bluuebunny/crossref_metadata_2025 huggingface dataset:
p35/Cyclin-Dependent Kinase 5 Phosphorylation of Ras Guanine Nucleotide Releasing Factor 2 (RasGRF2) Mediates Rac-Dependent Extracellular Signal-Regulated Kinase 1/2 Activity, Altering RasGRF2 and Microtubule-Associated Protein 1b Distribution in Neurons
Authors: Kesavapany Sashi , Amin Niranjana , Zheng Ya-Li , Nijhara Ruchika , Jaffe Howard , Sihag Ram , Gutkind J. Silvio , Takahashi Satoru , Kulkarni Ashok , Grant Philip , Pant Harish C.
Published: May 2004

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed kinase the activity of which is dependent on association with its neuron-specific activators, p35 and p39. Cdk5 activity is critical for the proper formation of cortical structures and lamination during development. In the adult nervous system, Cdk5 function is implicated in cellular adhesion, dopamine signaling, neurotransmitter release, and synaptic activity. In addition, Cdk5 is also involved in “cross-talk” with other signal transduction pathways. To further examine its involvement in cross-talk with other pathways, we identified proteins that interacted with p35 using the yeast two-hybrid system. We report here that p35 associates with Ras guanine nucleotide releasing factor 2 (RasGRF2) in coimmunoprecipitation and colocalization studies using transfected cell lines as well as primary cortical neurons. Additionally, Cdk5 phosphorylates RasGRF2 bothin vitroandin vivo, leading to a decrease in Rac–guanidine exchange factor activity and a subsequent reduction in extracellular signal-regulated kinase 1/2 activity. We show that p35/Cdk5 phosphorylates RasGRF2 on serine737, which leads to an accumulation of RasGRF2 in the neuronal cell bodies coinciding with an accumulation of microtubule-associated protein 1b. The membrane association of p35 and subsequent localization of Cdk5 activity toward RasGRF2 and Rac provide insights into important cellular signaling processes that occur at the membrane, resulting in downstream effects on signal transduction cascades.
Changes in Intracellular Chloride after Oxygen–Glucose Deprivation of the Adult Hippocampal Slice: Effect of Diazepam
Authors: Galeffi Francesca , Sah Renu , Pond Brooks B. , George Amanda , Schwartz-Bloom Rochelle D.
Published: May 2004

Ischemic injury to the CNS results in loss of ionic homeostasis and the development of neuronal death. An increase in intracellular Ca2+is well established, but there are few studies of changes in intracellular Cl–([Cl–]i) after ischemia. We used anin vitromodel of cerebral ischemia (oxygen–glucose deprivation) to examine changes in [Cl–]iand GABAAreceptor-mediated responses in hippocampal slices from adult rats. Changes in [Cl–]iwere measured in area CA1 pyramidal neurons using optical imaging of 6-methoxy-N-ethylquinolinium chloride, a Cl–-sensitive fluorescent indicator. Oxygen–glucose deprivation induced an immediate rise in [Cl–]i, which recovered within 20 min. A second and more prolonged rise in [Cl–]ioccurred within the next hour, during which postsynaptic field potentials failed to recover. The sustained increase in [Cl–]iwas not blocked by GABAAreceptor antagonists. However, oxygen–glucose deprivation caused a progressive downregulation of the K+–Cl–cotransporter (KCC2), which may have contributed to the Cl–accumulation. The rise in [Cl–]iwas accompanied by an inability of the GABAAagonist muscimol to cause Cl–influx.In vivo, diazepam is neuroprotective when given early after ischemia, although the mechanism by which this occurs is not well understood. Here, we added diazepam early after oxygen–glucose deprivation and prevented the downregulation of KCC2 and the accumulation of [Cl–]i. Consequently, both GABAAresponses and synaptic transmission within the hippocampus were restored. Thus, after oxygen–glucose deprivation, diazepam may decrease neuronal excitability, thereby reducing the energy demands of the neuron. This may prevent the activation of downstream cell death mechanisms and restore Cl–homeostasis and neuronal function.
Conditioning Injury-Induced Spinal Axon Regeneration Fails in Interleukin-6 Knock-Out Mice
Authors: Cafferty William B. J. , Gardiner Natalie J. , Das Partha , Qiu Jin , McMahon Stephen B. , Thompson Stephen W. N.
Published: May 2004

Regeneration of injured adult sensory neurons within the CNS is essentially abortive, attributable in part to lesion-induced or revealed inhibitors such as the chondroitin sulfate proteoglycans and the myelin inhibitors (Nogo-A, MAG, and OMgp). Much of this inhibition may be overcome by boosting the growth status of sensory neurons by delivering a conditioning lesion to their peripheral branches. Here, we identify a key role for the lesion-induced cytokine interleukin-6 (IL-6) in mediating conditioning lesion-induced enhanced regeneration of injured dorsal column afferents. In adult mice,conditioning injury to the sciatic nerve 1 week before bilateral dorsal column crush resulted in regeneration of dorsal column axons up to and beyond the injury site into host CNS tissue. This enhanced growth state was accompanied by an increase in the expression of the growth-associated protein GAP43 in preinjured but not intact dorsal root ganglia (DRGs). Preconditioning injury of the sciatic nerve in IL-6 –/– mice resulted in the total failure in regeneration of dorsal column axons consequent on the lack of GAP43 upregulation after a preconditioning injury. DRGs cell counts and cholera toxin β subunit labeling revealed that impaired regeneration in knock-out mice was unrelated to cell loss or a deficit in tracer transport.In vitro, exogenous IL-6 boosted sensory neuron growth status as evidenced by enhanced neurite extension. This effect required NGF or NT-3 but not soluble IL-6 receptor as cofactors. Evidence of conditioning lesion-enhanced growth status of DRGs cells can also be observedin vitroas an earlier and enhanced rate of neurite extension; this phenomenon fails in IL-6 –/– mice preinjured 7 din vivo. We suggest that injury-induced IL-6 upregulation is required to promote regeneration within the CNS. Our results indicate that this is achieved through a boosted growth state of dorsal column projecting sensory neurons.
Four Classes of Intercellular Channels between Glial Cells in the CNS
Authors: Altevogt Bruce M. , Paul David L.
Published: May 2004

Astrocytes form extensive gap junctions with other astrocytes and with oligodendrocytes. Junctional communication between CNS glia is likely of critical importance because loss of the gap junction channel-forming proteins, connexins Cx32 and Cx47, result in severe demyelination. However, CNS glia express at least six connexins, and the cellular origins and relationships of these proteins have not been determined. We produced a Cx29 reporter mouse in which the connexin coding sequence was replaced with a histological marker, which was used to demonstrate that Cx29, Cx32, and Cx47 are expressed specifically in oligodendrocytes. To determine the relationships between astrocyte and oligodendrocyte connexins, we used double- and triple-immunofluorescence microscopy using semithin sections (<1 μm) of adult mouse spinal cord. Astrocytes form two distinct classes of gap junctions with each other; those composed of Cx26 and those composed of Cx43 and Cx30. In addition, astrocytes establish two classes of intercellular channels with oligodendrocytes, heterotypic Cx26–Cx32 channels and heterotypic Cx30/Cx43–Cx47 channels that may also be heteromeric. In contrast, Cx29 does not colocalize with any of the other five connexins. The data provide the firstin vivodemonstration of heterotypic intercellular channels and reveal an unexpected complexity in the composition of glial gap junctions.
Memory Impairment in Aged Primates Is Associated with Focal Death of Cortical Neurons and Atrophy of Subcortical Neurons
Authors: Smith David E. , Rapp Peter R. , McKay Heather M. , Roberts Jeffrey A. , Tuszynski Mark H.
Published: May 2004

Mechanisms of cognitive decline with aging remain primarily unknown. We determined whether localized cell loss occurred in brain regions associated with age-related cognitive decline in primates. On a task requiring the prefrontal cortex, aged monkeys were impaired in maintaining representations in working memory. Stereological quantification in area 8A, a prefrontal region associated with working memory, demonstrated a significant 32 ± 11% reduction in the number of Nissl-stained neurons compared with young monkeys. Furthermore, the number of immunolabeled cholinergic neurons projecting to this region of cortex from the nucleus basalis was also reduced by 50 ± 6%. In contrast, neuronal number was strikingly preserved in an adjoining prefrontal cortical region also associated with working memory, area 46, and in the component of the nucleus basalis projecting to this region. These findings demonstrate extensive but highly localized loss of neocortical neurons in aged, cognitively impaired monkeys that likely contributes to cognitive decline. Cell degeneration, when present, extends transneuronally.
Mitogen- and Stress-Activated Protein Kinase 1 Mediates cAMP Response Element-Binding Protein Phosphorylation and Activation by Neurotrophins
Authors: Simon J. , Arthur C. , Fong Amy L. , Dwyer Jami M. , Davare Monika , Reese Ed , Obrietan Karl , Impey Soren
Published: May 2004

Activation of the transcription factor cAMP response element-binding protein (CREB) by neurotrophins is believed to regulate the survival, differentiation, and maturation of neurons in the CNS and PNS. Although phosphorylation of Ser133 is critical for the expression of CREB-regulated genes, the identity of neurotrophin-regulated Ser133 kinases has remained controversial. We show here that neurotrophin-induced CREB phosphorylation in CNS neurons depends exclusively on the extracellular signal-regulated kinase 1/2-activated kinase mitogen- and stress-activated protein kinase 1 (MSK1). Small interfering RNA directed against ribosomal S6 kinase 1 (RSK1) and RSK2 reduced phosphorylation of a RSK substrate but did not effect CREB-dependent transcription. However, expression of a selective inhibitory MSK1 mutant markedly attenuated BDNF-stimulated CREB phosphorylation and CREB-mediated transcription. Moreover, the ability of neurotrophins to stimulate CREB phosphorylation was abolished in CNS neurons from MSK1 knock-out mice. Consistent with a role for MSK1 in Ser133 phosphorylation, neurotrophin-induced expression of CREB-regulated genes was attenuated in MSK-deficient neurons. These results indicate that MSK1 is the major neurotrophin-activated Ser133 kinase in CNS neurons.
CD4-Positive T Cell-Mediated Neuroprotection Requires Dual Compartment Antigen Presentation
Authors: Byram Susanna C. , Carson Monica J. , DeBoy Cynthia A. , Serpe Craig J. , Sanders Virginia M. , Jones Kathryn J.
Published: May 2004

Our laboratory discovered that CD4-positive (CD4+) T cells of the immune system convey transitory neuroprotection to injured mouse facial motoneurons (FMNs) (Serpe et al., 1999, 2000, 2003). A fundamental question in the mechanisms responsible for neuroprotection concerns the identity of the cell(s) that serves as the antigen-presenting cell (APC) to activate the CD4+T cells. Here, we first establish that CD4+T cells reactive to non-CNS antigen fail to support FMN survival and, second, demonstrate a two-compartment model of CD4+T cell activation. Mouse bone marrow (BM) chimeras were developed that discriminate between resident antigen-presenting host cell and BM-derived antigen-presenting donor cell expression of major histocompatibility complex II within central and peripheral compartments, respectively. After facial nerve transection, neither compartment alone is sufficient to result in activated CD4+T cell-mediated FMN survival. Rather, CD4+T cell-mediated neuroprotection appears to depend on both resident microglial cells in the central compartment and a BM-derived APC in the peripheral compartment. This is the firstin vivoreport demonstrating a neuroprotective mechanism requiring APC functions by resident (i.e., parenchymal) microglial cells.
Expression of a Poly-Glutamine-Ataxin-3 Transgene in Orexin Neurons Induces Narcolepsy–Cataplexy in the Rat
Authors: Beuckmann Carsten T. , Sinton Christopher M. , Williams S. Clay , Richardson James A. , Hammer Robert E. , Sakurai Takeshi , Yanagisawa Masashi
Published: May 2004

The sleep disorder narcolepsy has been linked to loss of hypothalamic neurons producing the orexin (hypocretin) neuropeptides. Here, we report the generation of transgenic rats expressing a human ataxin-3 fragment with an elongated polyglutamyl stretch under control of the humanprepro-orexinpromoter (orexin/ataxin-3rats). At 17 weeks of age, the transgenic rats exhibited postnatal loss of orexin-positive neurons in the lateral hypothalamus, and orexin-containing projections were essentially undetectable. The loss of orexin production resulted in the expression of a phenotype with fragmented vigilance states, a decreased latency to rapid eye movement (REM) sleep and increased REM sleep time during the dark active phase. Wakefulness time was also reduced during the dark phase, and this effect was concentrated at the photoperiod boundaries. Direct transitions from wakefulness to REM sleep, a defining characteristic of narcolepsy, occurred frequently. Brief episodes of muscle atonia and postural collapse resembling cataplexy were also noted while rats maintained the electroencephalographic characteristics of wakefulness. These findings indicate that theorexin/ataxin-3transgenic rat could provide a useful model of human narcolepsy.
Interplay between Local GABAergic Interneurons and Relay Neurons Generates γ Oscillations in the Rat Olfactory Bulb
Authors: Lagier Samuel , Carleton Alan , Lledo Pierre-Marie
Published: May 2004

Olfactory stimuli have been known for a long time to elicit oscillations in olfactory brain areas. In the olfactory bulb (OB), odors trigger synchronous oscillatory activity that is believed to arise from the coherent and rhythmic discharges of large numbers of neurons. These oscillations are known to take part in encoding of sensory information before their transfer to higher subcortical and cortical areas. To characterize the cellular mechanisms underlying γ (30–80 Hz) local field potential (LFP) oscillations, we simultaneously recorded multiunit discharges, intracellular responses, and LFP in rat OB slices. We showed that a single and brief electrical stimulation of olfactory nerve elicited LFP oscillations in the mitral cell body layer lasting >1 sec. Both action potentials and subthreshold oscillations of mitral/tufted cells, the bulbar output neurons, were precisely synchronized with LFP oscillations. This synchronization arises from the interaction between output neurons and granule cells, the main population of local circuit inhibitory interneurons, through dendrodendritic synapses. Interestingly enough, the synchronization exerted by reciprocal synaptic interactions did not require action potentials initiated in granule cell somata. Finally, local application of a GABAAreceptor antagonist at the mitral cell and external plexiform layers confirmed the exclusive role of the granule cell reciprocal synapses in generating the evoked oscillations. We concluded that interneurons located in the granule cell layer generate synaptic activity capable of synchronizing activity of output neurons by interacting with both their subthreshold and spiking activity.
Protease-Activated Receptor 2 Sensitizes the Capsaicin Receptor Transient Receptor Potential Vanilloid Receptor 1 to Induce Hyperalgesia
Authors: Amadesi Silvia , Nie Jingjiang , Vergnolle Nathalie , Cottrell Graeme S. , Grady Eileen F. , Trevisani Marcello , Manni Chiara , Geppetti Pierangelo , McRoberts James A. , Ennes Helena , Davis John B. , Mayer Emeran A. , Bunnett Nigel W.
Published: May 2004

Inflammatory proteases (mast cell tryptase and trypsins) cleave protease-activated receptor 2 (PAR2) on spinal afferent neurons and cause persistent inflammation and hyperalgesia by unknown mechanisms. We determined whether transient receptor potential vanilloid receptor 1 (TRPV1), a cation channel activated by capsaicin, protons, and noxious heat, mediates PAR2-induced hyperalgesia. PAR2was coexpressed with TRPV1 in small- to medium-diameter neurons of the dorsal root ganglia (DRG), as determined by immunofluorescence. PAR2agonists increased intracellular [Ca2+] ([Ca2+]i) in these neurons in culture, and PAR2-responsive neurons also responded to the TRPV1 agonist capsaicin, confirming coexpression of PAR2and TRPV1. PAR2agonists potentiated capsaicin-induced increases in [Ca2+]iin TRPV1-transfected human embryonic kidney (HEK) cells and DRG neurons and potentiated capsaicin-induced currents in DRG neurons. Inhibitors of phospholipase C and protein kinase C (PKC) suppressed PAR2-induced sensitization of TRPV1-mediated changes in [Ca2+]iand TRPV1 currents. Activation of PAR2or PKC induced phosphorylation of TRPV1 in HEK cells, suggesting a direct regulation of the channel. Intraplantar injection of a PAR2agonist caused persistent thermal hyperalgesia that was prevented by antagonism or deletion of TRPV1. Coinjection of nonhyperalgesic doses of PAR2agonist and capsaicin induced hyperalgesia that was inhibited by deletion of TRPV1 or antagonism of PKC. PAR2activation also potentiated capsaicin-induced release of substance P and calcitonin gene-related peptide from superfused segments of the dorsal horn of the spinal cord, where they mediate hyperalgesia. We have identified a novel mechanism by which proteases that activate PAR2sensitize TRPV1 through PKC. Antagonism of PAR2, TRPV1, or PKC may abrogate protease-induced thermal hyperalgesia.
Boar seminal immunosuppressive fraction attenuates the leptin concentration and restores the thymus mass during pregnancy in mice
Authors: Veselský L , Holáň V , Dostál J , Železná B
Published: May 2004

The immunosuppressive fraction (ISF) of boar seminal vesicle fluid was recently demonstrated to inhibit production of T helper (Th)1 cytokines and enhance production of Th2 cytokines. The present study shows the effect of the ISF on leptin concentrations in blood plasma and adipose tissue in mice during pregnancy. The ISF effect on thymus weight during pregnancy is also demonstrated. The leptin concentration in blood plasma and adipose tissue increased and remained high in the latter half of pregnancy. ISF treatment at the beginning of pregnancy significantly lowered the leptin concentration both in blood plasma and adipose tissue of pregnant mice. Thymus involution has been described previously in pregnant mice. ISF treatment compensated for the loss of thymus mass during the whole pregnancy in the ISF-treated mice. The treatment of pregnant mice with ISF did not affect pregnancy and litter size.
Endocrine mechanisms of intrauterine programming
Authors: Fowden A L , Forhead A J
Published: May 2004

Epidemiological findings and experimental studies in animals have shown that individual tissues and whole organ systems can be programmedin uteroduring critical periods of development with adverse consequences for their function in later life. Detailed morphometric analyses of the data have shown that certain patterns of intrauterine growth, particularly growth retardation, can be related to specific postnatal outcomes. Since hormones regulate fetal growth and the development of individual fetal tissues, they have a central role in intrauterine programming. Hormones such as insulin, insulin-like growth factors, thyroxine and the glucocorticoids act as nutritional and maturational signals and adapt fetal development to prevailing intrauterine conditions, thereby maximizing the chances of survival bothin uteroand at birth. However, these adaptations may have long-term sequelae. Of the hormones known to control fetal development, it is the glucocorticoids that are most likely to cause tissue programmingin utero. They are growth inhibitory and affect the development of all the tissues and organ systems most at risk of postnatal pathophysiology when fetal growth is impaired. Their concentrationsin uteroare also elevated by all the nutritional and other challenges known to have programming effects. Glucocorticoids act at cellular and molecular levels to alter cell function by changing the expression of receptors, enzymes, ion channels and transporters. They also alter various growth factors, cytoarchitectural proteins, binding proteins and components of the intracellular signalling pathways. Glucocorticoids act, directly, on genes and, indirectly, through changes in the bioavailability of other hormones. These glucocorticoid-induced endocrine changes may be transient or persist into postnatal life with consequences for tissue growth and development both before and after birth. In the long term, prenatal glucocorticoid exposure can permanently reset endocrine systems, such as the somatotrophic and hypothalamic–pituitary–adrenal axes, which, in turn, may contribute to the pathogenesis of adult disease. Endocrine changes may, therefore, be both the cause and the consequence of intrauterine programming.
Acrosomal integrity and capacitation are not influenced by sperm cryopreservation in the giant panda
Authors: Spindler R E , Huang Y , Howard J G , Wang P , Zhang H , Zhang G , Wildt D E
Published: May 2004

Sperm cryopreservation and artificial insemination are important management tools for giant panda breeding and the preservation of extant genetic diversity. This study examined the influence of freeze–thawing on sperm function, specifically capacitation. Sperm from nine giant pandas were assessed before and after rapid (− 40 and − 100 °C/min) cryopreservation by incubation in HEPES-buffered Ham’s F10 medium with and without the capacitation accelerators, 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP). At 0, 3 and 6 h of exposure, aliquots were assessed for sperm motility traits and capacitation, defined as the proportion of sperm with intact acrosomes following exposure to solubilised zonae pellucidae (ursid or felid) or calcium ionophore subtracted from the proportion of sperm with intact acrosomes before exposure. Although mean±s.e.m.sperm motility post-thaw (56.1 ± 3.9% at 0 h) was less (P< 0.05) than pre-freeze (71.7 ± 6.0%), there was no difference (P> 0.05) in the proportion of acrosome-intact sperm (fresh, 93.0 ± 1.7% versus cryopreserved–thawed, 81.7 ± 4.7% at 0 h). Incidence of capacitation was greater (P< 0.05) in fresh sperm incubated with capacitation accelerators IBMX and dbcAMP (9 h: 50.9 ± 1.1) compared with fresh sperm incubated without accelerators (9 h: 41.2 ± 1.1%). Frozen–thawed sperm preincubated without accelerators underwent capacitation (49.6 ± 1.1%) to a greater extent (P< 0.05) compared with these fresh counterparts. Thawed samples with (9 h: 45.9 ± 1.4%) and without accelerators (9 h: 41.2 ± 1.1%) did not differ (P> 0.05) during the 9-h incubation. We conclude that giant panda spermatozoa (1) undergo capacitationin vitrowith or without chemical accelerators and (2) withstand a rapid cryopreservation protocol, including retaining normal acrosomal integrity and functional capacitation ability.
Birth of lambs of a pre-determined sex after in vitro production of embryos using frozen–thawed sex-sorted and re-frozen–thawed ram spermatozoa
Authors: Hollinshead F K , Evans G , Evans K M , Catt S L , Maxwell W M C , O'Brien J K
Published: May 2004

The characteristics and functional capacity of ram spermatozoa frozen–thawed prior to and after flow cytometric sorting was assessed after incubation (37 °C; 6 h),in vitrofertilisation (IVF), and transfer of fresh and vitrifiedin vitroproduced embryos. Frozen-thawed spermatozoa from two rams were allocated to four treatment groups: (i) non-sorted (Control); (ii) sorted (FS); (iii) sorted then re-frozen (FSF) and (iv) re-frozen control (FCF). Frozen-thawed samples were separated into X- and Y-chromosome bearing spermatozoa using a high-speed sperm sorter after density gradient centrifugation (X: 88 ± 1.5% and Y: 87 ± 1.1% purity). After 6 h incubation (37 °C), the percentage of motile spermatozoa was higher (P< 0.001) for FS (84 ± 2.0%) compared with all other treatments (Control: 36 ± 3.3%, FSF: 28 ± 3.1%, FCF: 20 ± 2.0%). In a sperm migration test greater numbers of FS spermatozoa penetrated 5 mm into the artificial cervical mucus compared with spermatozoa from all other treatments (152 ± 39.4 vs 31 ± 9.2 spermatozoa respectively;P< 0.05). Fertilisation and cleavage rates were higher (P< 0.05) forin vitromatured oocytes inseminated with Control compared with FSF spermatozoa. However, the Day 7 blastocyst development rate was higher for oocytes inseminated with FSF (62.2%) than FS and Control spermatozoa (52.7 and 50.0%;P< 0.05). The number of ewes pregnant (Day 60), lambing and thein vivoembryo survival rate was greater (P< 0.01) after the transfer of fresh embryos rather than vitrified embryos derived from X- and Y-spermatozoa (67.6, 64.7 and 41.2% vs 29.6, 25.9 and 14.8% respectively). Twenty-six of the 30 (86.7%) lambs derived from sex-sorted spermatozoa were of the correct sex. These results demonstrate that frozen–thawed ram spermatozoa can be sex-sorted for immediate or future use after re-cryopreservation and, in conjunction with IVF and embryo transfer, can be used to efficiently produce offspring of pre-determined sex.
Angiotensin II stimulates cAMP production and protein tyrosine phosphorylation in mouse spermatozoa
Authors: Mededovic Samra , Fraser Lynn R
Published: May 2004

Angiotensin II (AII), found in seminal plasma, has been shown to stimulate capacitation in uncapacitated mammalian spermatozoa. The present study investigated the location of AII receptors on spermatozoa and AII’s mechanism of action. AT1 type receptors for AII are present on the acrosomal cap region and along the whole of the flagellum of both mouse and human spermatozoa. Because combinations of low concentrations of AII and either calcitonin or fertilization-promoting peptide (FPP), both known to regulate the adenylyl cyclase (AC)/cAMP signal transduction pathway, elicited a significant response, this study investigated the hypothesis that these peptides act on the same pathway. AII was shown to significantly stimulate cAMP production in both uncapacitated and capacitated mouse spermatozoa and this was associated with increases in protein tyrosine phosphorylation. Using an anti-phosphotyrosine antibody to visualize the location of tyrosine phosphoproteins within individual cells, AII significantly stimulated phosphorylation within 20 min in both the head, especially in the acrosomal cap region, and the flagellum, especially in the principal piece, of uncapacitated mouse spermatozoa; combined AII + FPP was stimulatory within 5 min. In addition, Western blotting revealed that AII stimulation increased phosphorylation in a number of tyrosine phosphoproteins in both uncapacitated and capacitated mouse spermatozoa, with some being altered only in the latter category of cells. These results support the hypothesis that AII stimulates AC/cAMP in mammalian spermatozoa.
Insulin increases 17β-estradiol production by the dominant follicle of the first postpartum follicle wave in dairy cows
Authors: Butler S T , Pelton S H , Butler W R
Published: May 2004

Prolonged anovulation following parturition has a negative impact on fertility in dairy cows. Insulin plays an important role in ovarian function in many species, and is profoundly depressed in dairy cows during early lactation. We hypothesized that hypoinsulinemia during early lactation represents a key indicator of nutritional status, resulting in delayed ovulation. Holstein cows (n = 10) were subjected to either a hyperinsulinemic–euglycemic clamp (INS) or saline infusion (CTL) for 96 h, beginning on day 10 after parturition during the first postpartum follicular wave. Insulin was infused continuously (0.3 μg/kg body weight per h) via a jugular catheter, and euglycemia was maintained by infusion of glucose. Circulating insulin concentrations were elevated 2.6-fold in INS cows compared with CTL cows (0.73 ± 0.026 vs 0.28 ± 0.026 ng/ml; P < 0.001). Insulin treatment did not affect (P > 0.05) luteinizing hormone (LH) pulse frequency, pulse amplitude or mean circulating LH. Circulating estradiol was elevated in INS cows (P < 0.01) and circulating testosterone also tended to be higher. The ratio of testosterone to estradiol was not different between treatments for the initial 30 h of infusion, but was significantly reduced thereafter in response to insulin (P < 0.01), suggesting that hyperinsulinemia increased follicular aromatase activity. Insulin treatment also resulted in reduced circulating nonesterified fatty acids, and increased circulating total and free insulin-like growth factor-I concentrations. Insulin infusion increased estradiol secretion by the dominant follicle of the first postpartum follicular wave in dairy cows, and this effect appears not to be mediated through changes in pulsatile LH release.
Methods for quantifying follicular numbers within the mouse ovary
Authors: Myers M , Britt K L , Wreford N G M , Ebling F J P , Kerr J B
Published: May 2004

Accurate estimation of the number of ovarian follicles at various stages of development is an important indicator of the process of folliculogenesis in relation to the endocrine signals and paracrine/autocrine mechanisms that control the growth and maturation of the oocytes and their supporting follicular cells. There are 10-fold or greater differences in follicular numbers per ovary at similar ages and/or strains reported in earlier studies using various methods, leading to difficulties with interpretation of ovarian function in control vs experimental conditions. This study describes unbiased, assumption-free stereological methods for quantification of early and growing follicular numbers in the mouse ovary. A fractionator approach was used to sample a defined fraction of histological sections of adult wild-type ovaries. Primordial and primary follicles were counted independently with the optical and physical disector methods. The fractionator/disector methods, which are independent of follicular size or shape, gave estimations of 1930 ± 286 (S.E.M.) and 2227 ± 101 primordial follicles, and 137 ± 25 and 265 ± 32 primary follicles per ovary at 70 and 100 days of age respectively. From exact counts on serial sections, secondary and later follicular numbers at 100 days of age were estimated at 135 per ovary. Remnants of zona pellucidae (a marker of previous follicular atresia) were estimated using a fractionator/physical disector approach and were approximately 500 per ovary. The application of the quantitative methods described will facilitate an improved understanding of follicular dynamics and the factors that mediate their growth and maturation and allow for a better comparison between different studies.
Equine seminal plasma reduces sperm binding to polymorphonuclear neutrophils (PMNs) and improves the fertility of fresh semen inseminated into inflamed uteri
Authors: Alghamdi A S , Foster D N , Troedsson M H T
Published: May 2004

Seminal plasma (SP) is known to have immunosuppressive properties in several species. Equine SP has been reported to reduce or inhibit chemotaxis, phagocytosis and complement activityin vitro. The type and amount of the SP component that suppresses sperm–polymorphonuclear neutrophil (PMN) bindingin vitrowas determined, and the effect of such suppression on the fertility of mares inseminated in the presence of uterine inflammation, was analyzed. Sperm cells were suspended in either SP, semen extender or a mixture of both, and each was mixed with PMN-rich uterine secretions collected at 12 h after artificial insemination (AI). SP reduced binding between spermatozoa and PMNs significantly (P< 0.05). Fertile spermatozoa were suspended in SP or semen extender and used to inseminate mares 12 h after the induction of uterine inflammation. The pregnancy rate was normal (77%) when spermatozoa were suspended in SP, but was dramatically reduced to only 5% when spermatozoa were suspended in extender. The proteins from SP, blood plasma (BP) and a skim-milk-based semen extender (skim milk extender, SME) were precipitated by ammonium sulfate, resuspended in PBS and dialyzed. The effect of the precipitated proteins on sperm–PMN binding was compared with fresh, untreated SP. Both fresh SP, and isolated SP proteins reduced sperm–PMN binding (P< 0.001). Conversely, proteins isolated from either BP or SME did not reduce sperm–PMN binding. The different concentrations of SP proteins used showed a dose-dependent suppression of sperm–PMN binding. Concentrations of 1 mg/ml SP protein significantly reduced sperm–PMN binding and 6 mg/ml reduced the binding to a level similar to that observed with fresh whole SP (P< 0.001). Finally, SP protein digested with proteinase K resulted in the complete loss of SP suppressive activity confirming that the effective component is a proteinaceous substance.
Components of litter size in mice after 110 generations of selection
Authors: Holt M , Vangen O , Farstad W
Published: May 2004

The aim of the present study was to evaluate how ovulation rate and survival rate through pregnancy had been affected by more than 110 generations of upwards selection on litter size in mice. The mean number of pups born alive was 22 in the high line (selected line) and 11 in the control line (an increase in 2.6 standard deviations). Selection on litter size increased ovulation rate by 4.6 standard deviations, and it is suggested that selection also increased embryonic mortality in late pregnancy. Embryo survival from ovulation until birth was 66% in the selected line and 69% in the control line, and the observed loss in litter size from day 16 of pregnancy until birth was possibly higher in the high line compared with the control line. Selection for higher litter size has significantly increased body weight in both males and females, as the mean weight at mating for the females was 46 g in the high line and 33 g in the control line respectively.
Concepts in sperm heterogeneity, sperm selection and sperm competition as biological foundations for laboratory tests of semen quality
Authors: Holt William V , Van Look Katrien J W
Published: May 2004

Stringent selection mechanisms, in both internal and external fertilisation systems, reject all but a significant minority of the spermatozoa released at ejaculation. Sperm competition theory provides circumstantial evidence that the selection process involves mechanisms by which the quality of the fertilising spermatozoon is controlled, thereby ensuring that females and their offspring receive high quality genetic material. In this review we examine some of these selection processes to see whether they could be exploited for the improvement of laboratory tests of sperm quality. Such tests are not only required for clinical and agricultural purposes, but are increasingly needed in fields such as reproductive and environmental toxicology where the species requirement is much broader. Despite many years of research, sperm quality assessment methods continue to provide imprecise data about fertility; here we suggest that this may be a consequence of using tests that focus on the spermatozoa that would normally be unable to fertilise under natural conditions.

To achieve fertilisation a spermatozoon must be capable of responding appropriately to external signalling stimuli; those involving protein kinase-regulated flagellar function seem especially influential in governing effects ranging from non-Mendelian inheritance in mammals to sperm chemotaxis in sea urchins. Examination of the elicited responses reveals considerable heterogeneity in all species. Here we propose that this level of heterogeneity is meaningful both in terms of understanding how spermatozoa from some individuals possess fertility advantages over spermatozoa from their rivals in sperm competition, and in that the heterogeneity should be exploitable in the development of more accurate laboratory tests.
Intravascular neutrophils partially mediate the endometrial endothelial cell proliferative response to oestrogen in ovariectomised mice
Authors: Heryanto B , Girling J E , Rogers P A W
Published: May 2004

The aim of this study was to investigate the role of intravascular neutrophils in initiating endothelial cell proliferation following oestrogen treatment in ovariectomised mouse endometrium. Uterine tissues were collected from ovariectomised C57/CBA female mice 24 h after oestrogen treatment with or without systemic neutrophil depletion. Neutropenia was achieved with either an in-house anti-neutrophil serum (ANS) or Gr-1 monoclonal antibody. All mice received an i.p. injection of bromodeoxyuridine (BrdU) 4 h prior to dissection to allow visualisation of proliferating cells using immunocytochemistry. Endometrial sections were immunostained for BrdU, vascular endothelial growth factor (VEGF), and neutrophils (using ANS). Oestrogen treatment of ovariectomised mice significantly increased the number of intravascular neutrophils, whereas induction of neutropenia with either ANS or Gr-1 in conjunction with oestrogen treatment prevented this increase. Oestrogen treatment of ovariectomised mice also significantly increased the number of intravascular VEGF-positive cells; however, whereas induction of neutropenia with ANS significantly reduced this increase, Gr-1 did not. In both studies, neutropenia significantly reduced, but did not eliminate, the amount of endometrial endothelial cell proliferation. These results suggest a role for neutrophils in endometrial angiogenesis following acute oestrogen treatment; however, the presence of VEGF-positive cells even after induction of neutropenia suggests that more than one type of leukocyte may be involved.
Association between ovarian follicle development and pregnancy rates in dairy cows undergoing spontaneous oestrous cycles
Authors: Bleach Emma C L , Glencross Richard G , Knight Philip G
Published: May 2004

Ovarian follicle development continues in a wave-like manner during the bovine oestrous cycle giving rise to variation in the duration of ovulatory follicle development. The objectives of the present study were to determine whether a relationship exists between the duration of ovulatory follicle development and pregnancy rates following artificial insemination (AI) in dairy cows undergoing spontaneous oestrous cycles, and to identify factors influencing follicle turnover and pregnancy rate and the relationship between these two variables. Follicle development was monitored by daily transrectal ultrasonography from 10 days after oestrus until the subsequent oestrus in 158 lactating dairy cows. The cows were artificially inseminated following the second observed oestrus and pregnancy was diagnosed 35 days later. The predominant pattern of follicle development was two follicle waves (74.7%) with three follicle waves in 22.1% of oestrous cycles and four or more follicle waves in 3.2% of oestrous cycles. The interval from ovulatory follicle emergence to oestrus (EOI) was 3 days longer (P < 0.0001) in cows with two follicle waves than in those with three waves. Ovulatory follicles from two-wave oestrous cycles grew more slowly but were approximately 2 mm larger (P < 0.0001) on the day of oestrus.

Twin ovulations were observed in 14.2% of oestrous cycles and occurred more frequently (P < 0.001) in three-wave oestrous cycles; consequently EOI was shorter in cows with twin ovulations. Overall, 57.0% of the cows were diagnosed pregnant 35 days after AI. Linear logistic regression analysis revealed an inverse relationship between EOI and the proportion of cows diagnosed pregnant, among all cows (n = 158; P < 0.01) and amongst those with single ovulations (n = 145; P < 0.05). Mean EOI was approximately 1 day shorter (P < 0.01) in cows that became pregnant than in non-pregnant cows; however, pregnancy rates did not differ significantly among cows with different patterns of follicle development. These findings confirm and extend previous observations in pharmacologically manipulated cattle and show, for the first time, that in dairy cows undergoing spontaneous oestrous cycles, natural variation in the duration of post-emergence ovulatory follicle development has a significant effect on pregnancy rate, presumably reflecting variation in oocyte developmental competence.
A 24‐Hour Comparison of Serum Growth Hormone Concentrations in Patients with Heart Failure versus Healthy Controls
Authors: Duncan Brett , Moyna Niall M. , Heller Gary V. , McGill Carol , Katten Deborah , Finta Laurie , Velusamy Muthu , Kelsey Anita , Wieczorek Stacey , Wu Alan H. B. , White C. Michael
Published: May 2004

Study Objective. To compare endogenous serum growth hormone concentrations over a 24‐hour period in patients with chronic heart failure (CHF) and matched controls.

Design. Prospective, 24‐hour, endogenous concentration comparison.

Setting. Hospital research center.

Patients. Eight evaluable patients with nonischemic dilated cardiomyopathy and 10 healthy control subjects, matched for age and sex.

Intervention. Over a 24‐hour period, blood was drawn from the study participants every 20 minutes for determination of growth hormone.

Measurements and Main Results. For each patient, the area under the concentration‐time curve from time 0–24 hours (AUC0–24), maximum concentration (Cmax), and minimum concentration (Cnadir) of growth hormone were determined. The AUC0–24 and Cmax were 74% (p<0.05) and 62% (p<0.05) lower in patients with CHF than in controls, respectively. The Cnadir for all participants was 0 μg/L. Variability in growth hormone concentrations over the 24 hours was considerable for all study participants.

Conclusions. Growth hormone concentrations are suppressed over a 24‐hour period in patients with CHF versus healthy controls. Variability in levels throughout the day suggests that a single point evaluation cannot be used to determine deficiency or abundance of growth hormone.
Pharmacologic Management by Clinical Pharmacists of Behavioral and Psychological Symptoms of Dementia in Nursing Home Residents: Results from a Pilot Study
Authors: Rojas‐Fernandez Carlos H. , Eng Marty , Allie Nicole D.
Published: May 2004

A pharmacist‐based consulting service was developed for the pharmacologic management of behavioral and psychological symptoms of dementia (BPSD) in a nursing home setting. Patients were evaluated using the Behavioral Pathology in Alzheimer's Disease (BEHAVE‐AD) rating scale, and pharmacotherapy was selected using a structured approach. Eleven patients were evaluated and treated with various psychotropic drugs. The most commonly administered drug was trazodone at a mean dosage of 70 mg/day (range 50–100 mg/day). Nine of the patients demonstrated satisfactory treatment responses as shown by a decreased BEHAVE‐AD score of 30% or more (average BEHAVE‐AD scores at baseline and 1 month after treatment were 13 ± 4 and 4 ± 3, respectively), and no clinical side effects were observed. The service was well received by the facility staff and primary care providers. These preliminary results suggest that pharmacists can play an important role in the pharmacotherapy of BPSD with positive clinical outcomes.
Drug Record Discrepancies in an Outpatient Electronic Medical Record: Frequency, Type, and Potential Impact on Patient Care at a Hemodialysis Center
Authors: Manley Harold J. , Drayer Debra K. , McClaran Marcy , Bender Walter , Muther Richard S.
Published: May 2004

Background. Patients who require hemodialysis take many drugs. Electronic drug records may be discrepant with what patients are actually taking. Record discrepancies are a potential source of drug‐related problems. We sought to determine the extent to which drug record discrepancies occur in a hemodialysis population.

Methods. This was a prospective observational study of patients enrolled in a pharmacist clinic at an outpatient hemodialysis center from August‐December 2001. Patients participated in monthly drug interviews conducted by a pharmacist, during which patient drug use was determined. Data collected consisted of patient demographics, drug type, and number of drugs. Drug record discrepancies were classified and assigned a potential drug‐related problem. Results were compared with the electronic drug record. Patients with documented drug record discrepancies were compared with those patients for whom no discrepancy was identified.

Results. Over the 5‐month period, 215 drug interviews were conducted in 63 patients. One hundred thirteen drug record discrepancies were identified in 38 patients (60%). Discrepancies (mean ± SD 1.7 ± 1.3, range 1–7) were identified during 65 drug interviews (30.2%). Electronic drug records were discrepant by one drug record, two drug records, and more than two drug records 60.0%, 26.2%, and 13.8% of the time, respectively. Drug record discrepancies placed patients at risk for adverse drug events and dosing errors in 49.6% and 34.5%, respectively, of 113 discrepancies. Patient age negatively correlated with the number of drug record discrepancies identified (r=−0.27, p=0.04).

Conclusions. Drug record discrepancies occur frequently among patients undergoing hemodialysis. Incorporation of a pharmacist into the patient care team may increase the accuracy of the electronic drug record and avert unnecessary drug‐related problems.
Impact of Hospitalization on Blood Pressure Control in Italy: Results from the Italian Group of Pharmacoepidemiology in the Elderly (GIFA)
Authors: Onder Graziano , Gambassi Giovanni , Sgadari Antonio , Williamson Jeff D. , Cesari Matteo , Landi Francesco , Bernabei Roberto , Pahor Marco
Published: May 2004

Study Objectives. To evaluate whether hospitalization affects blood pressure control in hypertensive patients, and to identify factors associated with attainment of adequate blood pressure control and with aggressive pharmacologic treatment.

Design. Retrospective study.

Setting. Eighty‐one hospitals throughout Italy.

Patients. A total of 3304 patients (59% women, 41% men) with a diagnosis of hypertension and uncontrolled blood pressure values at hospital admission.

Measurements and Main Results. Patients' blood pressures were surveyed during study periods from 1988–1997. Controlled blood pressure was defined according to the sixth report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (systolic < 140 mm Hg, diastolic < 90 mm Hg). Aggressive pharmacologic treatment was defined as an increase in the number of antihypertensive drugs that the patient was receiving at hospital discharge compared with the number of drugs on admission. Adequate blood pressure control was achieved in only 767 (23.2%) patients, but the proportion increased in each subsequent survey. Age (odds ratio [OR] for 10‐year increase 0.82, 95% confidence interval [CI] 0.76–0.88) and male sex (OR 0.79, 95% CI 0.65–0.94) were associated with reduced likelihood of achieving adequate blood pressure control. In contrast, coronary heart disease (OR 1.38, 95% CI 1.14–1.66), year of survey (1995 vs 1988: OR 1.47, 95% CI 1.19–1.82; 1997 vs 1988: OR 1.27, 95% CI 1.02–1.42), length of hospital stay (medium vs lower tertile OR 1.20, 95% CI 0.96–1.49; higher vs lower tertile OR 1.42, 95% CI 1.13–1.77), and increase in number of antihypertensive drugs prescribed (OR 1.21, 95% CI 1.02–1.42 for one drug increase) were associated with improved blood pressure control. In 1753 (53.1%) patients, the number of antihypertensive drugs increased during their hospital stay. Younger age, fewer drugs on admission, lower comorbidity index, diagnosis of chronic heart failure, lengthy hospital stay, and increasing baseline values of systolic and diastolic blood pressure were associated with aggressive pharmacologic treatment.

Conclusion. Adequate blood pressure control was achieved in only 25% of patients with hypertension despite a trend toward improvement in recent years. Advanced age was one of the main factors associated with less aggressive pharmacologic treatment and with inadequate blood pressure control.