Identification of two type V myosins in fission yeast, one of which functions in polarized cell growth and moves rapidly in the cell.

We characterized the novel Schizosaccharomyces pombe genes myo4(+) and myo5(+), both of which encode myosin-V heavy chains. Disruption of myo4 caused a defect in cell growth and led to an abnormal accumulation of secretory vesicles throughout the cytoplasm. The mutant cells were rounder than normal, although the sites for cell polarization were still established. Elongation of the cell ends and completion of septation required more time than in wild-type cells, indicating that Myo4 functions in polarized growth both at the cell ends and during septation. Consistent with this conclusion, Myo4 was localized around the growing cell ends, the medial F-actin ring, and the septum as a cluster of dot structures. In living cells, the dots of green fluorescent protein-tagged Myo4 moved rapidly around these regions. The localization and movement of Myo4 were dependent on both F-actin cables and its motor activity but seemed to be independent of microtubules. Moreover, the motor activity of Myo4 was essential for its function. These results suggest that Myo4 is involved in polarized cell growth by moving with a secretory vesicle along the F-actin cables around the sites for polarization. In contrast, the phenotype of myo5 null cells was indistinguishable from that of wild-type cells. This and other data suggest that Myo5 has a role distinct from that of Myo4.

Schizosaccharomyces pombe rho2p GTPase regulates cell wall alpha-glucan biosynthesis through the protein kinase pck2p.

Schizosaccharomyces pombe rho1(+) and rho2(+) genes are involved in the control of cell morphogenesis, cell integrity, and polarization of the actin cytoskeleton. Although both GTPases interact with each of the two S. pombe protein kinase C homologues, Pck1p and Pck2p, their functions are distinct from each other. It is known that Rho1p regulates (1,3)beta-D-glucan synthesis both directly and through Pck2p. In this paper, we have investigated Rho2p signaling and show that pck2 delta and rho2 delta strains display similar defects with regard to cell wall integrity, indicating that they might be in the same signaling pathway. We also show that Rho2 GTPase regulates the synthesis of alpha-D-glucan, the other main structural polymer of the S. pombe cell wall, primarily through Pck2p. Although overexpression of rho2(+) in wild-type or pck1 delta cells is lethal and causes morphological alterations, actin depolarization, and an increase in alpha-D-glucan biosynthesis, all of these effects are suppressed in a pck2 delta strain. In addition, genetic interactions suggest that Rho2p and Pck2p are important for the regulation of Mok1p, the major (1-3)alpha-D-glucan synthase. Thus, a rho2 delta mutation, like pck2 delta, is synthetically lethal with mok1-664, and the mutant partially fails to localize Mok1p to the growing areas. Moreover, overexpression of mok1(+) in rho2 delta cells causes a lethal phenotype that is completely different from that of mok1(+) overexpression in wild-type cells, and the increase in alpha-glucan is considerably lower. Taken together, all of these results indicate the presence of a signaling pathway regulating alpha-glucan biosynthesis in which the Rho2p GTPase activates Pck2p, and this kinase in turn controls Mok1p.

Metronomic chemotherapy in the neoadjuvant setting: results of two parallel feasibility trials (TraQme and TAME) in patients with HER2+ and HER2- locally advanced breast cancer.

Neoadjuvant chemotherapy has practical and theoretical advantages over adjuvant chemotherapy strategy in breast cancer (BC) management. Moreover, metronomic delivery has a more favorable toxicity profile. The present study examined the feasibility of neoadjuvant metronomic chemotherapy in two cohorts [HER2+ (TraQme) and HER2- (TAME)] of locally advanced BC. Twenty patients were prospectively enrolled (TraQme, n=9; TAME, n=11). Both cohorts received weekly paclitaxel at 100 mg/m(2) during 8 weeks followed by weekly doxorubicin at 24 mg/m(2) for 9 weeks in combination with oral cyclophosphamide at 100 mg/day (fixed dose). The HER2+ cohort received weekly trastuzumab. The study was interrupted because of safety issues. Thirty-six percent of patients in the TAME cohort and all patients from the TraQme cohort had stage III BC. Of note, 33% from the TraQme cohort and 66% from the TAME cohort displayed hormone receptor positivity in tumor tissue. The pathological complete response rates were 55% and 18% among patients enrolled in the TraQme and TAME cohorts, respectively. Patients in the TraQme cohort had more advanced BC stages at diagnosis, higher-grade pathological classification, and more tumors lacking hormone receptor expression, compared to the TAME cohort. The toxicity profile was also different. Two patients in the TraQme cohort developed pneumonitis, and in the TAME cohort we observed more hematological toxicity and hand-foot syndrome. The neoadjuvant metronomic chemotherapy regimen evaluated in this trial was highly effective in achieving a tumor response, especially in the HER2+ cohort. Pneumonitis was a serious, unexpected adverse event observed in this group. Further larger and randomized trials are warranted to evaluate the association between metronomic chemotherapy and trastuzumab treatment.

Subcellular localization and possible function of actin, tropomyosin and actin-related protein 3 (Arp3) in the fission yeast Schizosaccharomyces pombe.

We investigated subcellular localizations and interactions of actin and two actin cytoskeleton-related proteins, Cdc8 tropomyosin and actin-related protein 3, Arp3, in the fission yeast Schizosaccharomyces pombe, using specific antibodies and by gene disruption. Actin was localized to the medial microfilamentous ring in the region of the septum during cytokinesis and to cortical patches by immunoelectron microscopy. F-actin cables were detected throughout the cell cycle by fluorescent staining with Bodipy-phallacidin. Cables were often linked to the patches and to the medial ring during its formation. Tropomyosin was localized to the medial ring and the cables. It was also distributed in the cell as patches, although co-localization with F-actin was not frequent. In cdc8ts mutant cells, F-actin cables were not observed although the F-actin patches were detected and cell polarity was maintained. These observations suggest that the F-actin cables may be involved in the formation of the medial ring, and that tropomyosin plays an important role in organizing both the ring and the cable, but is not involved in the F-actin patch formation or maintenance of cell polarity. Binding of Arp3 to actin was revealed by immunoprecipitation as well as by DNase I column chromatography. Arp3 seemed to form a complex with several proteins in the cell extracts, as previously reported for other organisms. Contrary to a previous report (McCollum et al., EMBO J. 15, 6438-6446, 1996), Arp3 was found to be concentrated in the medial region from early anaphase to late cytokinesis. Following arp3 gene disruption, F-actin patches were delocalized throughout the cell and cells did not undergo polarized growth, suggesting that Arp3 influences the proper localization of the actin patches in the cell and thereby controls the polarized growth of the cell.

Monoamine oxidase in rat ovary during the estrous cycle. A histochemical study by a new coupled peroxidatic oxidation method.

A new coupled peroxidatic oxidation method for histochemical detection of monoamine oxidase (MAO) was applied to rat ovary. With this new method, fixed tissues could be used, and two forms of MAO could be identified by use of selective inhibitors. MAO activity was observed in the corpora lutea, interstitial gland cells, and blood vessels. In the corpora lutea, no activity was detected during the first estrous cycle, but strong activity was observed in the next two cycles. MAO in blood vessels showed characteristic changes of activity during the estrous cycle. The results suggest that MAO activity might possibly be involved in ovulation and progesterone metabolism in the ovary. Like other organs, rat ovary was found to contain two types of MAO; type A MAO was predominant in the corpora lutea. On the other hand, only one type of MAO, type B, was found in the blood vessels.

Insulin production in a neuroectodermal tumor that expresses islet factor-1, but not pancreatic-duodenal homeobox 1.

We studied a 60-yr-old female with a brain tumor who showed severe symptoms of hypoglycemia (plasma glucose, 2.2 mmol/L) and hyperinsulinemia (1.28 nmol/L) after radiotherapy. The cystic brain tumor contained proinsulin and insulin at concentrations of 13.6 and 1.22 nmol/L, respectively. Immunohistochemical studies showed the tumor cells were ectodermal in origin but not endodermal, based on three diagnostic features of neuroectodermal tumors 1) pseudorosette formation noted under light microscopy, 2) finding of a small number of dense core neurosecretory granules on electron microscopy, and 3) positive immunostaining for both neuronal specific enolase and protein gene product 9.5. These cells also expressed the transcription factor, neurogenin-3, NeuroD/beta 2, and islet factor I, which are believed to be transcription factors in neuroectoderm as well as in pancreatic islet cells, but not pancreatic-duodenal homeobox 1, Pax4, or Nkx2.2. In addition, they did not express glucagon, somatostatin, or glucagon-like peptide-1. Our results show the presence of proinsulin in an ectoderm cell brain tumor that does not express the homeobox gene, pancreatic-duodenal homeobox 1, but expresses other transcription factors, i.e. neurogenin3, NeuroD/beta 2, and islet factor-1, which are related to insulin gene expression in the brain tumor.

Immunohistochemical localization of calretinin in the rat hindbrain.

The localization of calretinin in the rat hindbrain was examined immunohistochemically with antiserum against calretinin purified from the guinea pig brain. Calretinin immunoreactivity was found within neuronal elements. The distribution of calretinin-immunoreactive cell bodies and fibers is presented in schematic drawings and summarized in a table. Major calretinin-immunoreactive neurons were found in the lateral and medial geniculate nuclei, substantia nigra, ventral tegmental area, interpeduncular nucleus, periaqueductal gray, mesencephalic trigeminal nucleus, superior and inferior colliculi, pontine nuclei, parabrachial nucleus, dorsal and laterodorsal tegmental nuclei, cochlear nuclei, vestibular nuclei, medullary reticular nuclei, nucleus of the solitary tract, area postrema, substantia gelatinosa of the spinal trigeminal nucleus, and cerebellum. These results show that distinct calretinin-immunoreactive neurons are widely distributed in the rat hindbrain.

Hypothalamic galanin-immunoreactive neurons projecting to the posterior lobe of the rat pituitary: a combined retrograde tracing and immunohistochemical study.

UNLABELLED: To identify the galanin-immunoreactive neurons projecting to the posterior lobe of the pituitary in the rat hypothalamus, a retrograde tracer (complex of wheat germ agglutinin-enzymatically inactive horseradish peroxidase-colloidal gold) was injected into the posterior lobe of the pituitary. Sections of the hypothalamus were treated with a combination of silver enhancement of retrogradely transported tracer and immunohistochemistry of galanin. Of the total number of hypothalamic cells doubly labeled with retrograde tracing and galanin-immunostaining, 56-60% were found in the supraoptic nucleus, 18-23% in the retrochiasmatic nucleus, 8-10% in the lateral magnocellular portion of the paraventricular nucleus. The ratio of (number of doubly labeled cells/number of galanin-immunoreactive cells) in each of the above regions was similar to the ratio of (number of retrogradely labeled cells/number of Nissl-stained cells) in the supraoptic nucleus. Of all retrogradely labeled cells in the hypothalamus, 51-56% also contained galaninlike immunoreactivity. IN CONCLUSION: (1) galanin-immunoreactive fibers in the posterior lobe of the pituitary originate mainly in the supraoptic nucleus, retrochiasmatic nucleus, and lateral magnocellular portion of the paraventricular nucleus, (2) most of galanin-immunoreactive cells in these regions project to the posterior lobe of the pituitary, and (3) about half the neurons constituting the hypothalamo-neurohypophyseal system contain galaninlike immunoreactivity.

Topographic atlas of monoamine oxidase-containing neurons in the rat brain studied by an improved histochemical method.

The distribution of monoamine oxidase-containing neuronal somata was studied in the rat brain by using an improved enzyme histochemical technique of the coupled peroxidatic oxidation method applied to fixed free-floating sections. The majority of monoamine oxidase-containing neuronal somata appeared to correspond with well-known cell groups of monoamine-containing neurons with a few exceptions. The enzyme appeared to coexist also in histamine-containing neurons in the posterior hypothalamus. Furthermore, monoamine oxidase activity was localized in apparently non-monoaminergic cells in the mesencephalon, hypothalamus, thalamus and telencephalon.

Distribution of nitric oxide synthase in the central nervous system of Macaca fuscata: subcortical regions.

The distribution of nitric oxide synthase-immunoreactive neurons was studied in the Macaca fuscata by immunohistochemistry using antiserum against nitric oxide synthase. In the macaque lower brainstem, many nitric oxide synthase-containing cell bodies were found in the gigantocellular and parvocellular reticular nuclei, the nucleus of the spinal tract of trigeminal nerve, the cochlear nucleus, the prepositus hypoglossi and the nucleus of the solitary tract. Many nitric oxide synthase-immunoreactive perikarya were observed in the laterodorsal-pedunculopontine tegmental nucleus complex of the macaque pontine and midbrain tegmentum. In addition, there were many highly immunoreactive cell bodies in the superficial layers of the inferior and superior colliculi. In the forebrain, numerous cell bodies were observed in the caudate nucleus, putamen, nucleus accumbens, nucleus of the diagonal band, anterior perforated substance and amygdaloid complex. Whereas most of these neurons were labeled highly intense for nitric oxide synthase, there were also many lightly labeled nitric oxide synthase-immunoreactive neurons in the substantia innominata, globus pallidus, ansa peduncularis and lateral hypothalamic nucleus. The present observation indicated some species difference in the distribution of central nitric oxide synthase-containing neurons. Furthermore, the present neuroanatomical evidence that nitric oxide synthase is distributed in a variety of specific neuronal systems, with some differences in the patterns of cytoplasmic localization, further indicates the neural messenger role of nitric oxide in the central nervous system.

Calretinin distribution in the thalamus of the rat: immunohistochemical and in situ hybridization histochemical analyses.

The distribution of calretinin-containing cells was examined by in situ hybridization histochemistry and compared with the immunohistochemical mapping of calretinin in the thalamus of the rat. Results revealed a close correspondence between the immunohistochemical localization of cell bodies and the messenger RNA label produced by the calretinin oligonucleotide probe. Calretinin cells were most prominent in the midline (paraventricular, reuniens, rhomboid) and intralaminar (central medial, paracentral) nuclei and in a group of cells along the rostral central gray which appeared continuous with the caudal extent of the midline nuclei. A subpopulation of calretinin cell bodies was also identified in the reticular nucleus. The mediorostral lateral posterior nucleus, subparafascicular, lateral geniculate and habenular nuclei also contained calretinin messenger RNA probe label. In contrast, no positive cells were found in the anterior, ventral or posterior thalamic nuclei. The distribution of calretinin cells did not correspond directly with that of other histochemical markers. Thus, the in situ hybridization histochemical and immunohistochemical results revealed calretinin as a unique identifying marker for distinct sets of thalamic neurons.

Differential subcellular location of mitochondria in rat serotonergic neurons depends on the presence and the absence of monoamine oxidase type B.

Monoamine oxidase type A and type B are major neurotransmitter-degrading enzymes in the CNS. The type A is present on mitochondrial outer membranes in the whole extent of noradrenergic and dopaminergic neurons, including their axon terminals. The type B is present in serotonergic neurons, but its subcellular localization has not been elucidated. In the present study, we used both a double-labeling immunofluorescence method and electron microscopic immunohistochemistry to examine the subcellular localization of monoamine oxidase type B in serotonergic neurons projecting from the dorsal raphe nucleus to the suprachiasmatic nucleus in the rat brain. In the dorsal raphe nucleus, serotonin-positive neuronal cell bodies were clustered, and virtually all of these cell bodies were also positive for monoamine oxidase type B. By contrast, serotonin-negative neuronal cell bodies were mostly free of this enzyme. Within the neuronal cell bodies and dendrites that were positive for monoamine oxidase type B, most mitochondria contained this enzyme on their outer membranes, but a substantial proportion of mitochondria lacked this enzyme. In the suprachiasmatic nucleus, serotonin-positive varicosities were concentrated, but none of these varicosities exhibited monoamine oxidase type B. In this nucleus, mitochondria were found in almost all serotonin-positive axon terminals, but monoamine oxidase type B was not observed in any axon terminal that contained mitochondria. Our results show that there are two kinds of mitochondria in serotonergic neuronal cell bodies and dendrites: one containing monoamine oxidase type B on their outer membranes, and the other lacking this enzyme. In addition, mitochondria in serotonergic axon terminals do not possess monoamine oxidase type B. It is suggested in serotonergic neurons that only mitochondria lacking monoamine oxidase type B are transported by axonal flow up to axon terminals. It is also probable that mitochondria containing monoamine oxidase type B are transported along the axons, but that this enzyme undergoes a change, for example, conformational change, decomposition or removal from the membranes.

Combined use of silver staining of the retrograde tracer WGAapoHRP-Au and pre-embedding immunocytochemistry for electron microscopy: demonstration of dopaminergic terminals in synaptic contact with striatal neurons projecting to the substantia nigra in the rat.

We investigated the applicability of the pre-embedding immunoperoxidase technique to WGAapoHRP-Au retrograde tracing. After injection of the tracer into the substantia nigra of rat, the brain was fixed and cryostat sections were immunostained for dopamine. The sections were osmicated and silver-stained to amplify the colloidal gold particles. Products of both the immunoperoxidase staining and the silver staining could be detected and distinguished by electron microscopy at low magnification. The ultrastructure was so well preserved that synaptic characteristics could be investigated. Dopaminergic terminals were demonstrated to synapse with striatal neurons projecting to the substantia nigra.

Calbindin D28k and calretinin in oxytocin and vasopressin neurons of the rat supraoptic nucleus.A triple-labeling immunofluorescence study

The aim of the present study was to examine quantitatively whether two calcium-binding proteins, calbindin D28k and calretinin, are localized in oxytocin and vasopressin neurons of the supraoptic nucleus of the male rat. We used a triple-labeling immunofluorescence method with a confocal laser scanning microscope. Of the oxytocin-labeled cells, 70% were stained for both calbindin D28k and calretinin, 15% were stained for only calbindin D28k, 13% were stained for only calretinin, and 2% were stained for neither protein. Of the vasopressin-labeled cells, 73% were stained for neither calbindin D28k nor calretinin, 21% were stained for only calbindin D28k, 4% were stained for only calretinin, and 2% were stained for both proteins. Calbindin D28k and calretinin have been shown previously to contribute to calcium homeostasis by buffering [Ca(2+)](i). Therefore, these findings suggest that most of the oxytocin neurons may have a higher Ca(2+)-buffering capacity than most of the vasopressin neurons.

Evidence for a colocalization of oxytocin mRNA and galanin in magnocellular hypothalamic neurons: a study combining in situ hybridization and immunohistochemistry.

The possible colocalization of oxytocin (OT) and galanin (GAL) was studied by combining, on the same cryostat sections, in situ hybridization (ISH) for OT mRNA with a tritiated oligonucleotide probe and immunohistochemistry (ICC) of GAL. Many cells were either labelled by ISH (OT mRNA containing cells), or by ICC (GAL containing cells). Moreover, some magnocellular neurons in the supraoptic and paraventricular nuclei were labelled for both OT mRNA and GAL. These results demonstrate that some magnocellular neurons of the rat hypothalamus contain both GAL and OT. This approach is suitable for studying the intracellular distribution of OT gene expression and mature GAL under different physiological or experimental conditions.

Chemical features of monoaminergic and non-monoaminergic neurons in the brain of laboratory shrew (Suncus murinus) are changed by systemic administration of monoamine precursors.

5-Hydroxy-L-tryptophan (5-HTP) and L-3,4-dihydroxyphenylalanine (L-DOPA) were injected intraperitoneally (i.p.) into the laboratory shrew (Suncus murinus). Immunocytochemical and immunofluorescence studies were carried out on serial or same sections of the brain, which were reacted with specific antisera to dopamine (DA) or serotonin (5-HT) produced in our laboratory. We observed that cell bodies and nerve terminals of many catecholaminergic (CAnergic) neurons exhibited 5-HTP uptake and conversion of the precursor into 5-HT. However, the locus ceruleus showed scarcely any 5-HT immunoreactivity. This suggests that the precursor uptake mechanism may be different among various CAnergic groups. In contrast to these findings on CAnergic neurons, all serotoninergic (5-HTnergic) neurons after L-DOPA administration showed DA immunopositive reaction in their cell bodies and nerve terminals, suggesting that 5-HTnergic neurons may have the same capacity for precursor uptake. On the other hand, we observed that all aromatic L-amino acid decarboxylase (AADC)-only-positive neuron groups showed both DA and 5-HT immunoreactions after L-DOPA and 5-HTP administration, respectively, in the double-staining immunofluorescence method. From these results, AADC-only-positive neurons may be considered to belong to the amine precursor uptake and decarboxylation (APUD) system.

Ultrastructural localization of galanin immunoreactivity in the rat median eminence.

The aim of this study was to examine, by use of a pre-embedding immunoperoxidase technique, the ultrastructural localization of galanin immunoreactivity in the external layer of the rat median eminence. Galanin immunoreactivity was only observed in axonal profiles. Immunoreactive fibers were found in contact with the following non-immunoreactive structures: (1) axonal profiles that contain dense granular vesicles and clear vesicles, (2) axonal profiles that contain predominantly clear vesicles, (3) glial cell bodies, and (4) processes of tanycytes. Labeled terminals were also observed in the proximity of the perivascular space of the portal vessels. The results suggest possible interactions between galanin-immunoreactive terminals and other terminals containing peptide and/or other transmitters in the external layer of the median eminence.

Monoamine oxidase activity in noradrenaline neurons of the locus coeruleus of the rat. A double-labeling histochemical study.

The aim of the present study is to examine whether noradrenergic neurons of the locus coeruleus (LC) of the rat contain monoamine oxidase (MAO) activity. Sections were processed initially for MAO enzyme histochemistry using tyramine as a substrate, followed by fluorescence immunohistochemistry for tyrosine hydroxylase (TH). In the LC, virtually all TH-immunoreactive neurons (i.e., noradrenergic neurons) were also positive for MAO. No MAO activity was found in any TH-negative neurons. Neurons in the LC have previously been shown to form dopamine during noradrenaline biosynthesis and to produce serotonin from exogenously administered l-5-hydroxytryptophan. Moreover, dopamine- and serotonin-degrading MAO activity has also been found in LC neurons. Therefore, our results indicate that MAO activity is localized within noradrenergic neurons in the LC and is likely involved in the degradation of dopamine that is endogenously synthesized, and also in the elimination of serotonin that is produced from exogenous precursors.

Dopamine produced from L-DOPA is degraded by endogenous monoamine oxidase in neurons of the dorsal raphe nucleus of the rat: an immunohistochemical study.

The aim of the present study is to examine by immunohistochemistry whether dopamine produced from L-DOPA in serotonin neurons of the rat brain is degraded by endogenous monoamine oxidase (MAO). In rats that received intraperitoneally L-DOPA plus a peripheral decarboxylase inhibitor, carbidopa, a cluster of dopamine-immunoreactive neurons was found in the dorsal raphe nucleus (DR). In L-DOPA/carbidopa-injected rats that were pretreated with an intraperitoneal injection of a MAO inhibitor, pargyline, when compared with the L-DOPA/carbidopa-injected rats without the pargyline pretreatment, neurons of the cluster of the DR became much darker in dopamine staining. The distribution of dopamine-stained neurons in the DR of these rats corresponded very closely to the previously reported distribution of serotonin-immunoreactive neurons of normal rats. In normal or only pargyline-injected rats, dopamine-stained neurons were scarcely observed in the DR. We previously showed that serotonin neurons of the rat DR were induced to contain dopamine by the injection of L-DOPA plus carbidopa. These findings suggest that the newly produced dopamine from L-DOPA in serotonin neurons of the rat DR is degraded by endogenous MAO.

Catecholamine degradation by monoamine oxidase in locus coeruleus neurons of the rat. An immunohistochemical study.

We examined by immunohistochemistry the effects of monoamine oxidase (MAO) inhibition on the content of dopamine (DA) and noradrenaline (NA) in locus coeruleus (LC) neurons of the rat. In normal rats, clusters of DA- and NA-immunopositive neurons were identified in the LC. Rats treated with intraperitoneal injections of pargyline, an MAO inhibitor, showed significantly stronger DA- and NA-staining intensities in LC neurons compared to normal rats. In LC noradrenergic neurons, it is believed that DA is formed in the cytoplasm and then transported into the storage vesicles where it is converted to NA, and the secreted NA is recycled by a reuptake mechanism and transported back into storage vesicles via the cytoplasm. Furthermore, LC neurons of the rat have been shown to contain DA- and NA-degrading MAO activities on the outer membranes of the mitochondria. Therefore, our findings suggest that endogenous MAO degrades not only part of the DA formed in the cytoplasm of LC neurons, but also part of the secreted NA that has been transported back into the cytoplasm.