Expressão de genes imediatos induzidos por vocalizações em sagüis-comuns (Callithrix jacchus)

Immediate-early genes (IEGs) expression has been widely used as a valuable tool to investigate brain areas activated by specific stimuli. Studies of natural vocalizations, specially in songbirds, have largely benefited from this tool. Here we used IEGs expression to investigate brain areas activated by the hearing of conspecific common marmoset (Callithrix jacchus) vocalizations and/or utterance of antiphonal vocalizations. Nine adult male common marmosets were housed in sound-attenuating cages. Six animals were stimulated with playbacks of freely recorded natural long distance vocalizations (phee calls and twitters; 45 min. total duration). Three of them vocalized in response (O/V group) and three did not (O/n group). The control group (C) was composed by the remaining animals, which neither heard the playbacks nor spontaneously vocalized. After one hour of the stimulation onset (or no stimulation, in the case of the C group), animals were perfused with 0,9% phosphate-saline buffer and 4% paraformaldehyde. The tissue was coronally sectioned at 20 micro meter in a cryostat and submitted to immunohistochemistry for the IEGs egr-1 and c-fos. Marked immunoreactivity was observed in the auditory cortex of O/V and O/n subjects and in the anterior cingulate cortex, the dorsomedial prefrontal cortex and the ventrolateral prefrontal cortex of O/V subjects. In this study, brain areas activated by vocalizations of common marmosets were investigated using IEGs expression for the first time. Our results with the egr-1 gene indicate that potential plastic phenomena occur in areas related to hearing and uttering conspecific vocalizations.

Neurofisiologia hipocampal durante sessões de sono intercaladas por exploração espacial: Reverberação do passado ou predição do futuro?

The ability to predict future rewards or threats is crucial for survival. Recent studies have addressed future event prediction by the hippocampus. Hippocampal neurons exhibit robust selectivity for spatial location. Thus, the activity of hippocampal neurons represents a cognitive map of space during navigation as well as during planning and recall. Spatial selectivity allows the hippocampus to be involved in the formation of spatial and episodic memories, including the sequential ordering of events. On the other hand, the discovery of reverberatory activity in multiple forebrain areas during slow wave and REM sleep underscored the role of sleep on the consolidation of recently acquired memory traces. To this date, there are no studies addressing whether neuronal activity in the hippocampus during sleep can predict regular environmental shifts. The aim of the present study was to investigate the activity of neuronal populations in the hippocampus during sleep sessions intercalated by spatial exploration periods, in which the location of reward changed in a predictable way. To this end, we performed the chronic implantation of 32-channel multielectrode arrays in the CA1 regions of the hippocampus in three male rats of the Wistar strain. In order to activate different neuronal subgroups at each cycle of the task, we exposed the animals to four spatial exploration sessions in a 4-arm elevated maze in which reward was delivered in a single arm per session. Reward location changed regularly at every session in a clockwise manner, traversing all the arms at the end of the daily recordings. Animals were recorded from 2-12 consecutive days. During spatial exploration of the 4-arm elevated maze, 67,5% of the recorded neurons showed firing rate differences across the maze arms. Furthermore, an average of 42% of the neurons showed increased correlation (R>0.3) between neuronal pairs in each arm. This allowed us to sort representative neuronal subgroups for each maze arm, and to analyze the activity of these subgroups across sleep sessions. We found that neuronal subgroups sorted by firing rate differences during spatial exploration sustained these differences across sleep sessions. This was not the case with neuronal subgroups sorted according to synchrony (correlation). In addition, the correlation levels between sleep sessions and waking patterns sampled in each arm were larger for the entire population of neurons than for the rate or synchrony subgroups. Neuronal activity during sleep of the entire neuronal population or subgroups did not show different correlations among the four arm mazes. On the other hand, we verified that neuronal activity during pre-exploration sleep sessions was significantly more similar to the activity patterns of the target arm than neuronal activity during pre-exploration sleep sessions. In other words, neuronal activity during sleep that precedes the task reflects more strongly the location of reward than neuronal activity during sleep that follows the task. Our results suggest that neuronal activity during sleep can predict regular environmental changes

Investigação da relação entre conteúdo onírico e aprendizado de uma tarefa cognitiva complexa

Several lines of evidence indicate that sleep is beneficial for learning, but there is no experimental evidence yet that the content of dreams is adaptive, i.e., that dreams help the dreamer to cope with challenges of the following day. Our aim here is to investigate the role of dreams in the acquisition of a complex cognitive task. We investigated electroencephalographic recordings and dream reports of adult subjects exposed to a computer game comprising perceptual, motor, spatial, emotional and higher-level cognitive aspects (Doom). Subjects slept two nights in the sleep laboratory, a completely dark room with a comfortable bed and controlled temperature. Electroencephalographic recordings with 28 channels were continuously performed throughout the experiment to identify episodes of rapid-eye-movement (REM) sleep. Behaviors were continuously recorded in audio and video with an infrared camera. Dream reports were collected upon forced awakening from late REM sleep, and again in the morning after spontaneous awakening. On day 1, subjects were habituated to the sleep laboratory, no computer game was played, and negative controls for gamerelated dream reports were collected. On day 2, subjects played the computer game before and after sleep. Each game session lasted for an hour, and sleep for 7-9 hours. 9 different measures of performance indicated significant improve overnight. 81% of the subjects experienced intrusion of elements of the game into their dreams, including potentially adaptative strategies (insights). There was a linear correlation between performance and dream intrusion as well as for game improval and quantity of reported dreaming. In the electrophysiological analysis we mapped the subjects brain activities in different stages (SWS 1, REM 1, SWS 2, REM 2, Game 1 and Game 2), and found a modest reverberation in motor areas related to the joystick control during the sleep. When separated by gender, we found a significant difference on female subjects in the channels that indicate motor learning. Analysis of dream reports showed that the amount of gamerelated elements in dreams correlated with performance gains according to an inverted-U function analogous to the Yerkes-Dodson law that governs the relationship between arousal and learning. The results indicate that dreaming is an adaptive behavior

Sonhos antecipatórios: influência de um evento significativo da vigília na atividade onírica

In most cultures, dreams are believed to predict the future on occasion. Several neurophysiological studies indicate that the function of sleep and dreams is to consolidate and transform memories, in a cyclical process of creation, selection and generalization of conjectures about the reality. The aim of the research presented here was to investigate the possible adaptative role of anticipatory dreams. We sought to determine the relationship between dream and waking in a context in which the adaptive success of the individual was really at risk, in order to mobilize more strongly the oneiric activity. We used the entrance examination of the Federal University of Rio Grande do Norte (UFRN) as a significant waking event in which performance could be independently quantified. Through a partnership with UFRN, we contacted by e-mail 3000 candidates to the 2009 examination. In addition, 150 candidates were approached personally. Candidates who agreed to participate in the study (n = 94) completed questionnaires specific to the examination and were asked to describe their dreams during the examinaton period. The examination performance of each candidate in the entrance examination was provided by the UFRN to the researcher. A total of 45 participants reported dreams related to the examination. Our results show a positive correlation between performance on the examination and anticipatory dreams with the event, both in the comparison of performance on objective and discursive, and in final approval (in the group that not dreamed with the exam the rate of general approval, 22,45%, was similar to that found in the selection process as a whole, 22.19%, while for the group that dreamed with the examination that rate was 35.56%). The occurrence of anticipatory dreams reflectes increased concern during waking (psychobiological mobilization) related to the future event, as indicated by higher scores of fear and apprehension, and major changes in daily life, in patterns of mood and sleep, in the group that reported testrelated dreams. Furthermore, the data suggest a role of dreams in the determination of environmentally relevant behavior of the vigil, simulating possible scenarios of success (dream with approval) and failure (nightmares) to maximize the adaptive success of the individual

Atividade multimodal no córtex sensorial primário de ratos.

The currently accepted model of sensory processing states that different senses are processed in parallel, and that the activity of specific cortical regions define the sensorial modality perceived by the subject. In this work we used chronic multielectrode extracellular recordings to investigate to which extent neurons in the visual and tactile primary cortices (V1 and S1) of anesthetized rats would respond to sensory modalities not traditionaly associated with these cortices. Visual stimulation yielded 87% of responsive neurons in V1, while 82% of S1 neurons responded to tactile stimulation. In the same stimulation sessions, we found 23% of V1 neurons responding to tactile stimuli and 22% of S1 neurons responding to visual stimuli. Our data supports an increasing body of evidence that indicates the existence multimodal processing in primary sensory cortices. Our data challenge the unimodal sensory processing paradigm, and suggest the need of a reinterpretation of the currently accepted model of cortical hierarchy.

Expressão de zif268 no cérebro do lagarto Tropidurus Hispidus após exploração de um ambiente enriquecido.

In the present work, we investigated behavioral changes associated with the increase in Zif268 protein expression within telencephalic areas of the tropical lizard Tropidurus hispidus that correspond to the mammalian hippocampus (HC). We used 13 male individuals of this species, collected at the Federal Agrotechnical School of Rio Grande do Norte, under SISBIO license number 19561-1. Four animals had their brains removed and were submitted to a Western blot with antibodies for the Zif268 protein. The remaining animals were separated in two different groups: a control group (n=4) and an exploration group (n=5). Animals from the exploration group were exposed to an enriched environment with many sensory cues novel to them. Control group animals stayed in the environment they were already habituated to. After 90 min from the onset of exposure to the new environment, animals from both groups were submitted to intracardiac perfusion with fixative, and the brains were removed, cryoprotected and frozen. After that, brains were sectioned at 20 μm and the sections were subjected to immunohistochemistry for the Zif268 protein. We verified that the Zif268 protein is likely conserved in the brain of T. hispidus, which showed antigenicity for the antibody anti-Zif268 made in mammals. In animals from the exploration group, we detected an increase of the Zif268 protein in the Septum, Striatum, Dorsoventricular Area and in cortical areas corresponding to the HC. This increase was proportional to the amount of environmental exploration, with maximum positive correlation in the hippocampal subareas Medial Cortex (R = 0.94 and p = 0.004) and Dorsomedial Cortex (R = 0.92 and p = 0.006). The data corroborate the notion that the reptilian hippocampus, as well as the mammalian HC, plays an important role in spatial exploration.

Alterações do ciclo sono-vigília moduladas por Dopamina e suas consequências Mnemônicas

Neuroscience is on a rise of discoveries. Its wide interdisciplinary approach facilitates a more complex understanding of the brain, covering various areas in depth. However, many phenomena that fascinate human kind are far from being fully elucidated, such as the formation of memories and sleep. In this study we investigated the role of the dopaminergic system in the process of memory consolidation and modulation of the phases of sleep-wake cycle. We used two groups of animals: wildtype mice and hiperdopaminergic mice, heterozygous for the gene encoding the dopamine transporter protein. We observed in wild-type mice that the partial blockade of the D2 dopamine receptor by the drug haloperidol caused deficits in memory consolidation for object recognition, as well as a significant reduction in the duration of rapid eye movement sleep (REM). We also found a mnemonic deficit without pharmacological intervention in hiperdopaminergic animals; this deficit was reversed with haloperidol. The results suggest that dopamine plays a key role in memory consolidation for object recognition. The data also support a functional relationship between the dopaminergic system and the modulation of REM sleep

Alterações na evocação de uma memória aversiva ao liongo do ciclo estral de ratas: influências da transmissão gabaérgica na amígdala

GABAergic neurotransmission has been implicated in many aspects of learning and memory, as well as mood and anxiety disorders. The amygdala has been one of the major focuses in this area, given its essential role in modulating emotionally relevant memories. However, studies with male subjects are still predominant in the field. Here we investigated the consequences for an aversive memory of enhancing or decreasing GABAergic transmission in the basolateral nucleus of the amygdala (BLA). Wistar female rats were trained in the plus-maze discriminative avoidance task, in which they had to learn to avoid one of the enclosed arms where an aversive stimulus consisting of a bright light and a loud noise was given (day 1). Fifteen minutes before the test session (day 2) animals received 0,2 μL infusions of either saline solution, the GABAergic agonist muscimol (0,05 mg/ml), or the GABAergic antagonist bicuculine (0,025 mg/ml) bilaterally intra-BLA. On the test day, females in proestrous or estrous presented adequate retrieval and did not extinguish the task, while females in metestrous or diestrous presented impaired retrieval. In the first group, muscimol infusion impaired retrieval and bicuculline had no effect, suggesting naturally low levels of GABAergic transmission in the BLA of proestrous and estrous females. In the second group, muscimol infusion had no effect and bicuculline reversed retrieval impairment, suggesting naturally high levels of GABAergic transmission in the BLA of metestrous and diestous females. Additionally, proestrous and estrous females presented higher anxiety levels compared to metestrous and diestrous females, which could explain better performance of this group. On the other hand, BLA GABAergic system did not interfere with the innate fear response because drug infusions had no effect in anxiety. Thus, retrieval alterations caused by the GABAergic drugs were probably related specifically to memory processes

Investigação da influência do cochilo na aprendizagem escolar

There is a known positive effect of nocturnal sleep for brain plasticity and the consolidation of declarative and procedural memories, as well as for the facilitation of insight in problem solving. However, a possible pro-mnemonic effect of daytime naps after learning is yet to be properly characterized. The goal of this project was to evaluate the influence of daytime naps on learning among elementary and middleschool students, measuring the one-day (acute), and semester-long (chronic) effects of post-learning naps on performance. In the Acute Day-Nap condition, the elementary students were exposed to a class and then randomly divided into three groups: Nap (N), Game-based English Class (GBEC) and Traditional English Class (TEC). There were 2 multiple-choice follow-up tests to evaluate students performance in the short and long runs. In the short run, the N group outperformed the other two groups; and such tendency was maintained in the long run. In the chronic condition, the middle-school students were randomly separated into two groups: Nap (N) and Class (C) and were observed during one academic term. The N group had increased school performance in relation to the C group. In the statistical analyses, independent t-tests were applied considering the scores significant when p<0,05, expressed in terms of average ± average standard error. Results can be interpreted as an indication that a single daytime nap opportunity is not enough to ensure learning benefits. Therefore, more research is needed in order to advocate in favor of a daytime nap as a pedagogical means of promoting enhanced school performance

Correlato eletroencefalográfico do estado vibracional

O estado vibracional (EV) é descrito como uma sensação de vibração intensa por todo o corpo, em que o sujeito se mantém num estado de relaxamento psicofisiológico que pode ser gerado de forma espontânea ou autoinduzida. Pessoas que aplicam esta técnica relatam alterações do estado mental e emocional, tais como: relaxamento, disposição, limpidez de pensamento, equilíbrio emocional, melhoria do raciocínio, bemestar, entre outros. Estas são, entretanto observações subjetivas, sendo a mensuração deste fenômeno uma lacuna e um desafio para a ciência. O objetivo desta pesquisa é explorar sistematicamente o estado vibracional no âmbito da neurociência. Desta forma, medidas eletroencefalográficas (EEG) foram utilizadas para observar se a sensação subjetiva de EV é acompanhada por mudanças na atividade elétrica cerebral. Além disso, para avaliar se o EV provoca algum efeito positivo em funções cognitivas como atenção e memória, foi utilizado um teste de reconhecimento de palavras antes e depois da aplicação da técnica de EV. Foram também aplicados questionários de dados gerais socioeconômicos e de saúde, do perfil de estados de humor, de qualidade do sono, além de inventários psicológicos. O foco inicial do trabalho foi a análise estatística dos dados de EEG, ficando as outras análises para uma etapa posterior. Dois grupos de voluntários foram analisados, o primeiro formado por 14 sujeitos que praticam a técnica de EV há pelo menos 10 anos (Grupo Experiente - GEXP), e o segundo formado por 11 sujeitos que nunca haviam realizado a técnica (Grupo Controle - GCONT). O GCONT obteve instruções sobre a técnica de EV antes dos experimentos. Foram realizadas análises estatísticas dos registros eletroencefalográficos, para comparar os grupos, em quatro condições: Basal, Relaxamento, Não-EV (período em que o sujeito está engajado na tarefa, mas ainda não percebe o EV) e EV (período em que o sujeito percebe o EV). Uma vez que os sujeitos do GCONT relataram não ter conseguido atingir a condição de EV propriamente, a comparação entre grupos foi feita apenas nas três condições, Basal, Relaxamento e Não-EV. Para isso, foi usado o teste de Mann-Whitney U com um limiar estatístico de p<0,05. De forma geral, o GEXP apresentou maior potência na banda de frequência alfa 2 (9,5-11,0 Hz) em todas as condições. Durante o período Não-EV, o GEXP também apresentou uma maior potência na banda de frequência alfa 3 (11,5-13,0 Hz) na região temporal esquerda, e gama 1 (30,5-55,0 Hz) e gama 2 (65,0-80,0 Hz) em regiões central, parietal e temporal esquerda, mas menor potência na banda de frequência teta 1 (3,5 - 5,0 Hz), em regiões centro-parietais. Para a análise estatística intragrupo, entre as condições, utilizou-se o teste estatístico Wilcoxon pareado. Observaram-se diferenças significativas (p<0,005), principalmente em regiões centrais, em teta 1 (3,5-5,0 Hz), sendo maior no Relaxamento, quando comparado com as condições Basal e Não-EV, no GCONT, e com o Não-EV e EV, no GEXP. No GEXP, a potência de gama 1 (30,5-55,0 Hz) e gama 2 (65,0-80,0 Hz) foi difusamente maior durante o EV, se comparado às outras três condições. Para o GCONT, apenas a condição Basal apresentou maior potência de gama 1 (30,5-55,0 Hz) e gama 2 (65,0- 80,0 Hz), se comparado com o Relaxamento. O aumento de teta 1 no Relaxamento, principalmente no GCONT, pode estar associado a uma maior sonolência deste grupo durante esta condição. Já o aumento de alfa 2 durante o Não-EV e o EV, pode estar associado com processos de atenção e cognição (DOLPPERMAYR et al., 2002; FELL et al., 2010; KLIMESCH et al., 1999; RAY E COLE, 1985). Por outro lado, o aumento da potência de gama em sujeitos experientes na técnica de EV encontrado aqui e em trabalhos anteriores, em meditadores experientes (FELL et al., 2010; LEHMANN et al., 2001; LUTZ et al., 2004), poderia estar associado a alterações nos processos mentais e cognitivos destes praticantes, tais como atenção, memória operacional, aprendizagem e percepção consciente embora, análises adicionais devam ser realizadas para excluir a possibilidade de interferência de artefatos musculares nos dados de EEG. Estes resultados suscitam a hipótese de que no engajamento da tarefa do EV e durante o EV, os sujeitos do GEXP conseguem manter-se em um estado de alerta, porém com maior nível de relaxamento e concentração. Uma inspeção mais detalhada dos dados, além de outros experimentos com diferentes protocolos, um maior número de sujeitos e pesquisas longitudinais são necessários para que testar esta hipótese

Criar o próprio jogo didático ou apenas jogar? Efeitos de diferentes estratégias de ensino na motivação e aprendizado de ciências

The use of games as educational tools is common, however the effectiveness of games with educational purposes is still poorly known. In this study we evaluated three different low-cost teaching strategies make and play your own board game, just play an educational science game and make a poster to be exposed in the school regarding: (1) science learning; (2) use of deep learning strategies (DLS); and (3) intrinsic motivation. We tested the hypothesis that, in these three parameters evaluated, scores would be higher in the group that made and play their own game, followed respectively by the group that just played a game and the group that made a poster. The research involved 214 fifth-grade students from six elementary schools in Natal/RN. A group of students made and played their own science board game (N = 68), a second group played a science game (N = 75), and a third group made a poster to be exposed at school (N = 71). Our hypothesis was partly empirically supported, since there was no significant difference in science learning and in the use of DLS between the group that made their own game and the group that just played the game; however, both groups had significantly higher scores in science learning and in use of DLS than the group that made the poster. There was no significant difference in the scores of intrinsic motivation among the three experimental groups. Our results indicate that activities related to non-digital games can provide a favorable context for learning in the school environment. We conclude that the use of games for educational purposes (both making a game and just playing a game) is an efficient and viable alternative to teach science in Brazilian public school

Congenital diaphragmatic hernia in the preterm infant.

BACKGROUND: Congenital diaphragmatic hernia (CDH) remains a significant cause of death in newborns. With advances in neonatal critical care and ventilation strategies, survival in the term infant now exceeds 80% in some centers. Although prematurity is a significant risk factor for morbidity and mortality in most neonatal diseases, its associated risk with infants with CDH has been described poorly. We sought to determine the impact of prematurity on survival using data from the Congenital Diaphragmatic Hernia Registry (CDHR). METHODS: Prospectively collected data from live-born infants with CDH were analyzed from the CDHR from January 1995 to July 2009. Preterm infants were defined as <37 weeks estimated gestational age at birth. Univariate and multivariate logistic regression analysis were>performed. RESULTS: During the study period, 5,069 infants with CDH were entered in the registry. Of the 5,022 infants with gestational age data, there were 3,895 term infants (77.6%) and 1,127 preterm infants (22.4%). Overall survival was 68.7%. A higher percentage of term infants were treated with extracorporeal membrane oxygenation (ECMO) (33% term vs 25.6% preterm). Preterm infants had a greater percentage of chromosomal abnormalities (4% term vs 8.1% preterm) and major cardiac anomalies (6.1% term vs 11.8% preterm). Also, a significantly higher percentage of term infants had repair of the hernia (86.3% term vs 69.4% preterm). Survival for infants that underwent repair was high in both groups (84.6% term vs 77.2% preterm). Survival decreased with decreasing gestational age (73.1% term vs 53.5% preterm). The odds ratio (OR) for death among preterm infants adjusted for patch repair, ECMO, chromosomal abnormalities, and major cardiac anomalies was OR 1.68 (95% confidence interval [CI], 1.34-2.11). CONCLUSION: Although outcomes for preterm infants are clearly worse than in the term infant, more than 50% of preterm infants still survived. Preterm infants with CDH remain a high-risk group. Although ECMO may be of limited value in the extremely premature infant with CDH, most preterm infants that live to undergo repair will survive. Prematurity should not be an independent factor in the treatment strategies of infants with CDH.

Outcomes of truncal vascular injuries in children.

BACKGROUND: Pediatric truncal vascular injuries occur infrequently and have a reported mortality rate of 30% to 50%. This report examines the demographics, mechanisms of injury, associated trauma, and outcome of patients presenting for the past 10 years at a single institution with truncal vascular injuries. METHODS: A retrospective review (1997-2006) of a pediatric trauma registry at a single institution was undertaken. RESULTS: Seventy-five truncal vascular injuries occurred in 57 patients (age, 12 +/- 3 years); the injury mechanisms were penetrating in 37%. Concomitant injuries occurred with 76%, 62%, and 43% of abdominal, thoracic, and neck vascular injuries, respectively. Nonvascular complications occurred more frequently in patients with abdominal vascular injuries who were hemodynamically unstable on presentation. All patients with thoracic vascular injuries presenting with hemodynamic instability died. In patients with neck vascular injuries, 1 of 2 patients who were hemodynamically unstable died, compared to 1 of 12 patients who died in those who presented hemodynamically stable. Overall survival was 75%. CONCLUSIONS: Survival and complications of pediatric truncal vascular injury are related to hemodynamic status at the time of presentation. Associated injuries are higher with trauma involving the abdomen.

Treatment evolution in high-risk congenital diaphragmatic hernia: ten years' experience with diaphragmatic agenesis.

OBJECTIVE: The objective of this study was to evaluate the impact of newer therapies on the highest risk patients with congenital diaphragmatic hernia (CDH), those with agenesis of the diaphragm. SUMMARY BACKGROUND DATA: CDH remains a significant cause of neonatal mortality. Many novel therapeutic interventions have been used in these infants. Those children with large defects or agenesis of the diaphragm have the highest mortality and morbidity. METHODS: Twenty centers from 5 countries collected data prospectively on all liveborn infants with CDH over a 10-year period. The treatment and outcomes in these patients were examined. Patients were followed until death or hospital discharge. RESULTS: A total of 1,569 patients with CDH were seen between January 1995 and December 2004 in 20 centers. A total of 218 patients (14%) had diaphragmatic agenesis and underwent repair. The overall survival for all patients was 68%, while survival was 54% in patients with agenesis. When patients with diaphragmatic agenesis from the first 2 years were compared with similar patients from the last 2 years, there was significantly less use of ECMO (75% vs. 52%) and an increased use of inhaled nitric oxide (iNO) (30% vs. 80%). There was a trend toward improved survival in patients with agenesis from 47% in the first 2 years to 59% in the last 2 years. The survivors with diaphragmatic agenesis had prolonged hospital stays compared with patients without agenesis (median, 68 vs. 30 days). For the last 2 years of the study, 36% of the patients with agenesis were discharged on tube feedings and 22% on oxygen therapy. CONCLUSIONS: There has been a change in the management of infants with CDH with less frequent use of ECMO and a greater use of iNO in high-risk patients with a potential improvement in survival. However, the mortality, hospital length of stay, and morbidity in agenesis patients remain significant.

In vivo and in vitro characterization of the B1 and B2 zinc-binding domains from the acute promyelocytic leukemia protooncoprotein PML.

Acute promyelocytic leukemia (APL) has been ascribed to a chromosomal translocation event which results in a fusion protein comprising the PML protein and retinoic acid receptor alpha. PML is normally a component of a nuclear multiprotein complex which is disrupted in the APL disease state. Here, two newly defined cysteine/histidine-rich protein motifs called the B-box (B1 and B2) from PML have been characterized in terms of their effect on PML nuclear body formation, their dimerization, and their biophysical properties. We have shown that both peptides bind Zn2+, which induces changes in the peptides' structures. We demonstrate that mutants in both B1 and B2 do not form PML nuclear bodies in vivo and have a phenotype that is different from that observed in the APL disease state. Interestingly, these mutations do not affect the ability of wild-type PML to dimerize with mutant proteins in vitro, suggesting that the B1 and B2 domains are involved in an additional interaction central to PML nuclear body formation. This report in conjunction with our previous work demonstrates that the PML RING-Bl/B2 motif plays a fundamental role in formation of a large multiprotein complex, a function that may be common to those unrelated proteins which contain the motif.