From the Cover: Visually evoked activity in cortical cells imaged in freely moving animals

We describe a miniaturized head-mounted multiphoton microscope and its use for recording Ca²⁺ transients from the somata of layer 2/3 neurons in the visual cortex of awake, freely moving rats. Images contained up to 20 neurons and were stable enough to record continuously for >5 min per trial and 20 trials per imaging session, even as the animal was running at velocities of up to 0.6 m/s. Neuronal Ca²⁺ transients were readily detected, and responses to various static visual stimuli were observed during free movement on a running track. Neuronal activity was sparse and increased when the animal swept its gaze across a visual stimulus. Neurons showing preferential activation by specific stimuli were observed in freely moving animals. These results demonstrate that the multiphoton fiberscope is suitable for functional imaging in awake and freely moving animals.     

From the Cover: Optogenetic and pharmacological suppression of spatial clusters of face neurons reveal their causal role in face gender discrimination

Neurons that respond more to images of faces over nonface objects were identified in the inferior temporal (IT) cortex of primates three decades ago. Although it is hypothesized that perceptual discrimination between faces depends on the neural activity of IT subregions enriched with “face neurons,” such a causal link has not been directly established. Here, using optogenetic and pharmacological methods, we reversibly suppressed the neural activity in small subregions of IT cortex of macaque monkeys performing a facial gender-discrimination task. Each type of intervention independently demonstrated that suppression of IT subregions enriched in face neurons induced a contralateral deficit in face gender-discrimination behavior. The same neural suppression of other IT subregions produced no detectable change in behavior. These results establish a causal link between the neural activity in IT face neuron subregions and face gender-discrimination behavior. Also, the demonstration that brief neural suppression of specific spatial subregions of IT induces behavioral effects opens the door for applying the technical advantages of optogenetics to a systematic attack on the causal relationship between IT cortex and high-level visual perception.Face neurons in the inferior temporal (IT) cortex are classically defined as all neurons whose responses discriminate visual images of faces from images of nonface objects (1). By the spirit of this definition, an ideal “face neuron” would be a unit that responds to any image containing a face and does not respond to any image containing only nonface objects. Such a hypothetical face neuron could, in principle, directly support face-detection behavior (detecting images of faces with various poses, sizes, positions, and identities among other stimuli) but is not necessarily useful for other face-related behaviors such as face discrimination (distinguishing two different faces). Thus, the mechanistic relationship between “face (detector) neurons” and other face-related behaviors remains far from clear.Although previous human neuropsychology (2–4), transcranial magnetic stimulation (TMS) (5, 6), and electrophysiological (7, 8) work motivates the hypothesis that face (detector) neurons are causally involved in face discrimination, there is little direct evidence for it. In this study, we aimed to test this hypothesis for one face-discrimination task: Is face gender^* discrimination impaired by temporary silencing of IT subregions enriched with face (detector) neurons? Because distributed IT cortical populations show the computational capacity to support a wide range of invariant object discriminations (9, 10)^†, the main alternative hypothesis we considered is that discrimination of different faces (i.e., different objects) can be causally supported by IT neurons outside of the face-detector neural clusters. Beyond this specific scientific question, our study was also motivated by the larger goal of developing better tools for direct manipulation of high-level visual neural activity in primates. That is, although “correlational” analysis of patterns of neural activity can strongly infer a role for those neurons in supporting a behavior, the most direct way to test the “causal” role of the spiking activity of a subset of neurons in a given behavioral task is to directly perturb that neuronal activity and measure its effect on the behavior (11, 12).Previously, direct electrical perturbation of specific IT subregions had been used to show the causal role of face-detector neurons in face-detection behavior (12), a result that is consistent with the current operational definition of those neurons. Although anecdotal studies in humans reported perceptual distortion of faces after electrical stimulation of large parts of fusiform cortex in humans (13–15), and TMS studies revealed the impact of large-scale perturbation of functional MRI (fMRI)-defined face-selective cortical regions (16) in face recognition (5, 17), a direct causal link between spiking of IT face neurons and face-discrimination behavior has not been established.In this study, using standard electrophysiology techniques in macaque monkeys and the traditional operational definition of face (detector) neurons, we recorded extensively from central IT cortex (CIT) (18) to locate the largest known spatial cluster of face neurons (also known as middle face patch) (ref. 19; see SI Methods for more details). Then, using optogenetic tools, we directly suppressed the spiking activity of ∼1-mm (Fig. S1) subregions of IT cortex enriched with face-detector neurons as well as other nearby IT subregions and assessed the causal contribution of each subregion in face gender-discrimination behavior.In a separate set of experiments, using pharmacological intervention (muscimol microinjection), we aimed to replicate our main optogenetic findings. Although the lower spatial resolution and much lower temporal resolution of pharmacological tools does not allow fine comparison of small IT subregions (as is possible with optogenetics), its bigger spatial impact (∼3-mm diameter) was used to confirm the basic characteristics of our main finding with a well-established neural suppression method.     

From the Cover: Physical activity when young provides lifelong benefits to cortical bone size and strength in men

The skeleton shows greatest plasticity to physical activity-related mechanical loads during youth but is more at risk for failure during aging. Do the skeletal benefits of physical activity during youth persist with aging? To address this question, we used a uniquely controlled cross-sectional study design in which we compared the throwing-to-nonthrowing arm differences in humeral diaphysis bone properties in professional baseball players at different stages of their careers (n = 103) with dominant-to-nondominant arm differences in controls (n = 94). Throwing-related physical activity introduced extreme loading to the humeral diaphysis and nearly doubled its strength. Once throwing activities ceased, the cortical bone mass, area, and thickness benefits of physical activity during youth were gradually lost because of greater medullary expansion and cortical trabecularization. However, half of the bone size (total cross-sectional area) and one-third of the bone strength (polar moment of inertia) benefits of throwing-related physical activity during youth were maintained lifelong. In players who continued throwing during aging, some cortical bone mass and more strength benefits of the physical activity during youth were maintained as a result of less medullary expansion and cortical trabecularization. These data indicate that the old adage of “use it or lose it” is not entirely applicable to the skeleton and that physical activity during youth should be encouraged for lifelong bone health, with the focus being optimization of bone size and strength rather than the current paradigm of increasing mass. The data also indicate that physical activity should be encouraged during aging to reduce skeletal structural decay.Physical activity is recommended for skeletal health because bones adapt to elevated mechanical loading. However, a disparity exists between the time when the skeleton shows greatest plasticity to mechanical loads (during youth) and when it is most at risk for failure (during aging) (1, 2). Do the skeletal benefits derived from physical activity-related loading during youth persist with aging? A popular hypothesis is that physical activity increases peak bone mass to prime the skeleton against the bone loss occurring during aging (3). Prospective observational studies suggest some of the benefits in bone mass generated through physical activity during youth persist into early adulthood (4–9); however, the prospective assessment of lifelong benefits is not practically feasible. Instead, the lifelong skeletal benefits of physical activity during youth can be explored using cross-sectional studies comparing former athletes with controls. Although cross-sectional studies typically do not control for selection bias and secular variations in activity levels, current data suggest that cessation of physical activity after youth is associated with the eventual return of bone mass to control levels (10).Although the benefits in bone mass acquired through physical activity during youth may be lost, some of the benefits in bone size and strength may persist throughout life. For the purposes of the current work, “bone size” refers to total cross-sectional area, and “bone strength” refers to torsional rigidity. The torsional rigidity of a tubular bone is dependent on its polar moment of inertia, which is calculated from the radii of its outer periosteal (r_p) and inner endocortical (r_e) surfaces as π(r_p⁴ − r_e⁴)/2. This relationship demonstrates that a bone is stronger if its material is distributed further from its central axis and that periosteal surface changes have a greater influence on strength than changes on the endocortical surface. For example, assuming constant bone material properties and a typical r_p: r_e ratio of 1.8, a 5% increase in r_p (equating to 10% and 15% increases in bone size and mass, respectively) results in a 24% increase in strength. If the same mass of bone added to the periosteal surface was simultaneously removed from the endocortical surface, r_e would increase by 15%, but the bone would still be 16% stronger because of its 5% greater r_p (i.e., size). Because physical activity during youth preferentially deposits new bone on the outer periosteal surface to increase bone size (11–13), and bone loss during aging occurs primarily on the endocortical surface to decrease mass (14, 15), the benefits in bone size and strength acquired through physical activity during youth have the potential to remain independent of the maintenance of benefits in bone mass.Cross-sectional and prospective observational studies have suggested that some of the benefits in bone size and strength acquired through physical activity during youth persist into early adulthood (5, 8, 16, 17), but whether these benefits persist throughout life remains unanswered. We demonstrated that mechanical loading during a period of rapid skeletal growth conferred lifelong benefits in bone size and strength in rodent models (18, 19). To explore whether the same phenomenon occurs in humans, the current study used a uniquely controlled cross-sectional study design that compared differences in humeral diaphysis properties between the throwing and nonthrowing arms of professional Major (MLB) and Minor (MiLB) League Baseball players at different stages of their careers with the differences between the dominant and nondominant arms in age-matched controls. The use of baseball players was inspired by the bilateral asymmetry initially observed in tennis players by Jones et al. (20) and minimizes the influence of selection bias because the unilateral upper extremity loading and humeral adaptation associated with overhand throwing (21–23) enables the nonthrowing arm to serve as an internal control site for inherited and other systemic traits. Similarly, use of MLB/MiLB players reduces secular variations in physical activity levels because individuals who reached professional level baseball typically threw with high volume from a young age, with throwing being the primary unilateral dominant training modality. Also, the game of baseball has not changed in more than 100 y, and the ball weight and the distance and speed at which the ball is thrown have remained relatively constant across generations. Another distinct advantage of studying former MLB/MiLB players (compared with even tennis players) is that they often retire completely from throwing activities once they stop professional play, allowing us to explore the skeletal benefits of physical activity long after the subjects’ return to habitual loading. By comparing the differences between the throwing and nonthrowing arms in throwing athletes with the differences between the dominant and nondominant arms in age-matched controls, we isolated the skeletal benefits of throwing from the differences caused by the elevated habitual unilateral loading associated with simple arm dominance.Using professional baseball players as a model, we addressed: (i) the magnitude and location of adaptations associated with throwing-related physical activity; (ii) whether the skeletal benefits of throwing-related physical activity persist lifelong once throwing is ceased; and (iii) whether there are skeletal benefits of continued throwing-related physical activity in later adulthood.     

From The Cover: A role for Arabidopsis cryptochromes and COP1 in the regulation of stomatal opening

Cryptochromes (CRY) are blue light photoreceptors that mediate various light-induced responses in plants and animals. Arabidopsis CRY (CRY1 and CRY2) functions through negatively regulating constitutive photomorphogenic (COP) 1, a repressor of photomorphogenesis. Water evaporation and photosynthesis are regulated by the stomatal pores in plants, which are closed in darkness but open in response to blue light. There is evidence only for the phototropin blue light receptors (PHOT1 and PHOT2) in mediating blue light regulation of stomatal opening. Here, we report a previously uncharacterized role for Arabidopsis CRY and COP1 in the regulation of stomatal opening. Stomata of the cry1 cry2 double mutant showed reduced blue light response, whereas those of the CRY1-overexpressing plants showed hypersensitive response to blue light. In addition, stomata of the phot1 phot2 double mutant responded to blue light, but those of the cry1 cry2 phot1 phot2 quadruple mutant hardly responded. Strikingly, stomata of the cop1 mutant were constitutively open in darkness and stomata of the cry1 cry2 cop1 and phot1 phot2 cop1 triple mutants were open as wide as those of the cop1 single mutant under blue light. These results indicate that CRY functions additively with PHOT in mediating blue light-induced stomatal opening and that COP1 is a repressor of stomatal opening and likely acts downstream of CRY and PHOT signaling pathways.     

From the Cover: Anoctamin 1 (TMEM16A) is essential for testosterone-induced prostate hyperplasia

Benign prostatic hyperplasia (BPH) is characterized by an enlargement of the prostate, causing lower urinary tract symptoms in elderly men worldwide. However, the molecular mechanism underlying the pathogenesis of BPH is unclear. Anoctamin1 (ANO1) encodes a Ca²⁺-activated chloride channel (CaCC) that mediates various physiological functions. Here, we demonstrate that it is essential for testosterone-induced BPH. ANO1 was highly amplified in dihydrotestosterone (DHT)-treated prostate epithelial cells, whereas the selective knockdown of ANO1 inhibited DHT-induced cell proliferation. Three androgen-response elements were found in the ANO1 promoter region, which is relevant for the DHT-dependent induction of ANO1. Administration of the ANO1 blocker or Ano1 small interfering RNA, inhibited prostate enlargement and reduced histological abnormalities in vivo. We therefore concluded that ANO1 is essential for the development of prostate hyperplasia and is a potential target for the treatment of BPH.Benign prostatic hyperplasia (BPH) is characterized by the anatomical enlargement of the prostate gland and is one of the most common diseases among elderly men (1). More than 50% of men aged more than 60 suffer from lower urinary tract symptoms, including urinary hesitancy, weak stream, and nocturia, which are commonly caused by bladder obstruction (2). The availability of testosterone or dihydrotestosterone (DHT) is known to cause the development of histologically characterized BPH (3). Clinical reports on BPH have suggested a positive association between BPH and prostate cancer, with increased risk of and mortality from prostate cancer among BPH patients (4). However, some epidemiologic studies have reported that BPH is not a cause of prostate cancer (5). Despite the controversy on the association between prostate cancer and BPH, common risk factors for the two diseases include chronic inflammation, metabolic disturbance, and genetic variation (6, 7). Regardless of its association with prostate cancer, BPH is still a social issue for the elderly, but the etiologic mechanisms of its pathology remain unknown.Anoctamin1 (ANO1, also known as TMEM16A) encodes a Ca²⁺-activated chloride channel (CaCC) (8–10), and is widely expressed in secretory epithelia, including the salivary gland, trachea (11), and intestine, smooth muscle (12), and sensory neurons (13). ANO1 is known to mediate various physiological functions, such as fluid and electrolyte secretion, gut motility, vascular smooth muscle contraction, and thermal nociception (14).ANO1 has been suggested to be a regulator of cell proliferation and tumorigenesis, even before it was discovered as a CaCC, and is highly expressed in several carcinomas, including gastrointestinal stromal tumors (15), esophageal squamous cell carcinoma (16), head and neck squamous cell carcinoma (17), oral cancer (18), breast cancer (19), and prostate cancer (20). The disruption of Ano1 or the administration of a pharmacological ANO1 inhibitor impairs the proliferation of interstitial cells of Cajal (21) and numerous cancer cells (19, 20, 22). ANO1 promotes tumorigenesis and cancer progression by inducing epidermal growth factor receptor-activated mitogen-activated protein kinase (MAPK)/AKT signaling (19) and regulates tumor cell motility and metastasis via the ezrin/radixin/moesin protein family (23). Thus, ANO1 is considered to be a potential target for anticancer therapy (24).BPH and prostate cancer share common characteristics, such as testosterone-dependent growth and response to hormone therapy, which indicates a causal link between the two diseases (25). Notably, ANO1 is highly overexpressed in prostate cancer cells (20). Knockdown of Ano1 results in reduced cell proliferation and the suppression of tumor progression in the breast cancer model (19). Thus, it is conceivable that ANO1 may be involved in BPH, which may progress to prostate cancer. Therefore, this study was performed to determine whether ANO1 plays a key role in testosterone-dependent prostate hyperplasia.     

From the Cover: PNAS Plus: Genetic contributions to circadian activity rhythm and sleep pattern phenotypes in pedigrees segregating for severe bipolar disorder

Abnormalities in sleep and circadian rhythms are central features of bipolar disorder (BP), often persisting between episodes. We report here, to our knowledge, the first systematic analysis of circadian rhythm activity in pedigrees segregating severe BP (BP-I). By analyzing actigraphy data obtained from members of 26 Costa Rican and Colombian pedigrees [136 euthymic (i.e., interepisode) BP-I individuals and 422 non–BP-I relatives], we delineated 73 phenotypes, of which 49 demonstrated significant heritability and 13 showed significant trait-like association with BP-I. All BP-I–associated traits related to activity level, with BP-I individuals consistently demonstrating lower activity levels than their non–BP-I relatives. We analyzed all 49 heritable phenotypes using genetic linkage analysis, with special emphasis on phenotypes judged to have the strongest impact on the biology underlying BP. We identified a locus for interdaily stability of activity, at a threshold exceeding genome-wide significance, on chromosome 12pter, a region that also showed pleiotropic linkage to two additional activity phenotypes.Quantitative sleep and activity measures are hypothesized to be endophenotypes for bipolar disorder (BP). Disturbance of sleep and circadian activity typically precedes and may precipitate the initial onset of BP (1, 2). Decreased sleep and increased activity occur before and during manic and hypomanic episodes. Conversely, increased sleep and decreased activity characterize BP–depression. Extreme diurnal variation in mood features prominently in both mania and depression, whereas shifts in circadian phase (the time within the daily activity cycle at which periodic phenomena such as bed time or awakening occur) can induce mania and ameliorate symptoms of BP–depression (3).Twin studies have identified multiple heritable sleep and activity phenotypes, including sleep duration, sleep quality, phase of activity preference and sleep pattern, and sleep architecture variables [e.g., the amount of slow wave and rapid eye movement (REM) sleep (4) and polysomnography profiles during non-REM sleep (5)]. Euthymic BP individuals, compared with healthy controls, display trait-like alterations in several such phenotypes—for example, sleep time and time in bed, sleep onset latency, and periods of being awake after sleep onset (6). However, no prior investigations have assayed the heritability of such phenotypes in BP individuals and their relatives.We report here the delineation of sleep and activity BP endophenotypes through investigations of 26 pedigrees (n = 558) ascertained for severe BP (BP-I), from the genetically related populations of the Central Valley of Costa Rica (CR) and Antioquia, Colombia (CO) (7–9). Pedigrees ascertained for multiple cases of severe BP (BP-I) should be enriched for extreme values of quantitative traits that are BP endophenotypes, enhancing their utility for genetic mapping studies of such phenotypes. Additionally, such pedigrees derived from recently expanded founder populations are likely to show increased frequencies for many deleterious alleles—another feature that will enhance their utility for mapping these traits (10, 11). We previously described, in these pedigrees, multiple heritable and BP-associated phenotypes from the domains of temperament, neurocognition, and neuroanatomy (10).Actigraphy (activity measurement using wrist-worn accelerometers) can be conducted over prolonged periods without impinging on an individual’s usual activities, enabling assessment of sleep and activity on a scale sufficient for genetic investigations. Actigraphy data on sleep quality and duration correlate strongly with those obtained through polysomnography, the gold standard for sleep research (12). Using actigraphy, one can estimate the main circadian activity parameters, which follow a sinusoidal waveform with a ∼24-h period: phase, amplitude (the strength of circadian rhythms, as measured by the difference in the amount of activity between active and inactive moments), and coherence of the rhythm (the degree of consolidation and stability of activity, rest, and sleep). Finally, actigraphy enables quantification of BP-associated features, such as fragmentation of rest and activity within and between days, that do not fit a sinusoidal waveform and cannot be analyzed parametrically (13).We recorded activity in euthymic BP-I individuals and their non–BP-I relatives from the CR and CO pedigrees for, on average, 14 consecutive days. For each actigraphy phenotype, we evaluated association with BP-I, assessed the heritability of each trait, and performed genome-wide genetic linkage analyses on all significantly heritable traits.     

From the Cover: The extremely slow and variable activity of dihydrofolate reductase in human liver and its implications for high folic acid intake

Numerous clinical trials using folic acid for prevention of cardiovascular disease, stroke, cognitive decline, and neural tube defects have been completed or are underway. Yet, all functions of folate are performed by tetrahydrofolate and its one-carbon derivatives. Folic acid is a synthetic oxidized form not significantly found in fresh natural foods; to be used it must be converted to tetrahydrofolate by dihydrofolate reductase (DHFR). Increasing evidence suggests that this process may be slow in humans. Here we show, using a sensitive assay we developed, that the reduction of folic acid by DHFR per gram of human liver (n = 6) obtained from organ donors or directly from surgery is, on average, less than 2% of that in rat liver at physiological pH. Moreover, in contrast to rats, there was almost a 5-fold variation of DHFR activity among the human samples. This limited ability to activate the synthetic vitamer raises issues about clinical trials using high levels of folic acid. The extremely low rate of conversion of folic acid suggests that the benefit of its use in high doses will be limited by saturation of DHFR, especially in individuals possessing lower than average activity. These results are also consistent with the reports of unmetabolized folic acid in plasma and urine.     

From the Cover: A plant-specific calreticulin is a key retention factor for a defective brassinosteroid receptor in the endoplasmic reticulum

Mammalian calreticulin (CRT) is a multifunctional Ca²⁺-binding protein involved in more than 40 cellular processes in various subcellular compartments, such as Ca²⁺ storage and protein folding in the endoplasmic reticulum (ER). CRT homologues were discovered in plants almost 15 years ago, and recent studies revealed that many plant species contain 2 or more CRTs that are members of 2 distinct families, the CRT1/2 family and the plant-specific CRT3 family. However, little is known about their physiological functions. Here we report ebs2 (EMS-mutagenized bri1 suppressor 2) as an allele-specific suppressor of bri1–9, a dwarf Arabidopsis mutant caused by retention of a defective brassinosteroid receptor in the ER. EBS2 encodes the Arabidopsis CRT3 that interacts with ER-localized bri1–9 in a glycan-dependent manner. Loss-of-function ebs2 mutations compromise ER retention of bri1–9 and suppress its dwarfism, whereas EBS2 over-expression enhances its dwarf phenotype. In contrast, mutations of 2 other CRTs or their membrane-localized homologues calnexins had little effect on bri1–9. A domain-swapping experiment revealed that the positively charged C-terminal tail of CRT3 is crucial for its “bri1–9-retainer” function. Our study revealed not only a functional role for a plant-specific CRT, but also functional diversity among the 3 Arabidopsis CRT paralogues.     

From the Cover: Contactin-2/TAG-1-directed autoimmunity is identified in multiple sclerosis patients and mediates gray matter pathology in animals

Gray matter pathology is increasingly recognized as an important feature of multiple sclerosis (MS), but the nature of the immune response that targets the gray matter is poorly understood. Starting with a proteomics approach, we identified contactin-2/transiently expressed axonal glycoprotein 1 (TAG-1) as a candidate autoantigen recognized by both autoantibodies and T helper (Th) 1/Th17 T cells in MS patients. Contactin-2 and its rat homologue, TAG-1, are expressed by various neuronal populations and sequestered in the juxtaparanodal domain of myelinated axons both at the axonal and myelin sides. The pathogenic significance of these autoimmune responses was then explored in experimental autoimmune encephalitis models in the rat. Adoptive transfer of TAG-1–specific T cells induced encephalitis characterized by a preferential inflammation of gray matter of the spinal cord and cortex. Cotransfer of TAG-1–specific T cells with a myelin oligodendrocyte glycoprotein-specific mAb generated focal perivascular demyelinating lesions in the cortex and extensive demyelination in spinal cord gray and white matter. This study identifies contactin-2 as an autoantigen targeted by T cells and autoantibodies in MS. Our findings suggest that a contactin-2–specific T-cell response contributes to the development of gray matter pathology.     

基于RIP1/RIP3/NLRP3信号通路探讨祛瘀生新方治疗溃疡性结肠炎小鼠的作用机制

目的:研究祛瘀生新方对溃疡性结肠炎(UC)小鼠的治疗效果以及对RIP1/RIP3/NLRP3信号通路的调节作用.方法:将36只小鼠随机分为实验组(祛瘀生新组)、模型组、阳性对照组(美莎拉嗪组)、空白组、中药拆方1组(生新组)、中药拆方2组(祛瘀组),6只/组,采用3.5％葡聚糖硫酸钠盐(DSS)溶液自由饮水法制备UC小...     

Expression and functional characterization of the mt1 melatonin receptor from rat brain in Xenopus oocytes: evidence for coupling to the phosphoinositol pathway

Melatonin-sensitive receptors were expressed in Xenopus laevis oocytes following an injection of mRNA from rat brain. The administration of 0.1-100 micromol/L melatonin to voltage-clamped oocytes activates calcium-dependent chloride currents via a pertussis toxin-sensitive G protein and the phosphoinositol pathway. To determine which melatonin receptor type (mt1, MT2, MT3) is functionally expressed in the Xenopus oocytes, we used (i) agonists and antagonists of different receptor types to characterize the pharmacological profile of the expressed receptors and (ii) a strategy of inhibiting melatonin receptor function by antisense oligonucleotides. During pharmacological screening administration of the agonists 2-iodomelatonin and 2-iodo-N-butanoyl-5-methoxytryptamine (IbMT) to the oocytes resulted in oscillatory membrane currents, whereas the administration of the MT3 agonist 5-methoxycarbonylamino-N-acetyltryptamine (GR135,531) exerted no detectable membrane currents. The melatonin response was abolished by a preceding administration of the antagonists 2-phenylmelatonin and luzindole but was unaffected by the MT3 antagonist prazosin and the MT2 antagonist 4-phenyl-2-propionamidotetralin (4-P-PDOT). In the antisense experiments, in the control group the melatonin response occurred in 45 of 54 mRNA-injected oocytes (83%). Co-injection of the antisense oligonucleotide, corresponding to the mt1 receptor mRNA, caused a marked and significant reduction in the expression level (13%; P < 0.001). In conclusion, the results demonstrate that injection of mRNA from rat brain in Xenopus oocytes induced the expression of the mt1 receptor which is coupled to the phosphoinositol pathway.     

From the Cover: Loss of human Greatwall results in G2 arrest and multiple mitotic defects due to deregulation of the cyclin B-Cdc2/PP2A balance

Here we show that the functional human ortholog of Greatwall protein kinase (Gwl) is the microtubule-associated serine/threonine kinase-like protein, MAST-L. This kinase promotes mitotic entry and maintenance in human cells by inhibiting protein phosphatase 2A (PP2A), a phosphatase that dephosphorylates cyclin B-Cdc2 substrates. The complete depletion of Gwl by siRNA arrests human cells in G2. When the levels of this kinase are only partially depleted, however, cells enter into mitosis with multiple defects and fail to inactivate the spindle assembly checkpoint (SAC). The ability of cells to remain arrested in mitosis by the SAC appears to be directly proportional to the amount of Gwl remaining. Thus, when Gwl is only slightly reduced, cells arrest at prometaphase. More complete depletion correlates with the premature dephosphorylation of cyclin B-Cdc2 substrates, inactivation of the SAC, and subsequent exit from mitosis with severe cytokinesis defects. These phenotypes appear to be mediated by PP2A, as they could be rescued by either a double Gwl/PP2A knockdown or by the inhibition of this phos-phatase with okadaic acid. These results suggest that the balance between cyclin B-Cdc2 and PP2A must be tightly regulated for correct mitotic entry and exit and that Gwl is crucial for mediating this regulation in somatic human cells.     

HCV infection in haemodialysed patients: A role for serum IL-10 and TGF-β1 in liver damage?

BACKGROUND: Hepatitis C virus (HCV) infection is often clinically silent in haemodialysed (HD) patients and their immune response may modulate liver damage in HCV infection. IL-10 and TGF-beta1 could play a role in this setting as, IL-10 down-regulates hepatic fibrosis, while TGF-beta1 is a pro-fibrotic cytokine. AIM: To evaluate the role of IL-10 and TGF-beta1 in HD/HCV+ patients. PATIENTS: 71 HD/HCV+ patients (58 with normal [HD/HCV-N] and 13 with high serum transaminases [HD/HCV-H]), 40 non-uremic patients with chronic hepatitis C (HCV+), 56 HD anti-HCV- patients and 20 healthy volunteers (H). METHODS: IL-10 and TGF-beta1 serum levels were assessed using ELISA tests. Liver histology was assessed by Ishak's score. RESULTS: IL-10 serum levels were significantly higher in HD patients, both HCV+ (3.7+/-0.4 pg/ml; p<0.01) and HCV- (3.8+/-0.8 pg/ml; p<0.05) than in non-uremic HCV patients (2.3+/-0.4 pg/ml). Among the HD/HCV+ patients, IL-10 serum levels were similar in HD/HCV-N and in HD/HCV-H patients. Among the HD/HCV+ patients, IL-10 serum levels were similar in those with moderate histological damage compared to those with mild damage. TGF-beta1 serum levels were significantly lower in HD patients, both HCV+ (4.6+/-0.9 ng/ml) and HCV- (6.0+/-0.9 ng/ml) than in non-uremic HCV+ patients (8.1+/-1.1 ng/ml; p<0.001 and p<0.01, respectively), but similar to the values found in H (5.3+/-0.9 ng/ml; p=n.s.). No correlation was seen between IL-10 and TGF-beta1 serum levels in any of the groups considered. CONCLUSIONS: Patients on haemodialysis treatment to have high levels of IL-10, which remain high even when patients are anti-HCV+, whereas the opposite is true of TGF-beta1. The cytokine pattern observed in HD patients is compatible with the hypothesis explaining the relatively benign evolution of HCV-related liver disease in HD patients, and has a pathophysiological role.     

Targeting the PI3K/Akt signaling pathway in pancreatic β‐cells to enhance their survival and function: An emerging therapeutic strategy for type 1 diabetes

Type 1 diabetes (T1D) is an autoimmune disease caused by the destruction of the insulin‐producing β‐cells within the pancreas. Islet transplantation represents one cure; however, during islet preparation and post transplantation significant amounts of β‐cell death occur. Therefore, prevention and cure of T1D is dependent upon the preservation of β‐cell function and the prevention of β‐cell death. Phosphoinositide 3‐kinase (PI3K)/Akt signaling represents a promising therapeutic target for T1D due to its pronounced effects on cellular survival, proliferation, and metabolism. A growing amount of evidence indicates that PI3K/Akt signaling is a critical determinant of β‐cell mass and function. Modulation of the PI3K/Akt pathway, directly (via the use of highly specific protein and peptide‐based biologics, excretory/secretory products of parasitic worms, and complex constituents of plant extracts) or indirectly (through microRNA interactions) can regulate the β‐cell processes to ultimately determine the fate of β‐cell mass. An important consideration is the identification of the specific PI3K/Akt pathway modulators that enhance β‐cell function and prevent β‐cell death without inducing excessive β‐cell proliferation, which may carry carcinogenic side effects. Among potential PI3K/Akt pathway agonists, we have identified a novel parasite‐derived protein, termed FhHDM‐1 (Fasciola hepatica helminth defense molecule 1), which efficiently stimulates the PI3K/Akt pathway in β‐cells to enhance function and prevent death without concomitantly inducing proliferation unlike several other identified stimulators of PI3K/Akt signaling . As such, FhHDM‐1 will inform the design of biologics aimed at targeting the PI3K/Akt pathway to prevent/ameliorate not only T1D but also T2D, which is now widely recognized as an inflammatory disease characterized by β‐cell dysfunction and death. This review will explore the modulation of the PI3K/Akt signaling pathway as a novel strategy to enhance β‐cell function and survival.     

A functional role of mitogen-activated protein kinases, Erk1 and Erk2, in the differentiation of a human leukemia cell line, UT-7/GM: A possible key factor for cell fate determination toward erythroid and megakaryocytic lineages

The mitogen-activated protein (MAP) kinase cascade is a key regulator of mammalian cell proliferation and differentiation. In this study, we examined the roles of 2 members of the MAP kinase family, extracellular signal-regulated kinase 1 (Erk1) and Erk2, in erythropoietin (EPO)-induced erythroid differentiation and thrombopoietin (TPO)-induced megakaryocytic differentiation. UT-7/GM was used as a model system because this cell line is an erythroid/megakaryocytic bipotent cell line that can be induced to differentiate into the erythroid and megakaryocytic lineages by EPO and TPO, respectively. The kinetics of activation of Erk1 and Erk2 were examined during erythroid and megakaryocytic differentiation of UT-7/GM cells. EPO induced a transient activation of these kinases, peaking after 1 minute of stimulation and then declining quickly almost to the basal level. In contrast, TPO-induced activation of the kinases peaked at 10 minutes and persisted for up to 60 minutes, similar to the activation by granulocyte-macrophage colony-stimulating factor. The percentage of EPO-induced hemoglobin-positive cells was elevated by the addition of PD98059, a specific inhibitor of MEK1 (MAP kinase/ERK kinase 1). In contrast, PD98059 clearly reduced the amount of glycoprotein IIb/IIIa antigens induced by TPO on UT-7/GM cells. Thus, inactivation of Erk1 and Erk2 kinases promoted EPO-induced erythroid differentiation and suppressed TPO-induced megakaryocytic differentiation of UT-7/GM cells. In conclusion, the activation of Erk1 and Erk2 kinases may be a critical event in the determination of cell fate and the differentiation processes of the erythroid and megakaryocytic lineages.