The precursor protein of non-A beta component of Alzheimer's disease amyloid is a presynaptic protein of the central nervous system.

Non-A beta component of Alzheimer's disease amyloid (NAC) is the second component in the amyloid from brain tissue of patients affected with Alzheimer's disease. Its precursor protein (NACP) was shown to be a brain-specific protein. In rat brain, NACP was more abundant in the neocortex, hippocampus, olfactory bulb, striatum, thalamus, and cerebellum and less abundant in the brain stem. Confocal laser microscopy analysis revealed that anti-NACP immunostaining was colocalized with synaptophysin-immunoreactive presynaptic terminals. Ultrastructural analysis showed that NACP immunoreactivity was associated with synaptic vesicles. NACP sequence showed 95% identity with that of rat synuclein 1, a synaptic/nuclear protein previously identified in rat brain, and good homology with Torpedo synuclein from the electric organ synapse and bovine phosphoneuroprotein 14 (PNP-14), a brain-specific protein present in synapses. Therefore, NACP is a synaptic protein, suggesting that synaptic aberration observed in senile plaques might be involved in amyloidogenesis in Alzheimer's disease.

The role of N-methyl-D-aspartate receptors and nitric oxide in cochlear dopamine release.

Dopamine (DA) released from lateral olivocochlear (LOC) terminals may have a neuroprotective effect in the cochlea. To explore the role of N-methyl-d-aspartate (NMDA) receptors and nitric oxide (NO) in the modulation of a cochlear DA release, we measured the release of [3H]DA from isolated mouse cochlea in response to the application of NMDA. NMDA at 100 muM significantly increased the electrical-field stimulation-evoked and resting release of DA from the cochlea. The NO donor sodium nitroprusside enhanced the basal outflow of DA but failed to influence the evoked release. The administration of the nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) alone was ineffective, but it significantly inhibited the initial phase of the NMDA-induced elevation of DA outflow, which suggested the role of NO in the NMDA-induced DA release. The DA uptake inhibitor nomifensine increased the electrically evoked release of DA. Nomifensine failed to change the effect of NMDA on the resting or electrically-evoked DA release, which suggested that the uptake mechanism does not play a role in NMDA-evoked and NO-mediated DA release. In summary, we provide evidence that NO can modulate the release of DA from the cochlea following NMDA receptor activation, but does not affect the uptake of DA.

T lymphocytes are targets for platelet- and trophoblast-derived microvesicles during pregnancy.

Microvesicles (MVs) can derive from several cell types and their membranes contain cell surface elements. Their role is increasingly recognized in cell-to-cell communication, as they act as both paracrine and remote messengers, occurring in circulating form as well as in plasma. Successful pregnancy requires a series of interactions between the maternal immune system and the implanted fetus, such that the semi-allograft will not be rejected. These interactions occur at the materno-placental interface and/or at a systemic level. In the present study we identified for the first time the in vivo plasma pattern of the MVs of third-trimester, healthy pregnant women, their cellular origin, and their target cells using flow cytometry and confocal laser microscopy. We searched for the cellular target molecules of thrombocyte-derived MVs with the help of neutralizing antibodies. We examined the in vitro effects of MVs on STAT3 phosphorylation of primary lymphocytes and Jurkat cells. We found that both placental trophoblast-derived and maternal thrombocyte-derived MVs bind to circulating peripheral T lymphocytes, but not to B lymphocytes or NK cells. We were able to show that the P-selectin (CD62P)-PSGL-1 (CD162) interaction is one mechanism binding platelet-derived MVs to T cells. We were also able to demonstrate that MV-lymphocyte interactions induce STAT3 phosphorylation in T cells. Our findings indicate that both thrombocyte- and trophoblast-derived MVs may play an important role in the immunomodulation of pregnancy. We suggest that the transfer of different signals via MVs represents a novel form of communication between the placenta and the maternal immune system, and that MVs contribute to the establishment of stable immune tolerance to the semi-allograft fetus.

Inhibition of lipid peroxidation by heme-nonapeptide derived from cytochrome c.

Heme-nonapeptide, derived from cytochrome c, inhibited both the NADPH- and NADH-dependent lipid peroxidation of brain microsomes but, in the case of liver microsomes, this inhibitory effect manifested itself in the presence of SKF-525A (a specific blocker of cytochrome P-450) only. Heme-nonapeptide prevented the transient accumulation of lipid peroxides in microsomes during lipid peroxidation. The oxygen consumption of microsomes in the presence of NADPH or NADH was stimulated by heme-nonapeptide. From these results we concluded that, in vitro, there are two independent mechanisms of lipid peroxidation in liver microsomes. It is suggested that, in vivo, the heme-peptide-sensitive mechanism, observed in brain microsomes, is more important.

D2 autoreceptor inhibition reveals oxygen-glucose deprivation-induced release of dopamine in guinea-pig cochlea.

Dopamine (DA), released from the lateral olivocochlear (LOC) efferent terminals, the efferent arm of the short-loop feedback in the cochlea, is considered as a protective factor in the inner ear since it inhibits auditory nerve dendrite firing in ischemia- or noise-induced excitotoxicity leading to sensorineural hearing loss (SNHL). In the present study we investigated the effect of oxygen-glucose deprivation (OGD), an in vitro ischemia model, on guinea-pig cochlear [(3)H]DA release in a microvolume superfusion system. We found that OGD alone failed to induce a detectable elevation of [(3)H]DA level, but in the presence of specific D(2) receptor antagonists, sulpiride and L-741,626, it evoked a significant increase in the extracellular concentration of [(3)H]DA. D(2) negative feedback receptors are involved not exclusively in the regulation of synthesis and vesicular release of DA, but also in the activation of its reuptake. Thus, D(2) receptor antagonism interferes with the powerful reuptake of DA from the extracellular space. To explore the underlying mechanism of this DA-releasing effect we applied nomifensine and found that the effect of OGD on cochlear DA release in the presence of D(2) antagonists could be inhibited by this selective DA uptake inhibitor. This finding indicates that the OGD-evoked DA release was mainly mediated through the reverse operation of the DA transporter. The two structurally different D(2) antagonists also augmented the electrical field stimulation-evoked release of DA proving the presence of D(2) autoreceptors on dopaminergic LOC terminals. Our results confirm the presence and role of D(2) DA autoreceptors in the regulation of DA release from LOC efferents, and suggest a protective local mechanism during ischemia which involves the direct transporter-mediated release of DA. Increasing the release of the protective transmitter DA locally in the inner ear may form the basis of future new therapeutic strategies in patients suffering from SNHL.

Dancing the tight rope on the nanoscale--Calibrating a heat flux sensor of a scanning thermal microscope.

We report on a precise in situ procedure to calibrate the heat flux sensor of a near-field scanning thermal microscope. This sensitive thermal measurement is based on 1ω modulation technique and utilizes a hot wire method to build an accessible and controllable heat reservoir. This reservoir is coupled thermally by near-field interactions to our probe. Thus, the sensor's conversion relation V(th)(Q(GS)*) can be precisely determined. V(th) is the thermopower generated in the sensor's coaxial thermocouple and Q(GS)* is the thermal flux from reservoir through the sensor. We analyze our method with Gaussian error calculus with an error estimate on all involved quantities. The overall relative uncertainty of the calibration procedure is evaluated to be about 8% for the measured conversion constant, i.e., (2.40 ± 0.19) µV/µW. Furthermore, we determine the sensor's thermal resistance to be about 0.21 K/µW and find the thermal resistance of the near-field mediated coupling at a distance between calibration standard and sensor of about 250 pm to be 53 K/µW.

Ultrastructural localization of beta-arrestin-1 and -2 in rat lumbar spinal cord.

beta-arrestins play significant roles in agonist-mediated desensitization of G protein-coupled receptors. Although the presence of beta-arrestin subtypes, beta-arrestin-1 and(- 2) in rat brain has been studied extensively, their existence in the spinal cord has not been described. In the current study, we performed immunohistochemical analyses of beta-arrestins at both light and electron microscopic levels using rat lumbar 1-2 spinal cord segments. Intense immunoreactivity for beta-arrestin-1 was found in the motoneurons in lamina IX of the ventral horn and elongated cells in the dorsal nucleus of Clarke. Modest immunoreactivity was detected among the neurons of laminae V and VII/VIII, and weaker immunoreactivity in laminae III, IV, and X. beta-arrestin-2 immunoreactivity was also distributed through laminae III-X in the order of IX > dorsal nucleus of Clarke > V > VII/VIII > IV > III > X. Laminae I and II did not show immunoreactivity. At the electron microscopic level, both beta-arrestin-immunoreactive and nonimmunoreactive dendrites were observed, whereas axons and terminal boutons were devoid of immunoreactivity. In immunoreactive dendrites most beta-arrestin immunoreactivity was distributed throughout the cytoplasm, demonstrating their association with microtubules. In addition, strong immunoreactivity was often found at postsynaptic densities. Our results thus suggest beta-arrestins' possible involvement in both motor and sensory mechanisms at the postsynaptic level in rat lumbar spinal cord.

A1-receptor-mediated effect of adenosine on the release of acetylcholine from the myenteric plexus: role and localization of ecto-ATPase and 5'-nucleotidase.

No attempt has been made so far to classify the subtypes of presynaptic inhibitory adenosine receptors located in the myenteric plexus and to localize ecto-ATPase and 5'-nucleotidase in the intestine. The release of [3H]acetylcholine and smooth muscle responses to acetylcholine were measured and the effect of selective adenosine receptor ligands was studied using field-stimulated isolated longitudinal muscle strips of guinea-pig ileum. Release of ATP and its hydrolysis rate were also measured using the luciferin-luciferase technique. A histochemical method combined with electron microscopy was used for localization of ecto-ATPase and 5'-nucleotidase, enzymes responsible for destruction of extracellular ATP, ADP and AMP. Subtype-selective A1-receptor agonists and antagonists inhibited and enhanced, respectively, the release of acetylcholine associated with neuronal activity. A significant amount of ATP was released in response to electrical stimulation and administration of carbamylcholine. The release of ATP was inhibited by atropine and 4-diphenylacetoxy-N-methylpiperidine methiodide, an M3-receptor antagonist. Hydrolysis of ATP was rapid and resulted in an accumulation of extracellular adenosine involved in presynaptic A1-receptor-mediated inhibition of acetylcholine release. While the inhibitory effect of adenosine and ATP was significantly potentiated by dipyridamol, an adenosine uptake blocker, that of 2-ms ATP was not. The effect of ATP was not competitively antagonized by 8-cyclopentyl-1,3-dipropylxanthine, a selective A1-receptor antagonist. In conclusion, axon terminals of cholinergic interneurons are equipped with inhibitory A1- and P2 gamma-receptors. Therefore, both adenosine and ATP control the release of acetylcholine through these receptors. ATP is mainly released from the smooth muscle in response to stimulation of M3-muscarinic receptors by endogenous acetylcholine (cascade transmission [Vizi E. S. et al. (1992) Neuroscience 50, 455-465]) and is rapidly hydrolysed by ecto-ATPase localized on the surface of the smooth muscle and axon terminals producing ADP and AMP, and by 5'-nucleotidase present only on the surface of smooth muscle cells producing adenosine.

ATP acts as fast neurotransmitter in rat habenula: neurochemical and enzymecytochemical evidence.

The release of ATP and ADP, the putative central neurotransmitters, from the isolated habenula preparation was investigated in the rat, at rest and during electrical stimulation, using the luciferin-luciferase assay and the creatine phosphokinase assay. Electrical field stimulation (2 Hz, 360 pulses) released a considerable amount of ATP (2450 +/- 280 pmol/g wet tissue) from the tissue; inhibition of the voltage Na+ entry by tetrodotoxin (1 microM) reduced significantly the evoked release (by 66.25 +/- 6.65%), but not the resting release of ATP. Endogenous ADP also appeared in the effluent, but its amount differed during resting condition and after stimulation from that of ATP, suggesting that the majority of the released compound is ATP in response to stimulation. When ATP was added to the tissue, it readily decomposed to ADP and AMP (Km = 811.6 +/- 68.88 microM, vmax = 23.1 +/- 2.75 nmol/min per prep., measured by high-performance liquid chromatography combined with ultraviolet detection), indicating that the habenula contains ectoATPases. In addition, the inactivation of extracellular ATP by the ectoATPase enzyme was also visualized by electron microscopic enzyme cytochemistry. The ectoATPase enzyme was present on the membranes of the dendrites and nerve terminals and in the synapses of the habenula. Taking into account the fact that ATP is ubiquitous in excitable cells (storage) and the findings published by Edwards et al. in 1992 ("ATP receptor-mediated synaptic currents in the central nervous system", Nature, Vol. 359, pp. 144-147), our data provides evidence for the release by axonal stimulation and extracellular decomposition of ATP, all needed for an endogenous substance qualified as a transmitter.

Studies on the release and extracellular metabolism of endogenous ATP in rat superior cervical ganglion: support for neurotransmitter role of ATP.

The release of endogenous ATP, measured by the luciferin-luciferase assay, and the release of [3H]acetylcholine from the isolated superior cervical ganglion of the rat loaded with [3H]choline were studied simultaneously. Electrical field stimulation enhanced the release of endogenous ATP and acetylcholine in a [Ca2+]o-dependent manner. The Na+ channel blocker, tetrodotoxin (1 microM) inhibited the stimulation-evoked release of endogenous ATP and of [3H]acetylcholine, but did not change the resting release. The release of ATP was dependent on the frequency of stimulation between 2 and 10 Hz. when the number of shocks was kept constant (360 shocks), while acetylcholine was not released in a frequency-dependent fashion. Ten days after cutting of the preganglionic nerve of the superior cervical ganglion the stimulation-evoked release of acetylcholine and ATP was abolished and the uptake of [3H]choline was significantly reduced but not inhibited. Hexamethonium, (100 microM) a nicotinic acetylcholine receptor antagonist, significantly reduced the release of both acetylcholine and ATP, indicating a positive feedback modulation of ACh and ATP release. 8-Cyclopentyl-1,3-dipropylxanthine (10 nM), the selective A1-adenosine receptor antagonist exhibited similar effect on the release of ATP and acetylcholine: both of them were augmented, showing that the stimulation-evoked release of ATP and acetylcholine are under the inhibitory control of A1-adenosine receptors. When the temperature was reduced to 7 degrees C to inhibit carrier-mediated processes, the resting and stimulated release of acetylcholine was not changed. Conversely, the release of ATP in response to stimulation was reduced by 79.9 +/- 5.6%, and the basal release was also almost completely blocked. Carbamylcholine by itself was able to release ATP, but not acetylcholine, in a hexamethonium-inhibitable manner, even from ganglia whose preganglionic nerve had been cut 10 days prior to experiments, suggesting that ATP release can occur in response to nicotinic receptor stimulation of postsynaptic cells. The breakdown of ATP or AMP by superior cervical ganglion was measured by high performance liquid chromatography combined with UV detection. ATP and AMP, added to the tissues, were readily decomposed: the Km (apparent Michaelis constant) and Vmax (apparent maximal velocity) were 475 +/- 24 microM and 3.50 +/- 0.18 nmol/min per mg for ectoATPase and 1550 +/- 120 microM and 14.5 +/- 0.9 nmol/min per mg tissue for 5'-nucleotidase. In addition, by using electron microscopic enzyme histochemistry, the presence of ectoATPase was also shown in the superior cervical ganglion. It is concluded that endogenous ATP and acetylcholine are released simultaneously in response to stimulation of preganglionic nerve terminals in the superior cervical ganglion in a [Ca2+]o-dependent, tetrodotoxin-sensitive manner and is metabolized by ectoenzymes present in the tissue. The dissociation of the release of ATP and acetylcholine at different stimulation frequencies and temperatures shows that the release-ratio of acetylcholine and ATP can vary upon the condition of stimulation: this can reflect either the different composition of synaptic vesicles in the preganglionic nerve terminals or a significant contribution of non-exocytotic, carrier-mediated type of release of ATP to the bulk release.

Lipopolysaccharide treatment modifies pH- and cation-dependent ecto-ATPase activity of endothelial cells.

The aim of this study was to investigate the distribution pattern of Ca2+- and Mg2+-dependent ecto-ATPases on the surface of rat brain capillary endothelial cells (ECs) in control and lipopolysaccharide (LPS)-treated animals. Ecto-ATPases in the membrane of vascular endothelial cells are suggested to play a crucial role in thromboregulation. Loss of this enzyme activity after oxidative stress and upregulation of the enzyme chain hydrolyzing extracellular ATP after transient forebrain ischemia have also been reported. We used histochemistry to localize the activities of this enzyme on ECs and found pH- and cation-dependent changes in the localization of enzyme activity both in control and in LPS-treated animals. These findings suggest the presence of more than one ecto-ATPase enzyme on the surface of rat capillary ECs. The different behavior of ECs after LPS treatment is the target of further investigations. The increased ecto-nucleotidase activity might play a role in nucleotide-mediated cellular responses after bacterial infections.

Reversible ischemia increases levels of Alzheimer amyloid protein precursor without increasing levels of mRNA in the rabbit spinal cord.

In a rabbit spinal cord ischemia model (RSCIM), the time courses of neuropathological damage of the spinal cord and neurological impairment of the motor functions are well established, demonstrating that the extent of neuropathological damage and the severity of neurological impairment are closely correlated. We used the RSCIM to elucidate the effects of reversible (15 min) and irreversible (60 min) ischemia on the endogenous levels of amyloid protein precursors (APPs) at both the mRNA and protein levels in the caudolumbar/sacral region of the spinal cord. We speculate that endogenous APPs are induced by ischemia as either trophic factors or stress-induced proteins in the RSCIM. A 15-min occlusion transiently increased the APP protein levels in neurons, which returned to the original levels by the end of 60 min occlusion. The increase in APP protein levels during 15-min ischemic insult does not appear to involve regulation at the mRNA level. The increased level of APPs, particularly of the soluble form, could support the possibility that APPs play a neuroprotective role in the RSCIM as stress-induced proteins. In contrast, failure to maintain the increased APP protein levels or to increase the mRNA, as seen in the 60-min ischemia samples, may be one of the causal factors that induce necrosis and neuronal cell death leading to irreversible neurological impairment.

Cultured astrocytes react to LPS with increased cyclooxygenase activity and phagocytosis.

Phagocytosis and prostaglandin E(2) production were investigated in purified cultures of perinatal rat forebrain astrocytes. Light and electron microscopic data indicated that astrocytes respond to bacterial endotoxin, lipopolysaccharide (LPS) by increased phagocytosis and by activating the cyclooxygenase enzyme-pathway. LPS-inducible phagocytosis of astrocytes was demonstrated by electron microscopic studies on colloidal gold uptake and by photometric determination of fluorescent bead ingestion. The internalisation of fragments of the plasma membrane was shown by histochemical detection of membrane-bound ecto-ATPase activity within intracellular vesicles. Activation of the cyclooxygenase pathway, a characteristic reaction of immune cells under inflammatory conditions, was also detected in astroglial cells upon treatment with LPS. The increased prostaglandin E(2) (PGE(2)) production by astrocytes in response to LPS was reduced by the non-steroid anti-inflammatory drug, indomethacin. Our data indicate that astrocytes display some tissue-protective reactions in response to inflammation inducing factors, even in the absence of peripheral immune cells or central microglia. The role of inducible astrocytic phagocytosis in a non-immune protection-pathway is discussed.

Improved growth of cultured brain microvascular endothelial cells on glass coated with a biological matrix.

An improved method for culturing primary rat brain capillary endothelial cells on glass has been developed, using a corneal extracellular matrix coat. Since the collagen-coated plastic attachment surface conventionally used for primary cultures of brain microvascular endothelium gives a high level of background fluorescence in microfluorimetric studies, an alternative attachment surface was tested involving no plastic element. Five substrata combinations were examined and a new combination of glass and corneal endothelial extracellular matrix coat was found to provide excellent cell adhesion, culture growth and purity. Other established substrata combinations tested for comparison, either involved plastic, or used glass with collagen or carbodiimide and collagen coating but the last two gave poor endothelial cell adhesion and growth. Our method using this new attachment surface combination results in stable and pure endothelial cultures, as verified by immunocytochemistry, which are suitable for fluorimetric investigations.

Presynaptic ecto- and postsynaptic endo-calcium-adenosine-triphosphatases in synaptosomes: doubts about biochemical interpretation of localization.

Ecto- and endo-Ca-adenosine-triphosphatase (ATPase) activity was identified as electron-dense lead or cerium phosphate precipitate in the rat cortical synaptosomes by transmission electron microscopy and enzyme histochemistry. The formation of the deposit was dependent on the presence of ATP (the substrate), Ca (activator) and levamisole, quercetin or ouabain (inhibitors of different phosphatases and ATPases). Reaction products were found at the external surface of the presynaptic membrane, both surfaces of the postsynaptic membrane, in the synaptic cleft and in the free mitochondrial membranes. In the presence of ATP and the three inhibitors together, the quantity of the precipitate decreased markedly, but we still found some deposit on the external surface of the presynaptic membrane (this activity is probably due to the so-called ecto-ATPase) and on the internal surface of the postsynaptic one (endo-ATPase). The distinction between ecto- and endo-ATPases in biochemical fractions solely upon biochemical differential measurements must be interpreted with caution.

Dynamics and control of a multimode laser: Reduction of space-dependent rate equations to a low-dimensional system.

We suggest a quantitatively correct procedure for reducing the spatial degrees of freedom of the space-dependent rate equations of a multimode laser that describe the dynamics of the population inversion of the active medium and the mode intensities of the standing waves in the laser cavity. The key idea of that reduction is to take advantage of the small value of the parameter that defines the ratio between the population inversion decay rate and the cavity decay rate. We generalize the reduction procedure for the case of an intracavity frequency doubled laser. Frequency conversion performed by an optically nonlinear crystal placed inside the laser cavity may cause a pronounced instability in the laser performance, leading to chaotic oscillations of the output intensity. Based on the reduced equations, we analyze the dynamical properties of the system as well as the problem of stabilizing the steady state. The numerical analysis is performed considering the specific system of a Nd:YAG (neodymium-doped yttrium aluminum garnet) laser with an intracavity KTP (potassium titanyl phosphate) crystal.

Co-localization of P2Y1 receptor and NTPDase1/CD39 within caveolae in human placenta.

Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39) is the dominant ecto-nucleotidase of vascular and placental trophoblastic tissues and appears to modulate the functional expression of type-2 purinergic (P2) G-protein coupled receptors (GPCRs). Hence, this ectoenzyme could regulate nucleotide-mediated signalling events in placental tissue. This immunohistochemical and immuno-electron microscopic study demonstrates the expression of NTPDase1/CD39, P2Y1 and P2Y2 receptors in different cell types of human placenta. Specifically P2Y1 has an exclusive vascular distribution whereas P2Y2 is localized on trophoblastic villi. Co-localization of P2Y1 and NTPDase1/CD39 are observed in caveolae, membrane microdomains of endothelial cells. The differential localization of these P2 receptors might indicate their unique roles in the regulation of extracellular nucleotide concentrations in human placental tissues and consequent effects on vascular tone and blood fluidity.

Microencapsulation technology by nature: Cell derived extracellular vesicles with therapeutic potential.

Cell derived extracellular vesicles are submicron structures surrounded by phospholipid bilayer and released by both prokaryotic and eukaryotic cells. The sizes of these vesicles roughly fall into the size ranges of microbes, and they represent efficient delivery platforms targeting complex molecular information to professional antigen presenting cells. Critical roles of these naturally formulated units of information have been described in many physiological and pathological processes. Extracellular vesicles are not only potential biomarkers and possible pathogenic factors in numerous diseases, but they are also considered as emerging therapeutic targets and therapeutic vehicles. Strikingly, current drug delivery systems, designed to convey therapeutic proteins and peptides (such as liposomes), show many similarities to extracellular vesicles. Here we review some aspects of therapeutic implementation of natural, cell-derived extracellular vesicles in human diseases. Exploration of molecular and functional details of extracellular vesicle release and action may provide important lessons for the design of future drug delivery systems.

Compact device for cleaning scanner-mounted scanning tunneling microscope tips using electron bombardment.

Most scanning probe techniques rely on the assumption that both sample and tip are free from adsorbates, residues, and oxide not deposited intentionally. Getting a clean sample surface can be readily accomplished by applying ion sputtering and subsequent annealing, whereas finding an adequate treatment for tips is much more complicated. The method of choice would effectively desorb undesired compounds without reducing the sharpness or the general geometry of the tip. Several devices which employ accelerated electrons to achieve this are described in the literature. To minimize both the effort to implement this technique in a UHV chamber and the overall duration of the cleaning procedure, we constructed a compact electron source fitted into a sample holder, which can be operated in a standard Omicron variable-temperature (VT)-STM while the tip stays in place. This way a maximum of compatibility with existing systems is achieved and short turnaround times are possible for tip cleaning.