Artificial induction of exocytosis in bull sperm.

We have investigated an exocytotic event, the acrosome reaction (AR), induced by treatment of bovine sperm with vesicles composed of dilauroyl phosphatidylcholine (PC12). Cell membrane permeability barriers (dye exclusion), acrosomal status (pisum sativum (PSA) lectin binding), and intracellular Ca2+ (Fluo3 fluorescence) were evaluated utilizing flow cytometry and fluorescence microscopy. By these methods the AR is resolved into four kinetically distinct steps: (a) PC12 transfer to the sperm plasma membrane (PM); (b) increased permeability of the PM to extracellular Ca2+; (c) localized leakage of acrosomal contents at the anterior tip of the sperm; and (d) vesiculation of sperm membranes and complete exposure of acrosomal contents. Evidence for PC12 transfer to sperm includes transfer of a fluorescent PC12 analogue from vesicles to cells and the absence of detectable vesicle--cell fusion. The fusion inducing properties of PC12 appear to reside in the lipid head group as neither dilauroylphosphatidylethanolamine nor dilauroylphosphatidylglycerol stimulated the AR. The effect of PC chain length on AR induction closely parallels the aqueous phase solubility of the lipid tested. The rate and extent of the AR depend on the extracellular calcium concentration. Cells treated in the absence of calcium do not undergo the AR, but do so rapidly (less than 1 min) upon subsequent addition of calcium. This role of Ca2+ is partially filled by Sr2+, but not by Ba2+ or Mg2+. The rate of the AR decreases with decreasing temperature and the AR occurs very slowly below 27 degrees C. Simultaneous evaluation of intracellular calcium and acrosomal status reveals the kinetic relationship between Ca2+ influx and the exposure of acrosomal contents. N-Ethylmaleimide preincubation arrests PC12-treated sperm at an intermediate stage in the AR, characterized by punctate PSA binding over the tip of the sperm head. The AR, a developmentally regulated, receptor-mediated fusion event, synchronously induced here in vitro, provides a useful model for mechanistic studies of exocytosis.     

Developmental regulation of pectic polysaccharides in the root meristem of Arabidopsis

JIM 5, an antibody that recognizes a relatively unesterifiedpectic epitope, distinguishes between dividing (meristematic)and non-dividing (central cells of the quiescent centre) cellsin the Arabidopsis root tip, indicating that non-dividing cellwalls contain higher levels of relatively unesterified pectinthan dividing cells. JIM 7, an antibody that recognizes a relativelymethyl esterified epitope, labels all cell walls uniformly throughoutthe root, suggesting that there is little variation in the relativelymethyl esterified pectic component in the two cell types. Theseobservations suggest that the characteristics of cell wallsin the root tip result in part from modulations in the amountof unesterified and non-methyl esterified pectin. Key words: Pectin, quiescent centre, roots, Arabidopsis     

Regeneration of Medicago truncatula from protoplasts isolated from kanamycin-sensitive and kanamycin-resistant plants

Medicago truncatula (barrel medic) is an annual legume of agricultural and biological interest. In this report regeneration from isolated mesophyll protoplasts is described. A specifically developed, highly regenerable seed line is essential for regeneration. Other critical requirements for regeneration are the starting plant material, the use of agarose droplets incubated in a shallow layer of liquid medium, and protoplast density. Plants are grown in controlled environment conditions. Protoplasts are purified using a Percoll-based flotation procedure, then embedded in 100 l agarose droplets containing a basal medium plus 25 M NAA and 4 M BAP (the same medium as in the surrounding shallow liquid layer) to induce protoplast division. A protoplast density of 6–8×10⁵ ml^–1 is required for maximum colony formation. M. truncatula plants previously transformed for kanamycin resistance yielded embryogenic callus and also regenerated plants. Protoplasts from other annual Medicago (M.intertexta and M.scutellata) species readily form calli by the procedure we have described.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid     

Ethylene is a positive regulator of root hair development in Arabidopsis thaliana

Evidence is provided that ethylene is a positive regulator of hair cell development in the root epidermis of Arabidopsis thaliana. Treatment of seedlings with increasing concentrations of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC) results in progressively more root hair cells developing in positions normally occupied by non-hair cells. Consistent with these findings are observations that treatments that block either ethylene synthesis or its perception reduce the number of root hairs. A model is proposed in which either ethylene or ACC is a signal involved in specifying the pattern of cell differentiation in the Arabidopsis root epidermis.     

Area security unit in a psychiatric hospital.

Since 1974 a psychiatric hospital security unit, designed to serve the whole catchment area, has cared for mentally ill (mostly psychotic) patients with disturbed behaviour that cannot be managed in open wards. There are a few long-term dangerous patients but most stay only briefly. The admission of women to the unit was not followed by the expected reduction in violence. The unit has facilities for occupational therapy, physical recreation, work, and study, which are particularly important for those who are too dangerous to leave it. The unit''s calming influence depends as much on the supportive effect of the high staff ratio as on the use of tranquillisers. This type of unit is not suitable for patients with personality disturbances who "act out" or for mentally abnormal offenders; but it functions well as a crisis centre for the disturbed mentally ill, and there is an increasing demand for its services.     

Adenine deaminase of a eukaryotic animal cell,Crithidia fasciculata

Adenine deaminase (adenine aminohydrolase, EC 3.5.4.2) has been found to occur in Crithidia fasciculata with a specific activity higher than that of the same enzymes of bacteria and yeasts. It is remarkable for its stability to heat, exhibiting no appreciable loss of activity after 60 min at 55 °C. It occurs in the soluble portion of cell extracts but can be released into the suspending medium by osmotic and/or cold shock.     

Effect of dilauroylphosphatidylcholine liposomes on motility, induction of the acrosome reaction, and subsequent egg penetration of ram epididymal sperm

The effects of dilauroylphosphatidylcholine (PC12) on ram epididymal sperm motility, acrosome reaction (AR) induction, plasma membrane permeability, mitochondrial function, and sperm penetration into zona-free hamster eggs were determined. PC12 (50 microM) induced cell motility in caput and cauda sperm, as measured by subjective estimation and automated motility analysis. Motion parameters of treated caput sperm approached those of control ejaculated sperm. Flow cytometric analysis revealed that membrane permeability to propidium iodide and mitochondrial uptake of rhodamine 123 changed during epididymal transit. PC12 induced the AR in sperm from all epididymal regions relative to control incubated sperm (caput 17% vs. control 8%; corpus 29% vs. control 13%; proximal cauda 48% vs. control 4%; distal cauda 51% vs. control 9%). After PC12 treatment, egg penetration by sperm was increased for sperm from the corpus (corpus 7% vs. control 0%) and cauda (proximal 48% vs. control 0%; distal 51% vs. control 0%), but not for caput sperm (caput 0% vs. control 0%). These studies establish that some sperm in each region of the epididymis possess the capacity for movement and the AR. Caput sperm, however, were unique in that they could not penetrate eggs. Additional maturational changes must occur in the caput and/or corpus epididymidis before penetration capacity can be expressed.     

Methods for the complete analysis of 5-fluorouracil metabolites in cell extracts

Methodology that permits complete analysis of the intracellular metabolites of 5-fluorouracil (FUra) has been developed. A high-pressure liquid chromatography system that is capable of separating all metabolites of FUra found in acid-soluble cell extracts is described. In addition to the expected FUra metabolites, FUDP-hexoses were found to be present in large amounts in L121O cells treated with FUra. Improved procedures that permit quantitation of the FdUMP which is covalently bound to dTMP synthetase, as well as the total intracellular FdUMP levels are described; the latter is accomplished by dissociation of the FdUMP-dTMP synthetase complex in sonicated cell extracts followed by phosphatase treatment and subsequent high-pressure liquid chromatography analysis of FdUrd. An example of the integrated methodology in which all metabolites of FUra metabolism are analyzed over a 6-h exposure period of L1210 cells to [6-³H]FUra is provided.     

The pericardium as a source of prostacyclin in the dog, ox and rat

Cyclo-oxygenase products of arachidonic acid metabolism formed by the pericardium and epicardial surface of dog heart were identified and quantitated by radioimmunoassay after separation by high-pressure liquid chromatography. Pieces of pariental pericardium, of dog, ox and rat, when incubated produced mainly 6-keto-PGF_1α, with lesser amounts of PGE₂, PGF_2α and thromboxane B₂. Biosynthesis of all prostanoids increased during incubation of the pariental pericardium of each species with arachidonic acid, but 6-keto-PGF_1α was still the major metabolite. When slices of dog heart were incubated with arachidonic acid (1 μg/ml) the rates of 6-keto-PGF_1α formation by the pariental pericardium was much greater than that of the myocardium and endocardium. Epicardial slices appeared to be intermediate in 6-keto-PGF_1α formation. The hearts of anesthetized dogs were also irrigated with Krebs' solution, and during the first 5 min of epicardial irrigation the pericardial fluid leaving the heart again contained high levels of 6-keto-PGF_1α, with lesser amounts of the other prostanoids. Addition of arachidonic acid (3 μg/ml) to the irrigating fluid caused an increase in all measured prostanoid levels, although 6-keto-PGF_1α remained the predominant metabolite. In contrast, intravenous infusion of isoproterenol selectively increased the release of 6-keto-PGF_1α from the irrigated heart. It is concluded that the pericardium and epicardium continuously release prostacyclin into the pericardial fluid, and that the increased release of this substance observed when cardiac workload increases derives mainly from these membranous sources. This raises the interesting possibility that pericardial prostacyclin might influence coronary vascular tone and chemoreflexes which arise from the epicardium during myocardial ischemia.