Intermittency properties in a temporal lobe epilepsy model.

Neuronal synchronization is important for communication between brain regions and plays a key role in learning. However, changes in connectivity can lead to hyper-synchronized states related to epileptic seizures that occur intermittently with asynchronous states. The activity-regulated cytoskeleton-associated protein (ARC) is related to synaptic alterations which can lead to epilepsy. Induction of status epilepticus in rodent models causes the appearance of intense ARC immunoreactive neurons (IAINs), which present a higher number of connections and conductance intensity than non-IAINs. This alteration might contribute to abnormal epileptic seizure activity. In this work, we investigated how IAINs connectivity influences the firing pattern and synchronization in neural networks. Firstly, we showed the appearance of synchronized burst patterns due to the emergence of IAINs. Second, we described how the increase of IAINs connectivity favors the appearance of intermittent up and down activities associated with synchronous bursts and asynchronous spikes, respectively. Once the intermittent activity was properly characterized, we applied the optogenetics control of the high synchronous activities in the intermittent regime. To do this, we considered that 1% of neurons were transfected and became photosensitive. We observed that optogenetics methods to control synchronized burst patterns are effective when IAINs are chosen as photosensitive, but not effective in non-IAINs. Therefore, our analyses suggest that IAINs play a pivotal role in both the generation and suppression of highly synchronized activities.

Optimisation of Radiation Therapy in Bladder Preservation Therapy for Patients With Clinical Stage T2N0M0 Bladder Cancer.

AIMS: A novel bladder preservation therapy, the OMC (Osaka Medical College) regimen, which combines radiation therapy with balloon-occluded arterial infusion of anticancer agents, is a treatment option for patients with muscle-invasive bladder cancer (MIBC). We retrospectively analysed the effects of changes in radiation dose and irradiation field on treatment efficacy and adverse events.The purpose of this study is to use the results of this study to help determine a course of radiation therapy for bladder preservation therapy of cT2N0M0 MIBC. MATERIALS AND METHODS: We examined 352 patients with clinical stage T2N0M0 (cT2N0M0) MIBC classified into the following groups based on the irradiation method: group A, the whole pelvis (50 Gy/25 fractions) + local bladder (10 Gy/5 fractions); group B, the small pelvis (50 Gy/25 fractions) + local bladder (10 Gy/5 fractions); group C, the whole pelvis (40 Gy/20 fractions) + local bladder (10 Gy/5 fractions). RESULTS: The complete response rate, 3-year overall survival and progression-free survival rates in group A were 92.9%, 94.9% and 82.1%, respectively; in group B were 87.2%, 86.7% and 76.7%, respectively; and in group C were 95.2%, 92.6% and 71.1%, respectively. No significant differences between the groups were noted. The incidence of ≥grade 3 urinary tract and gastrointestinal toxicities were not significantly different among the groups (group A: 7.8%, 1.7%; B, 11.1%, 0%; C, 7.1%, 1.8%, respectively). The 3-year progression-free rates of the common iliac lymph node (CILN) region in patients who received whole-pelvis and small-pelvis irradiation were 99.0 and 89.0% (P < 0.01), respectively, with the latter group having significantly high lymph node recurrence in the CILN region. CONCLUSIONS: Our findings showed that the optimal radiation therapy for patients with cT2N0M0 MIBC undergoing the OMC regimen is whole-pelvis irradiation including the CILN region, with a total dose of 50 Gy/25 fractions.

Decoding cell signalling and regulation of oligodendrocyte differentiation.

Oligodendrocytes are fundamental for the functioning of the nervous system; they participate in several cellular processes, including axonal myelination and metabolic maintenance for astrocytes and neurons. In the mammalian nervous system, they are produced through waves of proliferation and differentiation, which occur during embryogenesis. However, oligodendrocytes and their precursors continue to be generated during adulthood from specific niches of stem cells that were not recruited during development. Deficiencies in the formation and maturation of these cells can generate pathologies mainly related to myelination. Understanding the mechanisms involved in oligodendrocyte development, from the precursor to mature cell level, will allow inferring therapies and treatments for associated pathologies and disorders. Such mechanisms include cell signalling pathways that involve many growth factors, small metabolic molecules, non-coding RNAs, and transcription factors, as well as specific elements of the extracellular matrix, which act in a coordinated temporal and spatial manner according to a given stimulus. Deciphering those aspects will allow researchers to replicate them in vitro in a controlled environment and thus mimic oligodendrocyte maturation to understand the role of oligodendrocytes in myelination in pathologies and normal conditions. In this study, we review these aspects, based on the most recent in vivo and in vitro data on oligodendrocyte generation and differentiation.

Inter-epidemic Rift Valley fever virus seroconversions in an irrigation scheme in Bura, south-east Kenya.

Rift Valley fever (RVF) is an acute mosquito-borne viral zoonosis whose outbreaks are often associated with prolonged rainfall and flooding, during which large numbers of vectors emerge. Recent studies into the inter-epidemic maintenance of RVF virus (RVFV) suggest that both vertical transmission in vectors and direct transmission between hosts act in combination with predisposing factors for persistence of the virus. A comparative longitudinal survey was carried out in Tana River County, Kenya, in irrigated, riverine and pastoral ecosystems from September 2014-June 2015. The objectives were to investigate the possibility of low-level RVFV transmission in these ecosystems during an inter-epidemic period (IEP), examine variations in RVFV seroprevalence in sheep and goats and determine the risk factors for transmission. Three hundred and sixteen small ruminants were selected and tested for immunoglobulin G antibodies against RVFV nucleoprotein using a competitive ELISA during six visits. Data on potential risk factors were also captured. Inter-epidemic RVFV transmission was evidenced by 15 seroconversions within the irrigated and riverine villages. The number of seroconversions was not significantly different (OR = 0.66, CI = 0.19-2.17, p = .59) between irrigated and riverine areas. No seroconversions were detected in the pastoral ecosystem. This study highlights the increased risk of inter-epidemic RVFV transmission posed by irrigation, through provision of necessary environmental conditions that enable vectors access to more breeding grounds, resting places and shade, which favour their breeding and survival.

Gender differences in treatment outcomes among 15-49 year olds with smear-positive pulmonary tuberculosis in Kenya.

OBJECTIVE: To determine gender differences in treatment outcomes among 15-49 year olds with smear-positive pulmonary tuberculosis (PTB) and factors associated with poor outcomes in Kenya. DESIGN: Retrospective descriptive cohort. RESULTS: Of 16 056 subjects analysed, 38% were female and 62% male. Females had a higher risk of poor treatment outcome than males (12% vs. 10%, P < 0.001; adjusted OR 1.29, 95%CI 1.16-1.44, P < 0.001). In the first multivariate model, restricting the analysis to human immunodeficiency virus (HIV) positive patients and adjusting for risk factors and clustering, females had a non-significantly lower risk of poor outcome (OR 0.99, 95%CI 0.86-1.13, P = 0.844). In the model restricted to HIV-negative patients, a non-significantly lower risk was found (OR 0.89, 95%CI 0.73-1.09, P = 0.267). In the second model, restricting analysis to patients on antiretroviral therapy (ART) and adjusting for risk factors and clustering, females had a non-significantly lower risk of poor PTB treatment outcomes (OR 0.98, 95%CI 0.84-1.14, P = 0.792). In the model restricted to HIV-positive patients not on ART, a non-significantly higher risk was found (OR 1.15, 95%CI 0.79-1.67, P = 0.461). CONCLUSION: Females of reproductive age are likely to have poorer treatment outcomes than males. Among females, not commencing ART during anti-tuberculosis treatment seemed to be associated with poor outcomes.

Antenatal care and pregnancy outcomes in a safe motherhood health voucher system in rural Kenya, 2007-2013.

SETTING: A rural private health facility, Ruby Medical Centre (RMC), participating in a safe motherhood health voucher system for poor women in Kiambu County, Kenya. OBJECTIVES: Between 2007 and 2013, to determine 1) the number of women who delivered at the RMC, their characteristics and pregnancy-related outcomes, and 2) the number of women who received an incomplete antenatal care (ANC) package and associated factors. DESIGN: Retrospective cross-sectional study using routine programme data. RESULTS: During the study period, 2635 women delivered at the RMC: 50% were aged 16-24 years, 60% transferred in from other facilities and 59% started ANC in the third trimester of pregnancy. Of the 2635 women, 1793 (68%) received an incomplete ANC package: 347 (13%) missed essential blood tests, 312 (12%) missed the tetanus toxoid immunisation and 1672 (65%) had fewer than four visits. Presenting late and starting ANC elsewhere were associated with an incomplete package. One pregnancy-related mortality occurred; the stillbirth rate was 10 per 1000 births. CONCLUSION: This first assessment of the health voucher system in rural Kenya showed problems in ANC quality. Despite favourable pregnancy-related outcomes, increased efforts should be made to ensure earlier presentation of pregnant women, comprehensive ANC, and more consistent and accurate monitoring of reproductive indicators and interventions.

Contexte : Une structure de santé privée rurale, le Ruby Medical Centre (RMC), participant à un système de bons de traitement de Maternité sans risques destiné à des femmes pauvres du conté de Kiambu au Kenya.Objectifs : Entre 2007 et 2013, déterminer 1) le nombre de femmes qui ont accouché au RMC, leurs caractéristiques et le devenir de leur grossesse, et 2) le nombre ne bénéficiant que d'un paquet de soins anténataux (ANC) incomplets et les facteurs associés.Schéma : Etude rétrospective transversale basée sur les données recueillies en routine dans les programmes.Résultats : Au cours de la période d'étude, 2635 femmes ont accouché au RMC : 50% étaient âgées de 16 à 24 ans, 60% avaient été transférées d'autres structures et 59% avaient débuté les ANC au cours du 3e trimestre. De ces 2635 femmes, 1793 (68%) avaient un paquet d'ANC incomplet : 347 (13%) ont manqué les principaux tests sanguins, 312 (12%) n'ont pas eu de vaccination anti-tétanique et 1672 (65%) ont eu moins de quatre consultations. Un démarrage tardif et des ANC débutés ailleurs étaient associés à un paquet d'ANC incomplet. Un décès lié à la grossesse est survenu et le taux de mortinatalité a été de 10/1000 naissances.Conclusion : Cette première évaluation du système de bons de traitement dans les zones rurales du Kenya a mis en évidence des problèmes de qualité des ANC. En dépit de l'évolution favorable des grossesses, il est nécessaire d'accroitre les efforts pour faire venir les femmes enceintes plus tôt, offrir des ANC complets et un suivi plus cohérent et précis des indicateurs et des interventions de santé reproductive.

Marco de referencia: El Ruby Medical Centre (RMC) es un centro de atención de salud privado en zona rural, que participa en el sistema de cupones por una maternidad sin riesgo en el condado de Kiambu, en Kenia.Objetivos: Determinar entre el 2007 y el 2013: 1) la cantidad de mujeres cuyo parto se atendió en el RMC, las características de las mujeres y los desenlaces relacionados con el embarazo; y 2) el número de mujeres que recibieron una atención prenatal (ANC) incompleta y los factores asociados con esta situación.Métodos: Fue este un estudio transversal retrospectivo a partir de los datos del programa corriente.Resultados: Durante el período del estudio, se atendió el parto de 2635 mujeres en el RMC, el 50% de las cuales tenía entre 16 y 24 años de edad, el 60% acudió como remisión de otros centros de atención y el 59% había comenzado la ANC durante el tercer trimestre del embarazo. De las 2635 mujeres, 1793 recibieron una ANC incompleta (68%) a saber: en 347 no se practicaron los principales exámenes sanguíneos (13%); 312 no recibieron la vacuna con el toxoide antitetánico (12%); y 1672 acudieron a menos de cuatro citas de control (65%). Los factores asociados con una ANC incompleta fueron una presentación tardía al programa y el inicio de la ANC en un centro diferente. Se presentó un caso de mortalidad relacionada con el embarazo y la tasa de mortinatalidad fue de 10 por 1000 nacimientos.Conclusión: El presente estudio es la primera evaluación del sistema de cupones por una maternidad sin riesgo en la zona rural de Kenia y puso en evidencia problemas en materia de calidad de la ANC. Pese a los desenlaces favorables del embarazo, se precisan iniciativas que fomenten una presentación más temprana de las embarazadas al programa, la ANC integral, y una vigilancia más regular y exacta de los indicadores y las intervenciones en materia de salud reproductiva.

Neonatal anoxia in rats: hippocampal cellular and subcellular changes related to cell death and spatial memory.

Neonatal anoxia in rodents has been used to understand brain changes and cognitive dysfunction following asphyxia. This study investigated the time-course of cellular and subcellular changes and hippocampal cell death in a non-invasive model of anoxia in neonatal rats, using Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling (TUNEL) to reveal DNA fragmentation, Fluoro-Jade® B (FJB) to show degenerating neurons, cleaved caspase-3 immunohistochemistry (IHC) to detect cells undergoing apoptosis, and transmission electron microscopy (TEM) to reveal fine ultrastructural changes related to cell death. Anoxia was induced by exposing postnatal day 1 (P1) pups to a flow of 100% gaseous nitrogen for 25 min in a chamber maintained at 37 °C. Control rats were similarly exposed to this chamber but with air flow instead of nitrogen. Brain changes following anoxia were evaluated at postnatal days 2, 14, 21 and 60 (P2, P14, P21 and P60). In addition, spatial reference memory following anoxia and control treatments was evaluated in the Morris water maze, starting at P60. Compared to their respective controls, P2 anoxic rats exhibited (1) higher TUNEL labeling in cornus ammonis (CA) 1 and the dentate gyrus (DG), (2) higher FJB-positive cells in the CA2-3, and (3) somato-dendritic swelling, mitochondrial injury and chromatin condensation in irregular bodies, as well as other subcellular features indicating apoptosis, necrosis, autophagy and excitotoxicity in the CA1, CA2-3 and DG, as revealed by TEM. At P14, P21 and P60, both groups showed small numbers of TUNEL-positive and FJB-positive cells. Stereological analysis at P2, P14, P21 and P60 revealed a lack of significant differences in cleaved caspase-3 IHC between anoxic and control subjects. These results suggest that the type of hippocampal cell death following neonatal anoxia is likely independent of caspase-3 activation. Neonatal anoxia induced deficits in acquisition and performance of spatial reference memory in the Morris water maze task. Compared to control subjects, anoxic animals exhibited increased latencies and path lengths to reach the platform, as well as decreased searching specifically for the platform location. In contrast, no significant differences were observed for swimming speeds and frequency within the target quadrant. Together, these behavioral results indicate that the poorer performance by anoxic subjects is related to spatial memory deficits and not to sensory or motor deficits. Therefore, this model of neonatal anoxia in rats induces hippocampal changes that result in cell losses and impaired hippocampal function, and these changes are likely related to spatial memory deficits in adulthood.

Screening for tuberculosis in pregnancy: do we need more than a symptom screen? Experience from western Kenya.

OBJECTIVES: 1) To explore the utility of tuberculosis (TB) symptom screening for symptoms of ≥2 weeks' duration in a routine setting, and 2) to compare differences in TB diagnosis between human immunodeficiency virus (HIV) infected and non-HIV-infected pregnant women in western Kenya. DESIGN: Comparative cross-sectional study among pregnant women with known HIV status screened for TB from 2010 to 2012, in Eldoret, western Kenya. RESULTS: Of 2983 participants, respectively 34 (1%), 1488 (50.5%) and 1461 (49.5%) had unknown, positive and negative HIV status. The median age was respectively 30 years (interquartile range [IQR] 26-35) and 26 years (IQR 24-31) in HIV-infected and non-infected participants. A positive symptom screen was found in respectively 8% (119/1488) and 5% (67/1461) of the HIV-infected and non-infected women. The median CD4 count at enrolment was 377 cells/µl (IQR 244-530) for HIV-infected women. One non-HIV-infected patient was sputum-positive. For HIV-infected women, TB was presumptively treated in 1% (16/1488) based on clinical symptoms and chest X-ray. Cumulatively, anti-tuberculosis treatment was offered to 0.6% (17/2949) of the participants. CONCLUSION: This study does not seem to demonstrate the utility of TB symptom screening questionnaires in a routine setting among pregnant women, either HIV-infected or non-infected, in western Kenya.

Influence of spontaneous calcium events on cell-cycle progression in embryonal carcinoma and adult stem cells.

Spontaneous Ca(2+) events have been observed in diverse stem cell lines, including carcinoma and mesenchymal stem cells. Interestingly, during cell cycle progression, cells exhibit Ca(2+) transients during the G(1) to S transition, suggesting that these oscillations may play a role in cell cycle progression. We aimed to study the influence of promoting and blocking calcium oscillations in cell proliferation and cell cycle progression, both in neural progenitor and undifferentiated cells. We also identified which calcium stores are required for maintaining these oscillations. Both in neural progenitor and undifferentiated cells calcium oscillations were restricted to the G1/S transition, suggesting a role for these events in progression of the cell cycle. Maintenance of the oscillations required calcium influx only through inositol 1,4,5-triphosphate receptors (IP(3)Rs) and L-type channels in undifferentiated cells, while neural progenitor cells also utilized ryanodine-sensitive stores. Interestingly, promoting calcium oscillations through IP(3)R agonists increased both proliferation and levels of cell cycle regulators such as cyclins A and E. Conversely, blocking calcium events with IP(3)R antagonists had the opposite effect in both undifferentiated and neural progenitor cells. This suggests that calcium events created by IP(3)Rs may be involved in cell cycle progression and proliferation, possibly due to regulation of cyclin levels, both in undifferentiated cells and in neural progenitor cells.

Connexin36, an essential element in the rod pathway, is highly expressed in the essentially rodless retina of Gallus gallus.

Electrical coupling provided by connexins (Cx) in gap junctions (GJ) plays important roles in both the developing and the mature retina. In mammalian nocturnal species, Cx36 is an essential component in the rod pathway, the retinal circuit specialized for night, scotopic vision. Here, we report the expression of Cx36 in a species (Gallus gallus) that phylogenetic development endows with an essentially rodless retina. Cx36 gene is very highly expressed in comparison with other Cxs previously described in the adult retina, such as Cx43, Cx45, and Cx50. Moreover, real-time PCR, Western blot, and immunofluorescence all revealed that Cx36 expression massively increased over time during development. We thoroughly examined Cx36 in the inner and outer plexiform layers, where this protein was particularly abundant. Cx36 was observed mainly in the off sublamina of the inner plexiform layer rather than in the on sublamina previously described in the mammalian retina. In addition, Cx36 colocalized with specific cell markers, revealing the expression of this protein in distinct amacrine cells. To investigate further the involvement of Cx36 in visual processing, we examined its functional regulation in retinas from dark-adapted animals. Light deprivation markedly up-regulates Cx36 gene expression in the retina, resulting in an increased accumulation of the protein within and between cone synaptic terminals. In summary, the developmental regulation of Cx36 expression results in particular circuitry-related roles in the chick retina. Moreover, this study demonstrated that Cx36 onto- and phylogenesis in the vertebrate retina simultaneously exhibit similarities and particularities.

Differential expression of connexins during histogenesis of the chick retina.

Gap junction (GJ) channels couple adjacent cells, allowing transfer of second messengers, ions, and molecules up to 1 kDa. These channels are composed by a multigene family of integral membrane proteins called connexins (Cx). In the retina, besides being essential circuit element in the visual processing, GJ channels also play important roles during its development. Herein, we analyzed Cx43, Cx45, Cx50, and Cx56 expression during chick retinal histogenesis. Cx exhibited distinct expression profiles during retinal development, except for Cx56, whose expression was not detected. Cx43 immunolabeling was observed at early development, in the transition of ventricular zone and pigmented epithelium. Later, Cx43 was seen in the outer plexiform and ganglion cell layers, and afterwards also in the inner plexiform layer. We observed remarkable changes in the phosphorylation status of this protein, which indicated modifications in functional properties of this Cx during retinal histogenesis. By contrast, Cx45 showed stable gene expression levels throughout development and ubiquitous immunoreactivity in progenitor cells. From later embryonic development, Cx45 was mainly observed in the inner retina, and it was expressed by glial cells and neurons. In turn, Cx50 was virtually absent in the chick retina at initial embryonic phases. Combination of PCR, immunohistochemistry and Western blot indicated that this Cx was present in differentiated cells, arising in parallel with the formation of the visual circuitry. Characterization of Cx expression in the developing chick retina indicated particular roles for these proteins and revealed similarities and differences when compared to other species.

Involvement of tumour necrosis factor-alpha in Clostridium perfringens beta-toxin-induced plasma extravasation in mice.

BACKGROUND AND PURPOSE: Clostridium perfringens beta-toxin, an important agent of necrotic enteritis, causes plasma extravasation due to the release of a tachykinin NK(1) receptor agonist in mouse skin. In this study, we investigated the role of cytokines in beta-toxin-induced plasma extravasation. EXPERIMENTAL APPROACH: Male Balb/c, C3H/HeN and C3H/HeJ mice were anaesthetized with pentobarbitone and beta-toxin was injected i.d. into shaved dorsal skin. SR140333, capsaicin, chlorpromazine and pentoxifylline were given as pretreatment when required before the injection of the toxin. Cytokines in the dorsal skin were measured by ELISA. KEY RESULTS: Injection (i.d.) of beta-toxin induced a dose-dependent increase in dermal TNF-alpha and interleukin (IL)-1beta levels with a concomitant increase in plasma extravasation, but not the release of IL-6. SR140333 and capsaicin significantly inhibited the toxin-induced release of TNF-alpha and IL-1beta. The plasma extravasation and the release of TNF-alpha induced by beta-toxin were significantly inhibited by chlorpromazine and pentoxifylline which inhibit the release of TNF-alpha. The toxin-induced plasma extravasation in mouse skin was attenuated by pretreatment with a monoclonal antibody against TNF-alpha, but not anti-IL-1beta. Furthermore, the toxin caused an increase in plasma extravasation in both C3H/HeN (TLR4-intact) and C3H/HeJ (TLR4-deficient) mice. In C3H/HeN mice, the toxin-induced leakage was not inhibited by pretreatment with anti-TLR4/MD-2 antibody. CONCLUSIONS AND IMPLICATIONS: These observations show that beta-toxin-induced plasma extravasation in mouse skin is related to the release of TNF-alpha via the mechanism involving tachykinin NK(1) receptors, but not via TLR4.

Lack of photoreceptor signaling alters the expression of specific synaptic proteins in the retina.

Synaptic modulation by activity-dependent changes constitutes a cellular mechanism for neuronal plasticity. However, it is not clear how the complete lack of neuronal signaling specifically affects elements involved in the communication between neurons. In the retina, it is now well established that both chemical and electrical synapses are essential to mediate the transmission of visual signaling triggered by the photoreceptors. In this study, we compared the expression of synaptic proteins in the retinas of wild-type (WT) vs. rd/rd mice, an animal model that displays inherited and specific ablation of photoreceptors caused by a mutation in the gene encoding the beta-subunit of rod cGMP-phosphodiesterase (Pde6brd1). We specifically examined the expression of connexins (Cx), the proteins that form the gap junction channels of electrical synapses, in addition to synaptophysin and synapsin I, which are involved in the release of neurotransmitters at chemical synapses. Our results revealed that Cx36 gene expression levels are lower in the retinas of rd/rd when compared with WT. Confocal analysis indicated that Cx36 immunolabeling almost disappeared in the outer plexiform layer without significant changes in protein distribution within the inner plexiform layer of rd/rd retinas. Likewise, synaptophysin expression remarkably decreased in the outer plexiform layer of rd/rd retinas, and this down-regulation was also associated with diminished transcript levels. Furthermore, we observed down-regulation of Cx57 gene expression in rd/rd retinas when compared with WT and also changes in protein distribution. Interestingly, Cx45 and synapsin I expression in rd/rd retinas showed no noticeable changes when compared with WT. Taken together, our results revealed that the loss of photoreceptors leads to decreased expression of some synaptic proteins. More importantly, this study provides evidence that neuronal activity regulates, but is not essential to maintain, the expression of synaptic elements.

Abdominal wound dehiscence in patients receiving long-term steroid treatment.

Steroids inhibit primary wound healing and delay the formation of granulation tissue, but it has been controversial whether long-term steroid treatment by itself increases the risk of abdominal wound dehiscence. The aim of this study was to determine whether the pre-operative dose and post-operative total dose of steroids influence abdominal wound dehiscence. Of 28 patients who had surgery while receiving long-term steroid treatment, seven had abdominal wound dehiscence and 21 did not have dehiscence. The two groups differed significantly in the post-operative dose of steroids (404.3 +/- 147.1 and 135.6 +/- 118.7 mg, respectively) and the duration of wound healing (57.3 +/- 18.0 and 12.4 +/- 3.8 days), but no other differences were found. Abdominal wound dehiscence may be influenced by the post-operative rather than the pre-operative steroid dose.

Autophagosome requires specific early Sec proteins for its formation and NSF/SNARE for vacuolar fusion.

Double membrane structure, autophagosome, is formed de novo in the process of autophagy in the yeast Saccharomyces cerevisiae, and many Apg proteins participate in this process. To further understand autophagy, we analyzed the involvement of factors engaged in the secretory pathway. First, we showed that Sec18p (N-ethylmaleimide-sensitive fusion protein, NSF) and Vti1p (soluble N-ethylmaleimide-sensitive fusion protein attachment protein, SNARE), and soluble N-ethylmaleimide-sensitive fusion protein receptor are required for fusion of the autophagosome to the vacuole but are not involved in autophagosome formation. Second, Sec12p was shown to be essential for autophagy but not for the cytoplasm to vacuole-targeting (Cvt) (pathway, which shares mostly the same machinery with autophagy. Subcellular fractionation and electron microscopic analyses showed that Cvt vesicles, but not autophagosomes, can be formed in sec12 cells. Three other coatmer protein (COPII) mutants, sec16, sec23, and sec24, were also defective in autophagy. The blockage of autophagy in these mutants was not dependent on transport from endoplasmic reticulum-to-Golgi, because mutations in two other COPII genes, SEC13 and SEC31, did not affect autophagy. These results demonstrate the requirement for subgroup of COPII proteins in autophagy. This evidence demonstrating the involvement of Sec proteins in the mechanism of autophagosome formation is crucial for understanding membrane flow during the process.

ATP synthase F(1) sector rotation. Defective torque generation in the beta subunit Ser-174 to Phe mutant and its suppression by second mutations.

Subunit gamma of the ATP synthase F(1) sector is located at the center of the alpha(3)beta(3) hexamer and rotates unidirectionally during ATP hydrolysis, generating the rotational torque of approximately 45 pN.nm. A mutant F(1) with the betaSer-174 to Phe substitution (betaS174F) in the beta subunit generated lower torque ( approximately 17 pN.nm), indicating that betaS174F is mechanically defective, the first such mutant reported. The defective rotation of betaS174F was suppressed by a second-site mutation, betaGly-149 to Ala, betaIle-163 to Ala, or betaIle-166 to Ala in the same subunit, but not by betaLeu-238 to Ala. These results suggest that the region between betaGly-149 and betaSer-174 plays an important role in the coupling between ATP hydrolysis and mechanical work.

Combined trisomy 9P and Shprintzen syndrome resulting from a paternal t(9;22).

The authors report on a female infant with partial trisomy 9 (pter-->q12) together with partial monosomy 22 (pter-->q11.23) that included DiGeorge critical region (DGCR), as a result of adjacent-2 disjunction. In addition to the clinical features characteristic of trisomy 9p syndrome, the patient had Truncus arteriosus type A2, bilateral hydronephrosis, palatal anomaly, retrognathia, and laryngeal hypotonia, which are likely to be attributed to 22q11.2 deletion. This patient appears to be the first reported case with such unbalanced translocation resulting from a paternal reciprocal translocation. For live birth, the risk for male carrier is 8.7-17.4%. It is important to consider this higher risk when counseling. Precise study concerning the presence of the DGCR can facilitate in the better understanding of the condition.

[Correlation between 5-fluorouracil (5-FU) sensitivity as measured by collagen gel droplet embedded culture drug sensitivity test (CD-DST) and expression of orotate phosphoribosyl transferase (OPRT), thymidylate synthase (TS), and dihydropyrimidine dehydrogenase (DPD) in colorectal cancer].

The levels of OPRT, DPD, and TS were determined in colorectal cancer tissue specimens, and 5-FU sensitivity was measured by CD-DST. The correlation between enzyme activity and 5-FU sensitivity was then studied. Six patients with colorectal carcinoma who had undergone surgical resection in our institution between May and August 2000 were studied. The CD-DST method was used to measure the sensitivity to 5-FU under three sets of conditions: 0.2 microgram/ml x 5 days (A), 1.0 microgram/ml x 1 day (B), and 10.0 micrograms/ml x 3 h (C). The coefficients of correlation of tumor sensitivity to 5-FU and OPRT activity were A: 0.8246, B: 0.7670, and C: 0.7856, and to DPD activity were A: 0.2525, B: 0.3928, and C: 0.4337, while the coefficients of correlation to TS enzyme levels were A: -0.5240, B: -0.4770, and C: -0.6131. These findings demonstrate a high degree of correlation between OPRT activity at the tumor site and tumor sensitivity to 5-FU under a variety of conditions, suggesting that OPRT activity can be a useful indicator in predicting the anti-tumor effectiveness of 5-FU for a specific tumor.

Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi network.

Autophagy is an intracellular bulk protein degradation system. Beclin is known to be involved in this process; however, its role is unclear. In this study, we showed that Beclin was co-immunoprecipitated with phosphatidylinositol (PtdIns) 3-kinase, which is also required for autophagy, suggesting that Beclin is a component of the PtdIns 3-kinase complex. Quantitative analyses using a cross-linker showed that all Beclin forms a complex with PtdIns 3-kinase, whereas approximately 50% of PtdIns 3-kinase remains free from Beclin. Indirect immunofluorescence microscopy demonstrated that the majority of Beclin and PtdIns 3-kinase localize to the trans-Golgi network (TGN). Some PtdIns 3-kinase is also distributed in the late endosome. These results suggest that Beclin and PtdIns 3-kinase control autophagy as a complex at the TGN.