bluuebunny/crossref_metadata_2025 huggingface dataset:
Coordinated development of identified serotonergic neurons and their target ciliary cells in Helisoma trivolvis embryos
Authors: Koss Ron , Diefenbach Thomas J. , Kuang Shihuan , Doran Shandra A. , Goldberg Jeffrey I.
Published: February 2003

Embryonic neuron C1s (ENC1s) are bilateral serotonergic neurons that function as cilioexcitatory motor neurons in embryonic development of the pond snail, Helisoma trivolvis. Recent experiments demonstrated that these neurons stimulate cilia‐driven embryo rotation in response to hypoxia. In the present study, a comprehensive anatomic analysis of these cells and their target ciliary structures was done to address the following questions: (1) Does ENC1 have a morphology consistent with an oxygen‐sensitive sensory cell; (2) Is the development of ENC1's neurite outgrowth pathway coordinated with the development of its target effectors, the pedal and dorsolateral ciliary bands; and (3) What is the anatomic basis of ENC1–ciliary communication? By using an array of microscopic techniques on live and serotonin‐immunostained embryos, we found that each ENC1 possessed an apical dendrite that was capped with an integral dendritic knob penetrating the embryo surface. The dendritic knobs contained both microvilli and nonmotile cilia that suggested a sensory transduction role. Each ENC1 also possessed a descending projection, whose development was characterized by the rapid formation of the primary neurite pathway between stages E13 and E15, with the primary neurite of the right ENC1 developing in advance of its contralateral homologue. Secondary neurite branches formed between stages E15 and E30 in a spatiotemporal pattern that closely matched the development of the dorsolateral and pedal bands of cilia. Both dorsolateral and pedal ciliated cells formed basal processes that contacted ENC1 neurites. Finally, gap junction profiles were observed at neurite–neurite, neurite–ciliary cell, and ciliary cell–ciliary cell apposition sites, whereas putative chemical synaptic profiles were observed at neurite–neurite and neurite–ciliary cell apposition sites. J. Comp. Neurol. 457:313–325, 2003. © 2003 Wiley‐Liss, Inc.
Pontine sources of norepinephrine in the cat cochlear nucleus
Authors: Thompson Ann M.
Published: February 2003

In the current study, the distribution of noradrenergic neurons in the pontine tegmentum that project to the cochlear nucleus was determined with retrograde tract tracing combined with neurotransmitter immunohistochemistry in the cat. Double‐labeled neurons were observed in all noradrenergic cell groups, in both the dorsolateral and the ventrolateral tegmentum. Half of the double‐labeled cells were located in the locus coeruleus complex. Most of these were situated in its ventral division. Most other double‐labeled cells were located in peribrachial regions, especially lateral to the brachium conjunctivum. Relatively few double‐labeled cells were observed in both the A4 and the A5 cell groups, 2% and 0.4%, respectively, of the total. Except for neurons in A5, which projected only contralaterally, the projections were bilateral, with an ipsilateral preponderance. The results indicate that neurons located in the ipsilateral dorsolateral tegmentum, namely, in the locus coeruleus complex and the peribrachial region, are the primary source of pontine noradrenergic afferents to the cochlear nucleus of the cat. J. Comp. Neurol. 457:374–383, 2003. © 2003 Wiley‐Liss, Inc.
Differential morphology of pyramidal tract‐type and intratelencephalically projecting‐type corticostriatal neurons and their intrastriatal terminals in rats
Authors: Reiner Anton , Jiao Yun , Del Mar Nobel , Laverghetta Antonio Vincent , Lei Wan Long
Published: February 2003

Two types of corticostriatal projection neurons have been identified: 1) one whose intrastriatal arborization arises as a collateral of a projection to the ipsilateral brainstem via the pyramidal tract (PT‐type); and 2) one that projects intratelencephalically to the cortex and striatum, in many cases bilaterally, but not extratelencephalically (IT‐type). To assess possible functional differences between these two neuron types, we characterized their laminar location in the cortex, their perikaryal size, and the morphology of their intrastriatal terminals. IT‐type neurons were retrogradely labeled by tetramethylrhodamine‐dextran amine (RDA)3k injection into the contralateral striatum, whereas their intrastriatal terminals were labeled anterogradely by biotinylated dextran amine (BDA)10k injection into the contralateral motor or primary somatosensory cortex. To label PT‐type neurons and their ipsilateral intrastriatal terminals retrogradely, BDA3k was injected into the pontine pyramidal tract. We found that IT‐type neuronal perikarya are medium‐sized (12–13 μm) and located in layer III and upper layer V, whereas PT‐type perikarya are larger (18–19 μm) and most commonly located in lower layer V. At the electron microscopic level, the intrastriatal terminals of both corticostriatal neuron types made asymmetric synaptic contact with spine heads and less frequently with dendrites. IT‐type axospinous terminals were characteristically small (0.4–0.5 μm) and regular in shape, whereas PT‐type terminals were typically large (0.8–0.9 μm) and often irregular in shape. Perforated postsynaptic densities were common for PT‐type terminals, but not IT‐type. The clear differences between these two corticostriatal neuron types in perikaryal size and laminar location in the cortex, and in the size and shape of their intrastriatal terminals, suggest that they may differ in the nature of their influence on the striatum. J. Comp. Neurol. 457:420–440, 2003. © 2003 Wiley‐Liss, Inc.
Visualization of S100B‐positive neurons and glia in the central nervous system of EGFP transgenic mice
Authors: Vives Virginie , Alonso Gérard , Solal Anne Cohen , Joubert Dominique , Legraverend Catherine
Published: February 2003

S100B, the EF‐hand Ca++‐binding protein with gliotrophic and neurotrophic properties implicated in the pathogenesis of Alzheimer's disease, is coined as a glial marker, despite its documented presence in rodent brain neurons. We have generated a transgenic mouse whose EGFP reporter, controlled by the −1669/+3106 sequence of the murine S100B gene, allows the direct microscopic observation of most S100B‐expressing cells in the central nervous system (CNS). From embryonic day 13 onward, EGFP expression was targeted to selected neuroepithelial, glial, and neuronal cells, indicating that cell‐specific expression of S100B is regulated at the transcriptional level during development. In adult mice, the highest level of EGFP expression was found in ependymocytes; astrocytes; and spinal, medullar, pontine, and deep cerebellar S100B neurons. Our results, thus, agree with earlier reports suggesting that S100B is not a CNS glial‐specific marker. In addition, we detected EGFP and S100B in forebrain neurons previously thought not to express S100B in the mouse, including neurons of primary motor and somatosensory neocortical areas, the ventral pallidum and prerubral field. Another interesting finding was the selected EGFP targeting to neonatal S100B oligodendrocytes and adult NG2 progenitors as opposed to mature S100B oligodendrocytes. This finding suggests that, except for oligodendrocytes at the last stage of myelin maturation, the −1669/+3106 sequence of the S100B gene is a useful reagent for driving expression of transgenes in most S100B‐expressing cells of mouse brain. J. Comp. Neurol. 457:404–419, 2003. © 2003 Wiley‐Liss, Inc.
Odor‐evoked activity is spatially distributed in piriform cortex
Authors: Illig Kurt R. , Haberly Lewis B.
Published: February 2003

Much data on the olfactory bulb (OB) indicates that structural characteristics of odorant molecules are encoded as ordered, spatially consolidated sets of active cells. New results with “genetic tracing” (Zou et al. [2001] Nature 414:173–179) suggest that spatial order is also present in projections from the OB to the olfactory cortex. For the piriform cortex (PC), results with this technique indicate that afferents conveying input derived from single olfactory receptors (ORs) are distributed to well‐defined patches in the anterior PC (APC) but that these patches are much larger than in the OB. We have used c‐fos induction to examine how input patterning for single ORs is translated into patterns of odor‐evoked cellular activity in the PC. The laminar distribution of labeled cells and dual‐immunostaining for γ‐aminobutyric acid (GABA)ergic markers indicated that activity was detected largely in pyramidal cells. In odor‐stimulated rats, labeled cells were present throughout the posterior PC (PPC) but were concentrated in prominent rostrocaudal bands in APC. Analysis of responses to different odorants and concentrations revealed that locations and shapes of bands conveyed no apparent information regarding odor quality, rather, they appeared to correspond to subregions of the APC distinguished by cytoarchitecture and connectivity. Small‐scale variations in labeling density were observed within APC bands and throughout the PPC that could reflect the presence of a complex topographical order, but discrete patches at consistent locations as observed by genetic tracing were absent. This finding suggests that as a result of afferent overlap and intracortical processing, odor‐quality information is represented by spatially distributed sets of cells. A distributed organization may be optimal for discriminating biologically relevant odorants that activate large numbers of ORs. J. Comp. Neurol. 457:361–373, 2003. © 2003 Wiley‐Liss, Inc.
Steroid‐triggered programmed cell death of a motoneuron is autophagic and involves structural changes in mitochondria
Authors: Kinch Ginger , Hoffman Kurt L. , Rodrigues Elizabeth M. , Zee Michele C. , Weeks Janis C.
Published: February 2003

Neuronal death occurs during normal development and disease and can be regulated by steroid hormones. In the hawkmoth, Manduca sexta, individual accessory planta retractor (APR) motoneurons undergo a segment‐specific pattern of programmed cell death (PCD) at pupation that is triggered directly and cell autonomously by the steroid hormone 20‐hydroxyecdysone (20E). APRs from abdominal segment six [APR(6)s] die by 48 hours after pupal ecdysis (PE; entry into the pupal stage), whereas APR(4)s survive until adulthood. Cell culture experiments showed previously that 20E acts directly on APRs to trigger PCD, with intrinsic segmental identity determining which APRs die. The APR(6) death pathway includes caspase activation and loss of mitochondrial function. We used transmission electron microscopy to investigate the ultrastructure of APR somata before and during PCD. APR(4)s showed normal ultrastructure at all stages examined, as did APR(6)s until approximately stage PE. During APR(6) death, there was massive accumulation of autophagic bodies and vacuoles, mitochondria became ultracondensed and aggregated into compact clusters, and ribosomes aggregated in large blocks. Nuclear ultrastructure remained normal, without chromatin condensation, until the nuclear envelope fragmented late in the death process. Light microscopic immunocytochemistry showed that dying APR(6)s were TUNEL‐positive, which is diagnostic of fragmented DNA. These observations indicate that the steroid‐induced, caspase‐dependent, cell‐autonomous PCD of APR(6)s is autophagic, not apoptotic, and support an early role for mitochondrial alterations during PCD. This system permits the study of neuronal death in response to its bona fide developmental signal, the rise in a steroid hormone. J. Comp. Neurol. 457:384–403, 2003. © 2003 Wiley‐Liss, Inc.
Laminar and cellular targets of individual thalamic reticular nucleus axons in the lateral geniculate nucleus in the prosimian primate Galago
Authors: Uhlrich Daniel J. , Manning Karen A. , Feig Sherry L.
Published: February 2003

The visual sector of the thalamic reticular nucleus is the source of the primary inhibitory projection to the visual thalamic relay nucleus, the dorsal lateral geniculate nucleus. The purpose of this study was to investigate laminar and cellular targets of individual thalamic reticular nucleus axons in the highly laminated lateral geniculate nucleus of the prosimian primate Galago to better understand the nature and function of this projection. Thalamic reticular axons labeled anterogradely by means of biotinylated dextran amine were examined by using light microscopic serial reconstruction and electron microscopic analysis in combination with postembedding immunohistochemical labeling for the neurotransmitter γ‐aminobutyric acid (GABA). The synaptic targets of labeled reticular terminal profiles were primarily GABA‐negative dendrites (79–84%) of thalamocortical cells, whereas up to 16% were GABA‐positive dendritic shafts or F2 terminals of interneurons. Reconstructed thalamic reticular nucleus axons were narrowly aligned along a single axis perpendicular to the geniculate laminar plane, exhibiting a high degree of visuotopic precision. Individual reticular axons targeted multiple or all geniculate laminae, with little laminar selectivity in the distribution of swellings with regard to the eye of origin or to the parvocellular, koniocellular, or magnocellular type neurons contained in the separate layers of the Galago lateral geniculate nucleus. These results suggest that cells in the visual thalamic reticular nucleus influence the lateral geniculate nucleus retinotopically, with little regard to visual functional streams. J. Comp. Neurol. 458:128–143, 2003. © 2003 Wiley‐Liss, Inc.
Dynamics of microglia in the developing rat brain
Authors: Dalmau Ishar , Vela José Miguel , González Berta , Finsen Bente , Castellano Bernardo
Published: February 2003

Entrance of mesodermal precursors into the developing CNS is the most well‐accepted origin of microglia. However, the contribution of proliferation and death of recruited microglial precursors to the final microglial cell population remains to be elucidated. To investigate microglial proliferation and apoptosis during development, we combined proliferating cell nuclear antigen (PCNA) immunohistochemistry, in situ detection of nuclear DNA fragmentation (TUNEL), and caspase‐3 immunohistochemistry with tomato lectin histochemistry, a selective microglial marker. The study was carried out in Wistar rats from embryonic day (E) 16 to postnatal day (P) 18 in cerebral cortex, subcortical white matter, and hippocampus. Proliferating microglial cells were found at all ages in the three brain regions and represented a significant fraction of the total microglial cell population. The percentage of microglia expressing PCNA progressively increased from the embryonic period (25–51% at E16) to a maximum at P9, when the great majority of microglia expressed PCNA (92–99%) in all the brain regions analyzed. In spite of the remarkable proliferation and expansion of the microglial population with time, the density of microglia remained quite constant in most brain regions because of the considerable growth of the brain during late prenatal and early postnatal periods. In contrast, apoptosis of microglia was detected only at certain times and was restricted to some ameboid cells in white matter and primitive ramified cells in gray matter, representing a small fraction of the microglial population. Therefore, our results point to proliferation of microglial precursors in the developing brain as a physiological mechanism contributing to the acquisition of the adult microglial cell population. In contrast, microglial apoptosis occurs only locally at certain developmental stages and thus seems less crucial for the establishment of the final density of microglia. J. Comp. Neurol. 458:144–157, 2003. © 2003 Wiley‐Liss, Inc.
Retinotopic pathways providing motion‐selective information to the lobula from peripheral elementary motion‐detecting circuits
Authors: Douglass John K. , Strausfeld Nicholas J.
Published: February 2003

Recordings from afferent channels from the medulla supplying deep neuropils of the fly's optic lobes reveal different filter properties among the three classes of afferent neurons: transmedullary cells, T2 neurons, and Y cells. Whereas transmedullary cells respond to local flicker stimuli without discriminating these from directional or oriented motion, the T2 afferent neurons show clear motion orientation selectivity, which corresponds closely with a morphological bias in the orientation of their dendrites and could also be influenced by systems of local recurrent neurons in the medulla. A Y cell having a clearly defined terminal in the lobula, but having dendrite‐like processes in the medulla and, possibly, the lobula plate, discriminates the direction of motion and its orientation. These results demonstrate unambiguously that the lobula receives information about motion and that the channels carrying it are distinct from those supplying wide‐field motion‐selective neurons in the lobula plate. Furthermore, recordings from a newly identified recurrent neuron linking the lobula back to the inner medulla demonstrate that the lobula discriminates nondirectional edge motion from flicker, thereby reflecting a property of this neuropil that is comparable with that of primary visual cortex in cats. The present findings support the proposal that elementary motion detecting circuits supply several parallel channels through the medulla, which segregate to, but are not shared by, the lobula and the lobula plate. The results are discussed in the context of other intracellular recordings from retinotopic neurons and with analogous findings from mammalian visual systems. J. Comp. Neurol. 457:326–344, 2003. © 2003 Wiley‐Liss, Inc.
Synaptic remodeling in the dorsal motor nucleus of the vagus nerve following vagal‐hypoglossal nerve anastomosis in the cat
Authors: Lan Chyn‐Tair , Hsu Jee‐Ching , Chang Chen‐Nen , Chuang Huo‐Li , Ling Eng‐Ang
Published: February 2003

Horseradish peroxidase (HRP) retrograde tracing techniques and morphometric analyses were performed to investigate synaptic remodeling associated with neuronal and glial changes in the dorsal motor nucleus of the vagus (DMV) of cats after vagal‐hypoglossal nerve anastomosis (VHA). At 25 days postoperation (dpo), in the early target‐reinnervation stage, there were 50% fewer presynaptic boutons containing round vesicles (R) or round and large dense‐cored synaptic vesicles (R+D) contacting HRP‐labeled DMV motoneurons. The loss of R boutons was maintained throughout the remaining postoperative intervals up to 500 dpo, whereas R+D boutons were further reduced at 123 dpo but were restored at 315 dpo, so that, by 500 dpo, 71.4% of them had gained access to the DMV motoneurons. Boutons containing pleomorphic synaptic vesicles (P) were completely disconnected from the DMV motoneurons at 25 dpo and did not reappear even in the long‐term reinnervation stage. Loss and recovery of presynaptic boutons occurred in parallel with changes in astroglial ensheathment of the DMV motoneurons. It is suggested that synaptic remodeling associated with astroglial ensheathment in the DMV may be influenced by some retrogradely transported factors/signals derived from the newly acquired target organ, viz. tongue skeletal musculature. Our results further suggest that the observed changes in boutonal configurations may be attributable to modified functions of the DMV motoneurons induced by VHA. J. Comp. Neurol. 458:195–207, 2003. © 2003 Wiley‐Liss, Inc.
Olfactory ensheathing cells: Time lapse imaging of cellular interactions, axonal support, rapid morphologic shifts, and mitosis
Authors: Van Den Pol Anthony N. , Santarelli Justin G.
Published: February 2003

Olfactory ensheathing cells (OECs) have considerable potential for facilitating axonal growth across regions of spinal cord and brain injury but in this context have been studied primarily in static images of fixed tissue from the olfactory system or after transplantation. In the present work, we studied the behavior of live OECs, and their interactions with neurons, Schwann cells, and astrocytes by using cells that express the reporter gene coding for green fluorescent protein (GFP); the work is based on combinations of fluorescence, phase contrast, digital time lapse imaging, and P75 immunocytochemical identification. Cultures, explants, and regions of olfactory system slices rich in OECs enhanced axonal growth of cerebellar granule cells or hippocampal neurons; axons grew parallel to the long axis of fusiform OECs. Neuron cell bodies and axons preferred OECs over artificial substrates. Axons and neuron cell bodies can take active or passive roles in extension and migration on underlying motile OECs and move from one OEC to another. Axon extension was facilitated to a similar degree by OECs and Schwann cells, whereas astrocytes were more likely to integrate with existing OECs than with Schwann cells. OECs showed a dramatic ability to rapidly change shape, size, and direction of migration and to undergo mitosis. Mitosis was characterized by a quick retraction of all processes, thereby forming a sphere that divided into spherical daughter cells within minutes. Progeny OECs might take on the parental or a non‐parental morphotype, with both daughter cells showing robust expression of GFP. Together these OEC data demonstrated a substantial plasticity and capability for relatively rapid changes in structure and support the view that OECs have multiple attributes favorable for enhancing axonal extension and neuronal migration after central nervous system injury. J. Comp. Neurol. 458:175–194, 2003. © 2003 Wiley‐Liss, Inc.
Insulin‐like growth factor‐II/mannose‐6‐phosphate receptor: Widespread distribution in neurons of the central nervous system including those expressing cholinergic phenotype
Authors: Hawkes Cheryl , Kar Satyabrata
Published: February 2003

The insulin‐like growth factor‐II/mannose‐6‐phosphate (IGF‐II/M6P) receptor is single transmembrane glycoprotein that plays a critical role in the trafficking of lysosomal enzymes and the internalization of circulating IGF‐II. At present, there is little information regarding the cellular distribution of the IGF‐II/M6P receptor within the adult rat brain. With the use of immunoblotting and immunocytochemical methods, we found that the IGF‐II/M6P receptor is widely but selectively expressed in all major brain areas, including the olfactory bulb, striatum, cortex, hippocampus, thalamus, hypothalamus, cerebellum, brainstem, and spinal cord. Intense IGF‐II/M6P receptor immunoreactivity was apparent on neuronal cell bodies within the striatum, deeper layers (layers IV and V) of the cortex, pyramidal and granule cell layers of the hippocampal formation, selected thalamic nuclei, Purkinje cells of the cerebellum, pontine nucleus and motoneurons of the brainstem as well as in the spinal cord. Moderate neuronal labeling was evident in the olfactory bulb, basal forebrain areas, hypothalamus, superior colliculus, midbrain areas, granule cells of the cerebellum and in the intermediate regions of the spinal gray matter. We also observed dense neuropil labeling in many regions, suggesting that this receptor is localized in dendrites and/or axon terminals. Double‐labeling studies further indicated that a subset of IGF‐II/M6P receptor colocalizes with cholinergic cell bodies and fibers in the septum, striatum, diagonal band complex, nucleus basalis, cortex, hippocampus, and motoneurons of the brainstem and spinal cord. The observed widespread distribution and colocalization of IGF‐II/M6P receptor in the adult rat brain provide an anatomic basis to suggest a multifunctional role for the receptor in a wide‐spectrum of central nervous system neurons, including those expressing a cholinergic phenotype. J. Comp. Neurol. 458:113–127, 2003. © 2003 Wiley‐Liss, Inc.
Distribution of dendrites of descending neurons and its implications for the basic organization of the cockroach brain
Authors: Okada Ryuichi , Sakura Midori , Mizunami Makoto
Published: February 2003

To determine precisely the brain areas from which descending neurons (DNs) originate, we examined the distribution of somata and dendrites of DNs in the cockroach brain by retrogradely filling their axons from the cervical connective. At least 235 pairs of somata of DNs were stained, and most of these were grouped into 22 clusters. Their dendrites were distributed in most brain areas, including lateral and medial protocerebra, which are major termination areas of output neurons of the mushroom body, but not in the optic and antennal lobes, the mushroom body, the central complex, or the posteroventral part of the lateral horn. The last area is the termination area of major types of olfactory projection neurons from the antennal lobe, i.e., uni‐ and macroglomerular projection neurons, so these neurons have no direct connections with DNs. The distribution of axon terminals of ascending neurons overlaps with that of DN dendrites. We propose, based on these findings, that there are numerous parallel processing streams from cephalic sensory areas to thoracic locomotory centers, many of which are via premotor brain areas from which DNs originate. In addition, outputs from the mushroom body, central complex, and posteroventral part of the lateral horn converge on some of the premotor areas, presumably to modulate the activity of some sensorimotor pathways. We propose, based on our results and documented findings, that many parallel processing streams function in various forms of reflexive and relatively stereotyped behaviors, whereas indirect pathways govern some forms of experience‐dependent modification of behavior. J. Comp. Neurol. 458:158–174, 2003. © 2003 Wiley‐Liss, Inc.
Troglitazone‐induced intracellular oxidative stress in rat hepatoma cells: A flow cytometric assessment
Authors: Narayanan Padma Kumar , Hart Timothy , Elcock Fiona , Zhang Cindy , Hahn Laura , McFarland David , Schwartz Lester , Morgan D. Gwyn , Bugelski Peter
Published: February 2003

Background

Troglitazone (TRO), a thiazolidinedione (TZD) peroxisome proliferator‐activated receptor γ agonist, was recently withdrawn from the market because of rare but serious hepatotoxicity. Previous studies investigating the cytotoxicity of TRO in cultured rat hepatocytes have conjectured about the role of oxidative stress in TRO‐induced hepatotoxicity. Therefore, we investigated whether TRO induces oxidative stress and, if so, the portion of the TRO molecule responsible for the induction of oxidative stress.

Methods

Novikoff rat hepatoma (N1S1) cells were incubated with TRO, troglitazone quinone (TQ), thiazolidinedione‐phenoxyacetic acid (TD‐PAA) or rosiglitazone (RSG). Membrane peroxidation, intracellular glutathione (GSH) content, and cellular viability were monitored simultaneously by multiparameter flow cytometry.

Results

TRO and TQ increased membrane peroxidation, decreased intracellular GSH, and decreased cell viability in a concentration‐dependent manner. In contrast, TD‐PAA and RSG neither increased membrane peroxidation nor induced loss of cell viability. In addition, TRO caused a concentration‐dependent increase in intracellular superoxide generation accompanied by a collapse in mitochondrial membrane potential.

Conclusion

Multiparameter flow cytometric evaluation of N1S1 cells indicated that the chromane ring of TRO, rather than the TZD moiety, may be responsible for oxidative stress and suggested that a direct effect on mitochondrial physiology may play a role in TRO‐mediated hepatotoxicity. Cytometry Part A 52A:28–35, 2003. © 2003 Wiley‐Liss, Inc.
Toluidine blue test for sperm DNA integrity and elaboration of image cytometry algorithm
Authors: Erenpreisa Jekaterina , Erenpreiss Juris , Freivalds Talivaldis , Slaidina Maija , Krampe Rasma , Butikova Jelena , Ivanov Andrey , Pjanova Dace
Published: February 2003

Background

Sperm DNA integrity is of paramount importance in the prognosis of fertility. We applied image cytometry to a toluidine blue (TB) test we recently proposed.

Methods

Sperm samples from 33 men were assayed for standard sperm parameters and classified as normal or abnormal. Sperm smears were subjected to the TB test, DNA denaturation testing with acridine orange (AO), and terminal deoxyuridine triphosphate biotin nick end labeling (TUNEL). In CCD image analysis, TB‐stained sperm cell heads were microscopically assigned to one of four color groups (dark, blue, light violet, and light blue). The optical densities of 6,600 cells in green and red CCD images were used to elaborate an algorithm for discrimination of these groups.

Results

The proportions of sperm in TB color groups, as estimated with the developed image cytometry algorithm, correlated with microscopic features. The number of TB dark cells correlated with the number of AO‐red and TUNEL+ cells. The proportion of TB dark cells in normal samples did not exceed 35%. Light‐blue sperm cell heads prevailed in normal samples, whereas dark and blue sperm cell heads dominated in abnormal samples.

Conclusions

The TB test was suitable for the assessment of sperm cell DNA integrity. The elaborated image cytometry algorithm can be used for this purpose and for finer determination of sperm nucleus status. Cytometry Part A 52A:19–27, 2003. © 2003 Wiley‐Liss, Inc.
Automated analysis of morphometric parameters for accurate definition of erythrocyte cell shape
Authors: Albertini Maria C. , Teodori Laura , Piatti Elena , Piacentini Maria P. , Accorsi Augusto , Rocchi Marco B. L.
Published: February 2003

Background

Modification of erythrocyte morphology is clinically important in hematology and medicine. Its detection is routinely performed by subjective microscopic evaluation, which is difficult and strongly dependent on the operator's expertise. We developed an original automated methodology to analyze erythrocyte cell shape modification to support and improve the operator's capability and expedite measurements.

Methods

We used morphometric parameters derived from optical microscope images elaborated with an image processing software (NIH Scion Image) to construct a new application for statistical multivariate discriminant analysis.

Results

For each cell type the elaboration of the morphometric parameters allowed us to develop a chromogenic index, a dimension index, a biconcavity index, and a density profile. The measurements of these indexes were used to construct a statistical methodology that could discriminate among erythrocyte morphologies according to Bessis. When applied casewise, the model effectively differentiated between discocytes, target cells, ovalocytes, macrocytes, and microcytes, with an agreement of 70% between actual and predicted classifications.

Conclusions

The results clearly demonstrated that a set of opportunely selected morphometric parameters derived from optical microscope images and statistically analyzed can effectively discriminate with a high degree of certainty among different shape modifications that red blood cells can undergo in various in vitro and in vivo conditions. This method represents the first attempt to automate the definition of erythrocyte morphology and may have important applications in cases in which the detection of erythrocyte cell shape changes is crucial. Cytometry Part A 52A:12–18, 2003. © 2003 Wiley‐Liss, Inc.
Corrigendum
Authors: Fauth Christine , Zink Daniele
Published: February 2003

The original article to which this Corrigendum refers was published in Cytometry (2001) 45(3) 214–224.
Fluorescence lifetime‐based discrimination and quantification of cellular DNA and RNA with phase‐sensitive flow cytometry
Authors: Cui H. Helen , Valdez Joseph G. , Steinkamp John A. , Crissman Harry A.
Published: February 2003

Background

Simultaneous measurement of cellular DNA and RNA content provides information for determination of the functional status of cells and, clinically, for the diagnosis and grading assessment of various tumors. Most current flow cytometric methods are based on resolving the fluorescence emission spectra of dyes that bind preferentially to either type of nucleic acid. However, several monochromatic nucleic acid–binding fluorochromes display resolvable differences in fluorescence lifetime when bound to DNA or RNA. The differences in the lifetime of one fluorescent probe provide an alternate means to distinguish the binding of one probe to these cellular macromolecules and to simultaneously measure their cellular contents.

Methods

Three nucleic acid intercalating dyes, propidium iodide, ethidium bromide, and ethidium homodimer 1, were selected to study differences in fluorescence lifetimes when bound to cellular DNA and RNA. Fixed HL‐60 cells were treated with specific nucleases to initially determine the lifetime values of each dye when bound to the cellular DNA, RNA, or both. The lifetime values were then used as the signatures to resolve the cellular DNA and RNA contents in untreated cells.

Results

All three dyes showed fluorescence lifetime differences when bound to RNase‐treated, DNase‐treated, or untreated cells. With these lifetime values, the fluorescence emissions from DNA, RNA, or DNA/RNA were resolved from untreated cells with the use of phase‐sensitive detection. The lifetime differences resulting from the binding to either type of nucleic acid depended on the dye, the staining concentration, and the analysis condition.

Conclusions

The lifetimes of the nucleic acid–binding fluorochromes were altered when binding to different macromolecules under different conditions. Phase‐sensitive flow cytometry provided a unique means for simultaneous discrimination and quantification of subcellular macromolecules with one fluorescent probe. The data demonstrated the capabilities for resolving relative cellular DNA and RNA contents based on fluorescence lifetime. Cytometry Part A 52A:46–55, 2003. Published 2003 Wiley‐Liss, Inc.
Comparison of proliferation and rapid cytokine induction assays for flow cytometric T‐cell epitope mapping
Authors: Tesfa Lydia , Volk Hans‐Dieter , Kern Florian
Published: February 2003

Background

T‐cell epitope mapping by flow cytometry based on rapid ex vivo peptide‐specific cytokine induction in T cells is very efficient and time saving compared with traditional assays. We investigated whether the same epitopes could be identified by proliferation studies.

Methods

An assay based on rapid interferon‐γ induction in T cells (6 h of ex vivo stimulation) was run in parallel with a proliferation assay based on the incremental loss of carboxy‐fluorescein diacetate succinimidyl ester staining in proliferating cells. The proliferation assay was chosen because it can be evaluated by high‐resolution modern multiparameter flow cytometry. In both cases, T cells were stimulated with the same cytomegalovirus‐derived peptides. The peptides identified by the rapid induction of interferon‐γ were compared with those inducing T‐cell proliferation.

Results

Most epitopes were identified by proliferation and rapid cytokine induction methods; however, each method also identified epitopes that the other one did not. In general, rapid cytokine induction was associated with considerably less background noise, making epitope identification easier, and, owing to the short stimulation time necessary, several identification steps could be carried out on material stored in the incubator.

Conclusions

Even though most epitopes were identified by both approaches, the rapid cytokine induction method had major logistic advantages. However, it may be best to use both assays, particularly in situations in which the identification of epitopes may depend on prior clonal T‐cell expansion. Cytometry Part A 52A:36–45, 2003. © 2003 Wiley‐Liss, Inc.
Quantitative image analysis of cellular protein translocation induced by magnetic microspheres: Application to the EGF Receptor
Authors: Brock Roland , Jovin Thomas M.
Published: February 2003

Background

The molecular analysis of intracellular signal transduction requires technologies that address quantitatively the activation of signaling proteins and formation of molecular complexes without disrupting cellular integrity.

Methods

Cells expressing the epidermal growth factor receptor (EGFR) in its endogenous form or fused to green fluorescent protein were incubated with 1‐μm microspheres covalently functionalized with EGF. The disposition of the plasma membrane about the microspheres was analyzed by high‐resolution confocal microscopy in combination with computational resolution enhancement and optimized fixation procedures. Receptor activation and translocation of signaling proteins to the microspheres was quantitated by image processing protocols for recovering the microsphere‐associated fluorescence and the fluorescence in the local environment.

Results

EGF‐functionalized microspheres were internalized in an activation‐dependent manner similar to that of the soluble growth factor. The correlation of receptor activation and recruitment of a signaling protein was analyzed quantitatively by isolating immunofluorescence signals from the microspheres and from their immediate environment.

Conclusions

The microsphere‐based approach provides a quantitative analysis of cellular signal transduction with subcellular resolution under conditions maintaining cellular integrity. The analysis of signaling‐induced (co)localization of proteins around a microsphere complements other technologies directly probing for molecular interactions such as fluorescence resonance energy transfer. Cytometry Part A 52A:1–11, 2003. © 2003 Wiley‐Liss, Inc.
The Strained η2‐NAmido−NPyridine Coordination of Aminopyridinato Ligands
Authors: Kempe Rhett
Published: February 2003

This review gives an account of the syntheses and structures of group‐3 (including lanthanides), ‐4 and ‐5 metal complexes stabilised by strained η2‐coordinated aminopyridinato ligands. Reactivity studies and catalytic applications of the coordination compounds are discussed. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
Cyclodextrin‐Encapsulated Iron Catalysts for the Polymerization of Ethylene
Authors: Armspach Dominique , Matt Dominique , Peruch Frédéric , Lutz Pierre
Published: February 2003

The synthesis and characterization of dichloroiron(II) complexes of tridentate nitrogen ligands based on 2,6‐bis(imino)pyridine‐capped α‐ and β‐cyclodextrin derivatives are described and their propensity to act as ethylene polymerization catalysts reported. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
TiIV Complexes with Novel, Chiral, Tridentate Amido‐Amidinate or Monoanionic, Didentate Amidinate Ligands
Authors: Li Jian‐Feng , Huang Shu‐Ping , Weng Lin‐Hong , Liu Dian‐Sheng
Published: February 2003

Reaction of LiL or a new type of dianionic ligand Li2L (2) {H2L = (1R,2R)‐(−)‐1‐[N(H)C(Ph)N(SiMe3)]‐2‐[N(H)SiMe3]C6H10} with TiCl2(OiPr)2 yielded the novel complexes [TiCl(L)(OiPr)2] (1) and [Ti(L)(OiPr)2] (3), respectively. Treatment of Na2L′ {H2L′ = (1R,2R)‐(−)‐1,2‐[N(H)C(Ph)N(SiMe3)]2C6H10]} with Ti(OiPr)2Cl2 unexpectedly gave 3 via a unusual rearrangement/elimination reaction; The structure of complex 3 shows both an η3‐π bond and σ‐bonds to the central metal. This is a new coordination model and is obtained for the first time for this type of amido‐amidinate complex. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
Tellurium(II) Dialkanethiolates: np(S)‐σ*(Te−S′) Orbital Interactions Determine the 125Te NMR Chemical Shift, and the Molecular and Crystal Structure
Authors: Fleischer Holger , Mitzel Norbert W. , Schollmeyer Dieter
Published: February 2003

Tellurium(II) dimethanethiolate, Te(SMe)2, and tellurium(II) diethanethiolate, Te(SEt)2, were synthesized by reaction of TeO2 and Te(OiPr)4 with HSMe and HSEt, respectively. In the solid state, Te(SMe)2 exhibits a cis‐conformation of the methyl groups with respect to the TeS2 plane − an unprecedented situation for nonfunctionalized organotrichalcogenides − whereas Te(SEt)2 shows a trans‐conformation. Ab initio calculations performed for Te(SMe)2 and Te(SEt)2 show that the cis‐ and trans‐conformers represent minima on the potential energy surface and are stabilized by intramolecular π‐type n(S)‐σ*(Te−S′) orbital interactions. In the solid state, the molecules of each compound are associated through two centrosymmetric Te2S2 units with two of their neighbors, resulting in tetracoordinate Te atoms with distorted trapezoidal configurations. While the intermolecular Te···S distance increases in the sequence R = Me < Et < iPr < tBu, the length of the covalent Te−S bond decreases in the same order, a result attributed to intermolecular σ‐type np(S)‐σ*(Te−S′) orbital interactions. The 125Te NMR chemical shift of Te(SR)2 largely depends on R (R = Me, Et, iPr, tBu) and shows a nearly linear correlation with the first ionization energy of the corresponding thiol HSR. Ab initio calculations of the 125Te NMR shifts for the model compound Te(SH)2 (C2 symmetry) reveal that it also depends strongly on the HSTeS torsion angle. These results can be explained by a model in which π‐type np(S)‐σ*(Te−S′) and np(Te)‐σ*(S−H) orbital interactions determine the paramagnetic shielding of the tellurium nucleus. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
Synthesis of Tetrahemispheraplexes with Ammonium, Alkylammonium or Alkaline Metal Ions as Exohedral Guests via Self‐Assembly or Guest Exchange
Authors: Saalfrank Rolf W. , Demleitner Bernhard , Glaser Horst , Maid Harald , Reihs Stefan , Bauer Walter , Maluenga Monica , Hampel Frank , Teichert Markus , Krautscheid Harald
Published: February 2003

To study the interplay between the steric demand of the guest and the space available at the host, we have prepared the tetraammonium complexes {(NH4)4∩[Mg4(L1/2)6]} 1, tetrakis(methylammonium) complexes {(Me‐NH3)4∩[Mg4(L1/2)6]} 2, tetrakis(ethylammonium) complex {(Et‐NH3)4∩[Mg4(L1)6]} 3, and mixed sodium tris(ethylammonium) complex {Na(Et‐NH3)3∩[Mg4(L2)6]} 4. The tetrahemispheraplexes 1−4 were available in one‐pot syntheses. The tetrakis(alkylammonium) clusters {(R1−NH3)4∩[Mg4(L1)6]} 5 and the mixed diammonium‐bis(p‐methoxybenzylammonium) cluster {(NH4)2(R2−NH3)2∩[Mg4(L1)6]} 6, however, were prepared by exchange of the ammonium ions of 1a by alkylammonium ions. Similarly, exchange of the four ammonium ions of 1b by alkaline metal cations afforded the tetraalkaline metal tetrahemispheraplexes {M4∩[Mg4(L2)6]} 7. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
para‐Functionalized NCN‐Pincer Palladium(II) Complexes: Synthesis, Catalysis and DFT Calculations
Authors: Dijkstra Harm P. , Slagt Martijn Q. , McDonald Aidan , Kruithof Cornelis A. , Kreiter Robert , Mills Allison M. , Lutz Martin , Spek Anthony L. , Klopper Wim , Klink Gerard P. M. van , Koten Gerard van
Published: February 2003

Several para‐substituted NCN‐pincer palladium(II) complexes (1a−g and 6a−g) {NCN = [C6H3(CH2NMe2)2‐2,6]−, para = 4‐position} have been prepared and the electronic influences of the para substituents were studied in catalysis as well as by DFT calculations (B3LYP/LANL2DZ). From DFT calculations, it was found that the para substituent exerts only a minor effect on the partial charge, investigated by means of the Mulliken population analysis, at the palladium(II) center. Also, when the para‐functionalized, cationic NCN−PdII complexes 6a−g were applied as Lewis acid catalysts in the double Michael reaction between methyl vinyl ketone and ethyl α‐cyanoacetate, only small differences in the activities of the various catalysts were observed. These results, when translated to immobilized multipincer catalysts, imply that various para functionalities can be used for immobilization of the pincer−metal complexes without affecting the catalytic activity of the individual sites. The application of a number of shape‐persistent nanosize (NCN−PdII)n complexes (7, n = 3; 8, n = 3; 9, n = 8; 10, n = 12) as homogeneous catalysts in the same Michael reaction, confirmed this expectation. For complexes 7, 8 and 9, the catalytic activity per PdII center was found to be the same as for the monopincer analogs. Only dodecakis(NCN−PdII) complex 10 showed an almost threefold enhancement in catalytic activity per PdII center, which is ascribed to the high catalyst concentration at the periphery of this material. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
Investigation of the Ligand Exchange Behavior of Square‐Planar Nickel(II) Complexes by X‐ray Absorption Spectroscopy and X‐ray Diffraction
Authors: Feth Martin P. , Klein Axel , Bertagnolli Helmut
Published: February 2003

The local structure of nickel and bromine in three square‐planar nickel(II) complexes [(2,2′‐bipyridyl)(bromo)(mesityl)nickel(II)] (1), [(bromo)(N,N‐diisopropyl‐1,2‐ethanediimine)(mesityl)nickel(II)] (2), [(bromo)(mesityl)bis(triphenylphosphane)nickel(II) (3)] and their ligand exchange behavior towards several solvents has been determined by means of X‐ray Absorption Near Edge Spectroscopy (XANES), Extended X‐ray Absorption Fine Structure Spectroscopy (EXAFS), X‐ray Diffraction (XRD), multinuclear NMR and UV/Vis spectroscopy. Several square‐planar and octahedral nickel(II) complexes were prepared and spectroscopically characterized as reference compounds. The exchange behavior strongly depends on the nature of the incoming donor ligand (solvent molecule). In some cases the substitution of one or more ligands was accompanied by conservation of the square‐planar geometry. In other solvents the geometry changed from square‐planar to octahedral. The structural information obtained is important to the further development of such nickel(II) complexes as catalysts for various applications. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
Regiospecific Cyclometalation of Diphenyl(2‐substituted phenyl)phosphane with Methyltetrakis(trimethylphosphane)cobalt(I)
Authors: Klein Hans‐Friedrich , Beck Robert , Flörke Ulrich , Haupt Hans‐Jürgen
Published: February 2003

The pre‐chelate molecules 2‐(diphenylphosphanyl)‐N,N‐dimethylaniline, [2‐(diphenylphosphanyl)benzyl]dimethylamine, 1‐(diphenylphosphanyl)‐2‐ethylbenzene, 1‐(diphenylphosphanyl)‐2‐isopropylbenzene, and 2‐(diphenylphosphanyl)benzonitrile, in a reaction with [CoMe(PMe3)4], eliminate methane to afford the selectively 6‐ortho‐metalated complexes 1−5 that contain four‐membered metallacycles. The molecular structure of 3 shows a tbp‐coordinated cobalt atom, with axial C and PMe3 donor groups. Metalation in the aliphatic side‐chain occurs with 2‐(diphenylphosphanyl)toluene, giving complex 6 that contains a five‐membered metallacycle. Benzyldiphenylphosphane is selectively ortho‐metalated in the benzyl group, affording 7. As shown by the molecular structures, complex 7 is a true ligand isomer of 6. Substitution of a trimethylphosphane group in compounds 4 and 6 by ethene gives the pentacoordinate complexes 8 and 9, respectively. The ethene ligand is π‐coordinated in the equatorial plane of a trigonal bipyramid. Under 1 bar of CO, complex 6 forms monocarbonyl complex 10. Carbonylation of complexes 3 and 4 proceeds by insertion of CO into the Co−C bond under ring expansion, affording the aroylcobalt complexes 11 and 12, respectively. Complex 6 reacts with iodomethane in an oxidative substitution reaction yielding a structurally characterized octahedral complex mer‐13, which eliminates a methyl group in THF at 20 °C to form a pentacoordinate cobalt(II) complex 14. Complex 3 oxidatively adds iodomethane in a stereoselective cis addition to give the cobalt(III) complex mer‐15, which retains its four‐membered metallacycle and the CoCH3 group. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)