Differential regulation of the Kar3p kinesin-related protein by two associated proteins, Cik1p and Vik1p.

The mechanisms by which kinesin-related proteins interact with other proteins to carry out specific cellular processes is poorly understood. The kinesin-related protein, Kar3p, has been implicated in many microtubule functions in yeast. Some of these functions require interaction with the Cik1 protein (Page, B.D., L.L. Satterwhite, M.D. Rose, and M. Snyder. 1994. J. Cell Biol. 124:507-519). We have identified a Saccharomyces cerevisiae gene, named VIK1, encoding a protein with sequence and structural similarity to Cik1p. The Vik1 protein is detected in vegetatively growing cells but not in mating pheromone-treated cells. Vik1p physically associates with Kar3p in a complex separate from that of the Kar3p-Cik1p complex. Vik1p localizes to the spindle-pole body region in a Kar3p-dependent manner. Reciprocally, concentration of Kar3p at the spindle poles during vegetative growth requires the presence of Vik1p, but not Cik1p. Phenotypic analysis suggests that Cik1p and Vik1p are involved in different Kar3p functions. Disruption of VIK1 causes increased resistance to the microtubule depolymerizing drug benomyl and partially suppresses growth defects of cik1Delta mutants. The vik1Delta and kar3Delta mutations, but not cik1Delta, partially suppresses the temperature-sensitive growth defect of strains lacking the function of two other yeast kinesin-related proteins, Cin8p and Kip1p. Our results indicate that Kar3p forms functionally distinct complexes with Cik1p and Vik1p to participate in different microtubule-mediated events within the same cell.

How well do skinfold equations predict percent body fat in elite soccer players?

The use of generic equations for estimating percent body fat from skinfold thicknesses can be criticised when applied to specific sports. The present aims were to compare existing methods of using skinfold data and to derive an equation for predicting body fat values in professional soccer players. Forty-five professional soccer players (24.2 +/- 5.0 years; 82.0 +/- 8.5 kg; 1.82 +/- 0.07 m) participated. Skinfold thicknesses were assessed at eight sites for the application of existing prediction equations. Skinfold data were also utilised to determine a novel soccer-specific equation. All players had a reference estimate of percent fat by dual-energy x-ray absorptiometry (DXA). The existing skinfold equations differed from the DXA-referenced values by varying degrees, the equation of Withers et al. (1987) demonstrating the lowest bias and highest relationship and agreement with DXA. Regression analysis resulted in an equation incorporating anterior thigh, abdominal, triceps and medial calf sites, accounting for 78.4% variance in DXA criterion values.

Stromal PTEN inhibits the expansion of mammary epithelial stem cells through Jagged-1.

This corrects the article DOI: 10.1038/onc.2016.383.

Stromal PTEN inhibits the expansion of mammary epithelial stem cells through Jagged-1.

Fibroblasts within the mammary tumor microenvironment are active participants in carcinogenesis mediating both tumor initiation and progression. Our group has previously demonstrated that genetic loss of phosphatase and tensin homolog (PTEN) in mammary fibroblasts induces an oncogenic secretome that remodels the extracellular milieu accelerating ErbB2-driven mammary tumor progression. While these prior studies highlighted a tumor suppressive role for stromal PTEN, how the adjacent normal epithelium transforms in response to PTEN loss was not previously addressed. To identify these early events, we have evaluated both phenotypic and genetic changes within the pre-neoplastic mammary epithelium of mice with and without stromal PTEN expression. We report that fibroblast-specific PTEN deletion greatly restricts mammary ductal elongation and induces aberrant alveolar side-branching. These mice concomitantly exhibit an expansion of the mammary epithelial stem cell (MaSC) enriched basal/myoepithelial population and an increase in in vitro stem cell activity. Further analysis revealed that NOTCH signaling, specifically through NOTCH3, is diminished in these cells. Mechanistically, JAGGED-1, a transmembrane ligand for the NOTCH receptor, is downregulated in the PTEN-null fibroblasts leading to a loss in the paracrine activation of NOTCH signaling from the surrounding stroma. Reintroduction of JAGGED-1 expression within the PTEN-null fibroblasts was sufficient to abrogate the observed increase in colony forming activity implying a direct role for stromal JAGGED-1 in regulation of MaSC properties. Importantly, breast cancer patients whose tumors express both low stromal JAG1 and low stromal PTEN exhibit a shorter time to recurrence than those whose tumors express low levels of either alone suggesting similar stromal signaling in advanced disease. Combined, these results unveil a novel stromal PTEN-to-JAGGED-1 axis in maintaining the MaSC niche, and subsequently inhibiting breast cancer initiation and disease progression.

An immunocytochemical study of the development of central serotoninergic neurons in the chick embryo.

The development of central serotoninergic neurons in the chick embryo has been investigated immunocytochemically by utilizing an antiserum to serotonin (5-HT). Immunoreactive neurons are first detected in the brainstem on embryonic day 4 (E4, stage 23), days earlier than 5-HT systems have been detected previously by biochemical techniques. The earliest 5-HT-containing cells at E4 appear rostral to the pontine flexure, yet by E5, 5-HT neuronal groups are observed throughout the brainstem from just caudal to the mesencephalic flexure to the cervical flexure. During this and subsequent phases of development, two distinct patterns of cellular migration seem to be involved in the formation of the various 5-HT neuronal groups. One pattern involves a ventral migration of 5-HT cells, which appears dependent upon the directional guidance of midline radial processes (formed by floor plate cells) that extend across the neuroepithelium. The other pattern involves a lateral migration of cells, followed by an aggregation and rearrangement of 5-HT neurons into distinct subgroups or clusters. Through these patterns of migration most components of the 5-HT neuronal system can be recognized as early as E12, with the mature organization of the 5-HT cell groups occurring by E17. One unexpected finding was the comparatively late appearance (between E9 and E12) of 5-HT neurons in the paraventricular organ of the hypothalamus. Thus, in comparison to the initial observation of the majority of brainstem 5-HT neurons at E4 to E5, the hypothalamic 5-HT cells appear after a delay of between 5 and 7 days. Such differences illustrate the fact that neurons sharing a common neurotransmitter phenotype do not necessarily share the same developmental timetable for the expression of that particular phenotype, or they may undergo neurogenesis during considerably different periods of embryogenesis.

Effects of hydrogen sulfide exposure on surface properties of lung surfactant.

Hydrogen sulfide is an irritant and chemical asphyxiant gas that exerts its primary toxic effects on the respiratory and neurological systems. Exposure to hydrogen sulfide above a threshold value of 200-300 ppm is characterized by the sudden onset of hemorrhagic pulmonary edema. The purpose of this study was to determine whether this response is associated with changes in the surface properties of pulmonary surfactant. Bronchoalveolar lavage fluid was retrieved from the lungs of Fischer 344 rats exposed to two concentrations of hydrogen sulfide or fresh air for 4 h. Surface tension-lowering properties were assayed using a captive bubble surface tensiometer. Lung injury was assessed by histopathology and measurements of total protein and lactate dehydrogenase activity in the lavagate. Marked abnormalities in surfactant activity were demonstrated in the lavagates from rats exposed to the highest concentration (300 ppm) of hydrogen sulfide. These involved the properties of adsorption to the air-water interface and surface tension lowering under quasi-static interfacial compression. Exposure to 200 ppm hydrogen sulfide had no effect on minimum surface tension despite a significant increase in protein and lactate dehydrogenase in the lavagate. This would suggest a threshold-type response for the inhibition of surfactant activity by hydrogen sulfide. In vitro studies using normal rat surfactant showed that the abnormalities in surfactant activity were due to inhibitors in the edema fluid and not to a direct effect of sulfide on surfactant. The pathophysiological consequences of increased alveolar surface tension after hydrogen sulfide exposure may need to be considered in the clinical setting.

Serotonin-immunoreactive nerve fibers of the rat pituitary: effects of anticatecholamine and antiserotonin drugs on staining patterns.

Serotonin-immunoreactive (5-HT-IR) nerve fibers observed in the pars intermedia and pars nervosa of the rat pituitary gland were examined after treatment of animals with antiserotonergic or anti-catecholaminergic drugs. p-Chlorophenylalanine, (PCPA), an inhibitor of 5-HT synthesis, eliminated staining in both areas of pituitary gland. p-Chloroamphetamine (PCA), a serotonin neurotoxin, did not produce a significant change in 5-HT-IR from control tissue. Unexpectedly, 6-hydroxydopamine (6-OHDA), a catecholamine neurotoxin, eliminated 5-HT staining in the pars intermedia, but not in the pars nervosa. These observations suggest that 5-HT present in fibers innervating the intermediate and neural lobes is synthesized in axons locally, or is taken up into fibers from extrinsic sources. In addition, catecholamines and 5-HT may co-exist in nerve terminals of both lobes of the pituitary gland.

Co-localization of tyrosine hydroxylase (TH)- and serotonin (5-HT)-immunoreactive innervation in the rat pituitary gland.

Adult male Sprague-Dawley rats were examined for possible co-localization of tyrosine hydroxylase (TH) and serotonin (5-HT) within innervation of the pituitary neurointermediate lobe. Use of sheep antiserum to TH and a secondary antibody coupled to fluorescein isothiocyanate (FITC), followed by rabbit anti-5-HT, then rhodamine-coupled second antibody, produced co-existent staining in some, but not all fibers of both the neural and intermediate lobes, using paraffin embedded tissues. Application of a combination of the two primary antibodies followed by sequential application of secondary antibody also resulted in co-localized antigens in selected fibers. Multiple 'classic' neurotransmitters within the same nerve terminals may modulate selected areas of endocrine tissue to control hormone release.

Two populations of tyrosine hydroxylase-positive cells occur in the spinal cord of the chick embryo and hatchling.

The existence of tyrosine hydroxylase (TH)-containing neurons in the spinal cord of the chick embryo was investigated by anti-TH immunocytochemistry. Two populations of intensely immunostained cells were observed along the entire extent of the cord, beginning late in chick embryogenesis. One group of TH-positive cells was particularly numerous and found ventral to the central canal. The other group, which was smaller in number, was located along the superficial and lateral border of the dorsal horn of the spinal cord. When examined by the glyoxylic acid histofluorescence technique, cells could be visualized only very infrequently ventral to the central canal, and not at all within the dorsal horn. However, after pretreatment of hatchlings with the catecholamine synthesis precursor L-DOPA, cells ventral to the canal were readily observed by histofluorescence, while the dorsally located cells seldom visualized. Since these populations of TH-positive cells appear to only partially express the catecholaminergic phenotype, these cells may provide a model in which factors regulating the expression of neurotransmitter phenotypes can be examined in neurons of the developing CNS.

Magnetic resonance lipid signals in rat brain after experimental stroke correlate with neutral lipid accumulation.

Proton magnetic resonance spectroscopy (MRS) signals from lipids in brain have been observed to increase after ischemic brain injury. However, neither the chemical identity nor the cellular location of these lipids has been established. The aim of the present study was to identify the origin of MRS lipid signals in rat brain after temporary (90 min) middle cerebral artery occlusion (MCAO). Fatty acyl proton signals were detected by short-echo one and two dimensional (1)H MRS in superfused brain slices from the infarcted hemisphere 1-5 days after MCAO. The intensities of these signals were strongly correlated with the amount of triacylglyceride and cholesterol ester in lipid extracts from the samples (r(2)=0.96, P<0.05) and were not correlated with the amount of free fatty acids in the tissue. Histological staining of tissue revealed the presence of neutral lipid droplets in infarcted regions. Dual labeling by immunohistochemistry demonstrated that these droplets were localized to microglia/macrophage (OX-42-labeled cells). These results strongly suggest that (1)H MRS lipid signals from brain after stroke arise from microglia/macrophage phagocytosis of cellular membranes.

Development of the serotonergic system in the rat embryo: an immunocytochemical study.

The development of central serotonergic neurons has been examined immunocytochemically utilizing an antiserum to serotonin (5-HT). Cells of the B4-B9 complex are first detected early on embryonic day 13 (E13; 7 mm crown rump length, CRL) and increase rapidly in number through E15 when they appear as bilateral columns situated from just caudal to the mesencephalic flexure to the pontine flexure. Aggregation of cells into subgroups is apparent soon after 5-HT neurons leave the ventricular zone, allowing the identification of certain subdivisions of the B4-B9 complex long before they assume their adult locations. The initial detection of 5-HT immunoreactive cells in the medulla occurs 1-2 days after the appearance of cells in the B4-B9 complex, although it has been reported that the time of origin of medullary raphe neurons (B1-B3) occurs before that of raphe neurons in the midbrain and pons (B4-B9). The first medullary 5-HT neurons, comprising the B3 subdivision occur ventro-laterally on E14 (10-11 mm CRL) at least 1-2 days before midline 5-HT neurons are visualized in the B1 and B2 groups. Thus, in contrast to cells in the B4-B9 complex, medullary 5-HT neurons complete much of their migration before they can be detected immunocytochemically, indicating that the time of onset of transmitter synthesis and storage may differ during differentiation of cells sharing a common neurotransmitter phenotype. The formation of ascending 5-HT fiber projections occurs rapidly from cells of the B4-B9 complex. Within 24 hours after the initial detection of 5-HT fiber immunoreactivity in such cells at E13, their axons are seen entering the caudal diencephalon (E14). These fibers have traversed the diencephalon and floor of the telencephalon by E15-E16 and reach the frontal neocortical pole by E17. The main ascending bundle of 5-HT axons courses through the diencephalon in the vicinity of the medial forebrain bundle, although some fibers also diverge and travel along certain pre-existing non-5HT pathways. However, examples are also found of acute directional changes in 5-HT fiber growth which do not appear to be associated with pre-formed non-5HT pathways. The pattern of ascending fiber outgrowth suggests a priority routing system which provides certain regions with 5-HT axons in a preferential sequence irrespective of the distance of these areas from 5-HT cell groups or from major bundles of ascending 5-HT fibers.

Serotonin immunocytochemistry in the adult and developing rat brain: methodological and pharmacological considerations.

An antiserum has been raised in rabbits against serotonin (5-HT) conjugated to the invertebrate protein hemocyanin (HC). This antiserum was characterized with respect to its cross-reactivity with related compounds and its immunocytochemical staining properties in brains of adult and developing rats and in animals pretreated with various pharmacological regimens. When compared to an antiserum raised against 5-HT/bovine serum albumin (BSA) conjugates [59], the 5-HT/HC conjugate elicited a more profound immune response which resulted in the production of a specific, high titer antiserum that could be used directly for immunocytochemistry without removal of antibodies to the invertebrate carrier molecule, HC. Immunoabsorption experiments to assess the specificity of this antiserum demonstrated a small degree of cross-reactivity with dopamine (which was greater than that with norepinephrine or epinephrine). However, no staining of catecholaminergic neurons was found in untreated adult or developing animals, nor in animals pretreated with L-DOPA or L-DOPA + the MAO inhibitor nialamide, indicating that this cross-reactivity is not manifested under normal staining conditions. No cross-reactivity of the 5-HT/HC antiserum was observed for any 5-HT precursors or metabolites tested, although both this antiserum and the 5-HT/BSA antiserum did exhibit a high degree of cross-reactivity to the related indoleamines 5-methoxytryptamine (5-MT) and tryptamine. However, based on the immunocytochemical staining patterns observed, and the fact that both 5-MT and tryptamine are found in very low quantities in the normal rat brain, it appears that 5-HT is the predominant indoleamine stained by both of these antisera in the untreated rat brain. In animals pretreated with L-tryptophan + nialamide, some light staining was found in the dopaminergic A9 and A10 cell groups using either antiserum. However, since this staining was not observed in L-DOPA + nialamide treated animals it is not thought to be due to cross-reactivity with dopamine. Rather, since the staining could be inhibited by pretreatment with the catecholaminergic uptake blocker desmethylimipramine, it is postulated that this effect may be due to either (1) the non-specific uptake of 5-HT or 5-hydroxytryptophan (5-HTP) into the dopaminergic cells of A9 and A10 due to elevated levels of these substances in the dense serotonergic axonal plexus passing through this region or (2) to an increased uptake of circulating L-tryptophan by these A9 and A10 cells followed by conversion of this amino acid to tryptamine by aromatic amine decarboxylase, an enzyme common to both 5-HT and dopaminergic neurons. This latter possibility suggests that caution should be exercised when interpreting immunocytochemical staining patterns obtained in animals pretreated with L-tryptophan + nialamide using 5-HT antisera, since other cross-reactive indoleamines could be elevated by this pharmacological manipulation.

Analysis of the change in number of serotonergic neurons in the chick spinal cord during embryonic development.

The existence of serotonin (5-HT)-containing neurons in the spinal cord of the chick embryo was examined by anti-5-HT immunocytochemistry. The first immunoreactive cells were observed in embryos at 7 days of incubation (E7) and were initially located within the floor plate of the early spinal cord. By E9, immunostained cells occurred throughout the length of the spinal cord and were frequently encountered in most transverse sections of the cord. When examined at later embryonic ages of E12, 17 and at hatching (E21 or 22), the 5-HT cells became progressively more difficult to find with the advancing age of the embryos. To determine if this population of spinal cord 5-HT neurons actually diminished during development, a detailed quantitative analysis was undertaken to estimate the number of 5-HT cells in the cord of chick embryos at different ages. The results of this investigation demonstrated that the size of the 5-HT neuronal population rose rapidly from E7 and plateaued (at approximately 3500 neurons) between E9 and E12. As anticipated, the number of 5-HT cells at E17 decreased at all cord levels. Surprisingly, however, the number of spinal cord 5-HT neurons at hatching increased (depending on the cord level) either back to, or above, the counts estimated for the earlier ages of E9 and E12. Therefore, cells expressing the 5-HT phenotype in the spinal cord of the chick embryo persist throughout the period of embryonic development, rather than appear transiently.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of the serotonin-uptake inhibitor, fluoxetine, on immunoreactive serotonin innervation in the rat pituitary gland.

Serotonin (5-HT)-immunoreactive innervation can be demonstrated in both the pars intermedia and pars nervosa of the rat pituitary gland. Fluoxetine hydrochloride, a 5-HT uptake inhibitor, was administered in single or multiple doses to adult male Sprague-Dawley rats to examine the possibility that 5-HT present in pituitary fibers may be due to uptake of the indoleamine. Paraffin-embedded sections of pituitaries from fluoxetine-treated rats revealed diminution or loss of 5-HT fiber staining in the intermediate, but not in the neural lobe, in all animals examined. Multiple doses of fluoxetine produced the most consistent effects on immunostaining. These observations along with results of our previous studies suggest that the intermediate lobe innervation immunoreactive for 5-HT may be due to uptake of indoleamine from extrinsic sources, while the pars nervosa terminals containing 5-HT most likely originate from 5-HT neuronal perikarya in the lower brainstem or hypothalamus.

In vivo and in vitro development of serotonergic neurons.

The monoamines are one of the earliest developing neurotransmitter systems in the mammalian brain. The first part of this paper describes the normal ontogeny of the serotonergic (5-HT) system in the rat brain as studied using long survival 3H-thymidine autoradiography (time of neuronal genesis, time of origin) and the Falck-Hillarp histofluorescence method, electron microscopy, and immunocytochemistry (anti-5-HT). Due to their early ontogeny relative to other brain regions, 5-HT neurons (as well as monoamine neurons in general) have been suggested to exert some type of "trophic" influence on brain development. Results of pharmacological experiments designed to inhibit 5-HT synthesis in the embryonic rat brain by maternal treatment with p-chlorophenylalanine (pCPA) at a time when this monoamine might exert such an influence are discussed with regard to effects on the time course of neuronal genesis (time of origin) of 5-HT neurons and their target cells. These results, which prompted us to propose that 5-HT might act as a "differentiation signal" for certain of its target cells, are now discussed in light of our more recent immunocytochemical-autoradiographic studies (anti-5-HT, 3H-thymidine) which morphologically demonstrate close associations between developing 5-HT neurons and proliferating neuroepithelial cells in the embryonic brain. Postnatal studies using this immunocytochemical-autoradiographic method also provide evidence for interactions of 5-HT axons with proliferating glioblasts in the developing cerebellum and with immature granule cells and their precursors in the hippocampus. These findings, in conjunction with the results of our pCPA experiments, further enhance the possibility that 5-HT neurons could exert an epigenetic influence on the development of less differentiated cells with which they come into contact. Finally, preliminary studies using dissociated cell cultures containing 5-HT neurons suggest that interactions between 5-HT neurons and glial elements may be important for the differentiation of these neurons in vitro. Whether 5-HT neurons in turn influence the development of glial or neuronal cells in these cultures remains to be determined. These studies are evaluated with regard to a possible pre-transmission role for 5-HT during key phases of neuronal and glial genesis.

Clinical significance of rare and benign side effects: propofol and green urine.

A 55-year-old man requiring airway protection for esophagogastroduodenoscopy was sedated with propofol. On the third day of propofol infusion his urine was dark green. Although he was afebrile and his white blood cell count was within normal limits, the green urine was suspected to be of infectious etiology. Laboratory tests were ordered and broad-spectrum antibiotics were considered. Antibiotics were avoided when propofol was recognized as a rare and benign potential cause of the green urine. Earlier recognition of this side effect may have averted unnecessary laboratory monitoring. Prompt recognition of such side effects is important in limiting medical expenditures, inordinate drug exposure, and distress among patients and clinicians.

Clinical and economic effects of pharmacy services in geriatric ambulatory clinic.

Pharmacy services were introduced in an established multidisciplinary geriatric ambulatory clinic. The pharmacist collaborated with primary care providers to optimize patients' drug regimens. Over 8 months there were 250 patient visits to the clinic. Traditional medical care was provided at 144 (57.6%) of these visits and traditional medical care plus pharmacist evaluation was provided at 106 (42.4%). The pharmacist identified 220 potential and actual drug-related problems. Acceptance of pharmacist-recommended changes in drug therapy was 98.6%. A mean reduction of 3.4 agents/patient was achieved in the intervention group (p<0.0001). Clinical outcomes of changes in drug therapy were neutral or positive in 99.5% of cases. Pharmacy services resulted in net savings of $7,788 annually.

Efficacy of cobalt chelates in the rabbit eye model for epithelial herpetic keratitis.

PURPOSE: A new class of antiviral agent, cobalt chelates (the CTC series), was evaluated for treating epithelial herpetic keratitis, consequent stromal disease being the major infectious cause of blindness in industrial nations. METHODS: Effects of CTC complexes were monitored in cell cultures and in a rabbit eye model, either infected with herpes simplex virus type 1 (HSV-1) or uninfected. Several antiviral concentrations of CTC complexes nontoxic to Vero cells were administered to rabbit eyes with HSV-1-induced keratitis. Corneal surface virus titers were measured, and corneal lesions of epithelial keratitis were monitored by slit-lamp microscopy and scored. Recovery rates and incidence were compared in eyes treated with CTC complexes, placebo, or clinically formulated trifluorothymidine (Viroptic), using nonparametric statistics. RESULTS: All CTC complexes inhibited HSV-1 replication in vitro, CTC-96 being best. CTC-96, CTC-23, and CTC-67 eliminated (<1 plaque-forming unit[pfu]) corneal surface HSV-1 (otherwise >10(5) pfu) in order of descending potency, but CTC-82 was ineffective. CTC-96 (either 5 microg/ml six times daily or 10 microg/ml five times daily) accelerated herpetic dendritic keratitis recovery better than or the same as trifluorothymidine (10 mg/ml nine times daily). CTC complexes were nontoxic to Vero cells continuously exposed to < or =25 microg/ml; 50 microg/ml of CTC 96 nine times daily did not irritate uninfected rabbit eyes. CONCLUSION: Topical CTC-96 applications were at least as effective as Viroptic in diminishing disease signs and corneal surface virus at concentrations less than one-thousandth that of Viroptic.

Sialadenosis in bulimia. A new treatment.

OBJECTIVE: To see whether a cholinomimetic medication would help resolve the parotid gland enlargement associated with self-induced vomiting in bulimic patients. SETTING/DESIGN: The medication was administered on an inpatient eating disorder unit to bulimic patients with refractory sialadenosis. INTERVENTION: Pilocarpine hydrochloride drops were administered orally at a dosage of 1.25 to 5.0 mg/d until the parotid gland enlargement was significantly reduced. RESULTS: There was a marked diminution in the size of the parotid gland enlargement. CONCLUSION: Pilocarpine has proved to be beneficial in the treatment of sialadenosis in bulimic patients.

Dens invaginatus: another use for the ultrasonic.

Dens invaginatus is an anomaly which has numerous and complex forms. Whenever endodontic therapy involving such a tooth becomes necessary, the clinician must be aware of the various treatment modalities available to facilitate proper care. Presented in this case report is a maxillary lateral incisor having the anomaly dens invaginatus in which both surgical and nonsurgical treatment was required. The surgical phase was facilitated through the use of ultrasonic instrumentation.