. Bioconfinement was again demonstrated at or near 100% under field conditions where four transgenic lines were grown in close proximity to male‐sterile tobacco, and 900–2100 seeds per male‐sterile line were analysed for transgenes. Based upon these results, we conclude EcoRI‐driven selective male sterility holds practical potential as a safe and reliable transgene bioconfinement strategy. Given the mechanism of male sterility, this method could be applicable to any plant species.     

Contact inhibition in tissue culture

Conclusions Contact inhibition of movement is here defined simply as the stopping of the continued locomotion of a cell in the direction which has produced a collision with another cell; so that one cell does not use another as a substratum. Amongst fibroblasts and epithelial cells this inhibition seems to be brought about by a mechanism which it is suggested consists essentially of a spasm of contraction in the region of the contact, set off by some signal from the cell contacted. Many other kinds of cells show the general phenomenon of contact inhibition; but there is no certainty that they have the same contractile mechanism. The survey of the literature which this review has entailed suggests that it might be useful to end with four somewhat negative points: (1) Contact inhibition as originally defined is not concerned with mitosis. It may of course become so. (2) Contact inhibition of movement is difficult to analyse reliably without quantitative estimations and deliberate experiments. Anecdotes are not enough. (3) Malignant cells are not properly described as being devoid of contact inhibition. It is suggested that they are defective as compared with their cells of origin. (4) From the point of view invasion interest. It is the heterologous inhibition of tumour cells by normal cells that is relevant.