Small enterprise opportunities in municipal solid waste management.

PIP: Most developing countries are rapidly urbanizing, with growing urban populations fueling demand for more and better urban services which many cities simply cannot provide given the current financial constraints. With the public sector unable to service the needs of expanding cities, small businesses are moving in to fill the vacuum. Such fledgling private sector initiatives have often prevented problems from becoming crises, while also demonstrating that private sector enterprises have an important role to play in meeting the demand for municipal services. Waste collection and processing is an area which could benefit from private sector involvement and greater public-private coordination. The authors examine the progress to date of an action-research initiative led by the Collaborative Group on Municipal Solid Waste Management in Low-income Countries which is developing best practice guidelines for expanding the involvement of micro- and small enterprises in municipal solid waste management.^ieng

Calcium homeostasis in mouse fibroblast cells: affected by U-73122, a putative phospholipase C beta blocker, via multiple mechanisms.

1. The inhibitory effects of the putative phospholipase C beta inhibitor, U-73122, on ligand-induced and thapsigargin-induced [Ca2+]i transients were investigated in mouse fibroblast cells (the L line). 2. Ca2+ release from intracellular stores was stimulated either by ATP (and also by UTP or ADP) working through the activation of a P2U receptor, or by lysophosphatidic acid, which elicited a more pronounced response. 3. U-73122 inhibited the Ca2+ mobilization produced by all the agonists in a dose-dependent manner, consistent with a mode of action involving phospholipase C inhibition. 4. In addition, however, U-73122 slowed the kinetics of intracellular Ca2+ release induced by the Ca(2+)-ATPase inhibitor, thapsigargin, and reduced the influx of Ca2+ across the plasma membrane, following stimulation of store-dependent influx by the latter. 5. We conclude that U-73122 has multiple sites of action, all of which can lead to a change in Ca2+ homeostasis. Thus, particular caution is recommended when employing this agent and when interpreting the results obtained.

Shear stress-induced [Ca2+]i transients and oscillations in mouse fibroblasts are mediated by endogenously released ATP.

The effects of ATP, U-73122, apyrase, and saline shear stress on [Ca2+]i homeostasis were studied in fura-2 loaded, mouse fibroblast cells (L929), both in suspension and plated on glass. Release of internal Ca2+ was induced by ATP, via a receptor identified pharmacologically as a P2U type. In single cells, low concentrations of ATP evoked [Ca2+]i oscillations. These events were blocked by the putative phospholipase C inhibitor, U-73122 (but not by the inactive analog U-73343) and by the ATP/ADPase, apyrase. In addition, both these agents reduced the [Ca2+]i of unstimulated cells, especially after stirring, and blocked spontaneously occurring [Ca2+]i oscillations, which suggested an already activated state of the ATP receptor, independent from exogenous stimulations. Moreover, it was found that stirring of the cells was correlated with a steady accumulation of inositol phosphates, also blockable by apyrase, and that [Ca2+]i mobilization could be induced by puffs of saline in single cells. The transition to a Ca(2+)-free environment also provoked [Ca2+]i oscillations, most likely via the increase in ATP4- concentration. This evidence suggests that endogenous ATP is released from L fibroblasts in response to fluid shear stress, and this results in an autocrine, tonic up-regulation of the phosphoinositide signaling system and an ensuing alteration in Ca2+ homeostasis. Up until now, such a response to shear stress was believed to be unique to endothelial cells.

Calcium homeostasis in dissociated embryonic neurons: a flow cytometric analysis.

1. Ca2+ homeostasis in freshly dissociated neurons from embryonic rat hypothalamus, cortex, and brain stem was investigated with flow cytometry. Cells were dissociated from embryonic brain by enzymatic and mechanical means and were incubated with the acetoxymethylester derivative of the Ca(2+)-sensitive dye indo-1. Neurons hydrolyzed and retained the dye as determined by the intensity of fluorescence emission, whereas similarly treated cultured astrocytes gave very low-level fluorescence. 2. The fluorescence of the indo-1 dye was measured at two wavelengths (405 and 485 nm) for each cell. Data were collected only from those cells (presumptive neurons) with high levels of fluorescence. Methods were developed to calibrate the level of intracellular free calcium ([Ca2+]i) as the ratio of fluorescence at 410 and 485 nm. The level of intracellular free Ca2+ was then calculated for each neuron. 3. A wide distribution of resting [Ca2+]i was found, with a median of approximately 90 nM. After addition of ionomycin to cells in Ca(2+)-free medium, there was a transient increase in [Ca2+]i, suggesting that all embryonic neurons had internal Ca2+ stores. The presence of active calcium extrusion mechanisms was demonstrated with the use of ionomycin in Ca(2+)-containing medium and with metabolic inhibitors. Furthermore, incubation in sodium-free medium resulted in a transient increase in [Ca2+]i and a reduced ability to eliminate elevated [Ca2+]i from the cytoplasm, suggesting that calcium homeostasis was dependent on the activity of the Na(+)-Ca2+ exchange mechanism. 4. Depolarization with K+ or veratrine increased [Ca2+]i in approximately 20% of the cells. This increase was blocked by eliminating extracellular free Ca2+ or adding Co2+, nifedipine, or verapamil, suggesting mediation by voltage-sensitive calcium channels. 5. Neurons were sorted on the basis of high [Ca2+]i and placed into dissociated culture. After 24 h, neurons in culture retained indo-1 fluorescence, suggesting that populations of neurons can be collected on the basis of their levels of [Ca2+]i. 6. These results demonstrate that flow cytometric analysis allows the characterization of a variety of Ca(2+)-regulatory mechanisms in populations of freshly dissociated embryonic neurons. Although only a proportion of embryonic day 17 neurons exhibit voltage-sensitive calcium channels, all neurons have developed the ability to sequester and extrude Ca2+.

Basic fibroblast growth factor regulates the ability of astrocytes to support hypothalamic neuronal survival in vitro.

The putative neurotrophic effects of basic fibroblast growth factor (bFGF) were tested on embryonic hypothalamic neurons in dissociated cell culture. Basic FGF dramatically increased the survival of embryonic hypothalamic astrocytes plated on a poly-L-lysine (PLL) substrate. Basic FGF treatment also increased the number of hypothalamic neurons surviving in vitro; however, no neurotrophic effects were observed when astrocyte proliferation was prevented by using serum-free N2 medium or by using the mitotic inhibitor cytosine arabinoside. In contrast to effects when PLL was used as a substrate, bFGF reduced the survival of hypothalamic neurons plated on a confluent, contact-inhibited monolayer of astrocytes. This effect appears to be due to the direct actions of bFGF on astrocytes: treatment of confluent astrocytes with 5 ng/ml bFGF caused the protoplasmic astrocytes to develop a fibrillar morphology and reduced the ability of the astrocyte monolayer to promote neuronal survival after a further 24 hr in bFGF-free medium. It is concluded that in addition to its mitogenic effects, bFGF acts as a differentiation factor for protoplasmic astrocytes in vitro, and these morphological and functional changes may reflect the process of normal astrocytic development and response to brain injury in vivo.

Interaction of estradiol, alpha-melanocyte-stimulating hormone, and dopamine in the regulation of sexual receptivity in the female rat.

Ovariectomized and adrenalectomized rats kept in a reversed lighting system received either 5 micrograms (once) or 1 microgram estradiol benzoate (EB) daily for 2, 3, or 4 days. These treatments induced sexual receptivity in a proportion of the rats, and alpha-melanocyte-stimulating hormone (alpha-MSH; 20 micrograms/rat s.c.) enhanced this proportion. In rats made receptive by 5 micrograms EB alone, the dopamine (DA) activity in preoptic area and arcuate nucleus was significantly reduced, but the alpha-MSH concentrations were not affected 54-56 h after EB treatment. Addition of alpha-MSH significantly increased the DA activity in ventromedial nucleus (VMN) and zona incerta, but this action is unlikely to account for its stimulation of sexual receptivity, since this effect was not blocked by 0.1 mg/kg haloperidol. Treatment with 100 mg/kg Dopa, which induces a presynaptic DA action, stimulated the receptivity in the EB-primed rats and selectively increased the concentration of alpha-MSH in the VMN, while 50 mg/kg Dopa plus 50 mg/kg benserazide, which induces a postsynaptic DA activity, inhibited the receptivity and reduced the alpha-MSH levels in the VMN. It is suggested that estradiol stimulates the female sexual receptivity by reducing the activity of a dopaminergic system in arcuate nucleus and preoptic area. alpha-MSH does not appear to affect this action, although it enhances the effect of steroids on the sexual behaviour, probably at the level of the VMN. The secretion of alpha-MSH may be under a dopaminergic inhibitory control, and the peptide may autoregulate its own secretion via this dopaminergic system which is sited in zona incerta and VMN.

Involvement of catecholaminergic systems in the zona incerta in the steroidal control of gonadotrophin release and female sexual behaviour.

Previous reports have shown that dopamine (DA) in the zona incerta (ZI) has a stimulatory effect on gonadotrophin release. We have now investigated the possibility that steroids exert their feedback effects on the release of luteinizing hormone (LH) via catecholamine systems in the ZI. Since the same steroid regimes also stimulate female sexual behaviour, the possibility that the ZI is also involved in the control of sexual activity was investigated. Lesions in the ZI increased proceptive and receptive behaviour in oestrogen-primed ovariectomised rats that exhibited a low level of lordosis in a pre-lesion test and had no effect in receptive animals. Turnover rates (TR) of DA, noradrenaline (NA) and adrenaline were measured in the ZI, preoptic area (POA), arcuate nucleus (ARC), median eminence (ME) and ventromedial nucleus in ovariectomised rats treated 54 h before with either oil, oestradiol benzoate (OB) at 5, 10 or 50 micrograms/rat, or 5 micrograms/rat OB followed by 0.5 mg/rat progesterone (P) 48 h later. The TR as measured from the decline in concentration after alpha-methyltyrosine and changes in DOPAC concentration were correlated with the effect of the steroids on plasma LH and lordotic activity. Confirming previous reports, NA turnover in the POA and ME was increased, and DA turnover in the ME was decreased by steroids when they enhanced LH release. DA turnover in the ARC and ME was reduced when lordosis behaviour increased. Treatment with OB plus P stimulated LH release and sexual receptivity and at the same time significantly increased DA and NA turnover in the ZI. There was no correlation between these parameters after OB alone. This report shows that the DA system in the ZI may mediate the stimulatory effect of P on LH release in OB-primed rats but is not involved in the feedback effects of oestradiol alone. NA activity in the ZI is increased after OB plus P and may therefore also be concerned with stimulating LH release. The ZI is involved in the control of sexual behaviour, as electrolytic lesions in this area enhance receptivity and proceptivity, but the catecholamine systems in the ZI do not appear to mediate this control.

Astrocyte topography and tenascin cytotactin expression: correlation with the ability to support neuritic outgrowth.

We have observed a heterogeneity in the ability of a monolayer of cultured rat astrocytes to support the attachment and growth of dissociated embryonic hypothalamic neurons in culture. Areas of the monolayer which have an uneven surface ('rocky' astrocytes) are poor substrates for neuronal attachment and neuritic outgrowth, while surrounding areas of the glial monolayer ('flat' astrocytes) support extensive neuronal growth. Astrocytes obtained from both neonatal cerebral cortex or hypothalamus displayed 'rocky' morphology. We utilized immunocytochemical techniques with antibodies directed against putative adhesion molecules to investigate the source of this heterogeneity. Antibodies against tenascin/cytotacin, fibronectin, laminin, N-CAM, thrombospondin, heparan sulfate proteoglycan, and the p185 protein product of the neu oncogene were employed in indirect-immunofluorescence experiments. We found that the difference in the surface properties of astrocytes appears to be correlated with the expression of the extracellular matrix molecule tenascin/cytotacin, but not with any of the other molecules we tested. Our data suggest that tenascin/cytotactin is inhibitory to neuronal attachment and process outgrowth in the developing nervous system.

Cultured rat neurons and astrocytes express immunologically related epitopes of the GABAA/benzodiazepine receptor.

gamma-Aminobutyric acid (GABA) is the major inhibitory transmitter in the mammalian central nervous system (CNS) and an understanding of the development of receptors for this compound is of considerable interest. In this study, 3 monoclonal antibodies directed against the GABAA/benzodiazepine (GABA/BDZ) receptor were utilized in immunocytochemical experiments to localize receptor sites on neurons in dissociated cultures from fetal rat hypothalamus. Immunoreactivity was restricted to distinct patches on the plasma membrane of neurons. Analogous patches were observed on the surface of type 1 astrocytes, but not on type 2 astrocytes or oligodendrocytes. In membrane extracts from whole brain these antibodies reacted with two proteins (50 and 55 kDa) whereas extracts of cultured astrocytes yielded a single protein of approximately 63 kDa. Thus the identity of the glial protein remains unclear, but this study provides evidence for the presence of shared epitopes of the GABA/BDZ-receptor on both neurons and glia, and suggests that the astrocytic receptors may be important to nervous system function. Moreover it indicates that in vitro GABA/BDZ-receptors may form aggregates which would account for the restricted patterns of GABA sensitivity reported in some electrophysiological studies.

The effect of selective D1 and D2 dopaminergic agents on sexual receptivity in the female rat.

A number of dopaminergic drugs was tested for their effect on female sexual receptivity in ovariectomised plus adrenalectomised rats primed with oestradiol benzoate. The selective D1 agonist (SKF 38393 5-40 mg/kg) and D1 antagonist (SCH 23390 0.1-10 mg/kg) had no significant effect on sexual behaviour. The results of administration of mixed dopaminergic agents (DOPA, 10-50 mg/kg with benserazide or 100-200 mg/kg alone and haloperidol 0.01-1.0 mg/kg) and selective D2 dopaminergic agents (LY 171555 2.5-800 micrograms/kg, BHT 920 0.01-1.0 mg/kg and sulpiride 5-80 mg/kg) indicate that a central dopaminergic system has an inhibitory control on female sexual activity exerted through D2 receptors. Any stimulatory effects of the exogenous agonists were probably due to an action on presynaptic D2 receptors. The predominance of the D2 pre- and post-synaptic receptor activity appears to be influenced by the sexual receptivity of the animal.

Helicoidal architecture of fish eggshell.

Previous publications show arced patterns in electron micrographs of either microfibrils or canals in sectioned fish eggshells, but these have been misinterpreted. We show here that such patterns in the inner layer of cod (Gadus morrhua), plaice (Pleuronectes platessa) and trout (Salmo gairdneri) eggs arise from a helicoidal structure. This consists of a laminate of protein microfibrils, with the direction of ply processing like the steps of a spiral staircase and with the same sense as a left-handed corkscrew. Mechanically, this is an ideal way to strengthen a spherical shell, to resist deforming forces equally from any direction. Radial canals which traverse this layer are forced into flattened and twisted ribbons. Both the helicoidal microfibrillar structure and the canal shape in fish eggshells show remarkable convergent evolution with similar structures in insect cuticles. Trout eggs were resistant to deforming forces as high as 380,000 N/m2. Fish eggshells, like those of many other organisms, are mechanically well designed.