The TORC1 inhibitors Nprl2 and Nprl3 mediate an adaptive response to amino-acid starvation in Drosophila.

Target of rapamycin complex 1 (TORC1) is a master regulator of metabolism in eukaryotes that integrates information from multiple upstream signaling pathways. In yeast, the Nitrogen permease regulators 2 and 3 (Npr2 and Npr3) mediate an essential response to amino-acid limitation upstream of TORC1. In mammals, the Npr2 ortholog, Nprl2, is a putative tumor suppressor gene that inhibits cell growth and enhances sensitivity to numerous anticancer drugs including cisplatin. However, the precise role of Nprl2 and Nprl3 in the regulation of metabolism in metazoans remains poorly defined. Here we demonstrate that the central importance of Nprl2 and Nprl3 in the response to amino-acid starvation has been conserved from single celled to multicellular animals. We find that in Drosophila Nprl2 and Nprl3 physically interact and are targeted to lysosomes and autolysosomes. Using oogenesis as a model system, we show that Nprl2 and Nprl3 inhibit TORC1 signaling in the female germline in response to amino-acid starvation. Moreover, the inhibition TORC1 by Nprl2/3 is critical to the preservation of female fertility during times of protein scarcity. In young egg chambers the failure to downregulate TORC1 in response to amino-acid limitation triggers apoptosis. Thus, our data suggest the presence of a metabolic checkpoint that initiates a cell death program when TORC1 activity remains inappropriately high during periods of amino-acid and/or nutrient scarcity in oogenesis. Finally, we demonstrate that Nprl2/3 work in concert with the TORC1 inhibitors Tsc1/2 to fine tune TORC1 activity during oogenesis and that Tsc1 is a critical downstream effector of Akt1 in the female germline.

Cyclin A associates with the fusome during germline cyst formation in the Drosophila ovary.

Regulated changes in the cell cycle underlie many aspects of growth and differentiation. Prior to meiosis, germ cell cycles in many organisms become accelerated, synchronized, and modified to lack cytokinesis. These changes cause cysts of interconnected germ cells to form that typically contain 2(n) cells. In Drosophila, developing germ cells during this period contain a distinctive organelle, the fusome, that is required for normal cyst formation. We find that the cell cycle regulator Cyclin A transiently associates with the fusome during the cystocyte cell cycles, suggesting that fusome-associated Cyclin A drives the interconnected cells within each cyst synchronously into mitosis. In the presence of a normal fusome, overexpression of Cyclin A forces cysts through an extra round of cell division to produce cysts with 32 germline cells. Female sterile mutations in UbcD1, encoding an E2 ubiquitin-conjugating enzyme, have a similar effect. Our observations suggest that programmed changes in the expression and cytoplasmic localization of key cell cycle regulatory proteins control germline cyst production.

Corpus callosum damage and interhemispheric transfer of information following closed head injury in children.

We evaluated the relationship of corpus callosum atrophy and/or lesions on magnetic resonance imaging (MRI) to functional hemispheric disconnection following closed head injury (CHI) in 51 pediatric patients, including mild CHI, moderate to severe CHI with extracallosal lesions, and moderate to severe CHI with callosal atrophy and/or lesions. Interhemispheric transfer of information was assessed using auditory, motor, tactile, and visual tests in patients and in 16 uninjured children. Total and regional callosal areas were measured from the midsagittal MRI slice by morphometry. The corpus callosum lesion group demonstrated a greater right ear advantage on verbal dichotic listening than all other groups. Areas of the posterior corpus callosum were negatively correlated with laterality indices of verbal dichotic listening performance and tachistoscopic identification of verbal material. The relationship of corpus callosum atrophy and/or lesions to asymmetry in dichotic listening is consistent with previous investigation of posttraumatic hemispheric disconnection effects in adults.

Cell cycle control of chorion gene amplification.

Over-replication of two clusters of chorion genes in Drosophila ovarian follicle cells is essential for rapid eggshell biosynthesis. The relationship of this amplification to the follicle cell cycles has remained unclear. To investigate the regulation of amplification, we developed a technique to detect amplifying chorion genes in individual follicle cells using BrdU incorporation and FISH. Amplification occurs in two developmental phases. One of the gene clusters begins to amplify periodically during S phases of follicle cell endocycles. Subsequently, after endocycles have ceased, both clusters amplify continuously during the remainder of oogenesis. In contrast to the early phase, late amplification commences synchronously among follicle cells. The pattern of Cyclin E expression mirrors these two phases. We present evidence that Cyclin E is required positively for amplification. We suggest that Cyclin E also acts negatively to inhibit refiring of most origins within a cycle, and that specific factors at chorion origins allow them to escape this negative rereplication control. Our findings suggest that chorion amplification is a model for understanding metazoan replicons and the controls that restrict replication to once per cell cycle.

Magnetic resonance imaging in relation to functional outcome of pediatric closed head injury: a test of the Ommaya-Gennarelli model.

OBJECTIVE: To characterize late neuropathological findings of pediatric closed head injury (CHI), to assess depth of brain lesion in relation to acute severity, and to assess long-term outcome to test the Ommaya-Gennarelli model. METHODS: Magnetic resonance imaging (MRI) at least 3 months postinjury in a prospective sample (n 5 169) and at least 3 years after CHI in a retrospective sample (n 5 82) was studied. Lesion volume was measured by planimetry. Acute CHI severity was measured by the Glasgow Coma Scale. Patients were classified according to the depth of the deepest parenchymal lesion into no lesion, subcortical, and deep central gray/brain stem groups. The outcomes were assessed by the Glasgow Outcome Scale and the Vineland Adaptive Behavior Scale, which were performed at the time of the MRI in the retrospective sample and up to 3 years postinjury in the prospective sample. RESULTS: Focal brain lesions were present in 55.4% of the total sample. Depth of brain lesion was directly related to severity of acute impairment of consciousness and inversely related to outcome, as measured by both the Glasgow Outcome Scale and the Vineland Adaptive Behavior Scale. A rostrocaudal gradient of hemispheric lesion frequency was observed, whereas the posterior lesions of the corpus callosum were particularly common. Total lesion volume could not explain the depth of lesion effect. CONCLUSION: Our findings extend support for the Ommaya-Gennarelli model to pediatric CHI, indicating that depth of brain lesion is related to functional outcome. The relative frequency of focal brain lesions revealed by late MRI is higher than that of previous findings using acute computed tomography. Future investigations could explore whether depth of lesion observed using late MRI is sensitive to neuroprotective interventions.

Germline cyst formation in Drosophila.

In a wide variety of organisms, gametes develop within clusters of interconnected germline cells called cysts. Four major principles guide the construction of most cysts: synchronous division, a maximally branched pattern of interconnection between cells, specific changes in cyst geometry, and cyst polarization. The fusome is a germline-specific organelle that is associated with cyst formation in many insects and is likely to play an essential role in these processes. This review examines the cellular and molecular processes that underlie fusome formation and cyst initiation, construction, and polarization in Drosophila melanogaster. The studies described here highlight the importance of cyst formation to the subsequent development of functional gametes.

The Drosophila endocycle is controlled by Cyclin E and lacks a checkpoint ensuring S-phase completion.

Early during Drosophila oogenesis the 16 interconnected cells of each germ-line cyst choose between two alternative fates. The single future oocyte enters meiosis, arrests, and becomes transcriptionally quiescent. The remaining 15 cells initiate a series of polyploid cell cycles to prepare for their role as nurse cells. Like many other polyploid and polytene cells, during nurse cell growth the major satellite DNAs become highly under-represented by a mechanism that has remained obscure. We implicate the cell-cycle regulator cyclin E in DNA under-representation by identifying a hypomorphic, female sterile cycE mutation, cycE01672, that increases the amount of satellite DNA propagated in nurse cells. In mutant but not wild-type endomitotic nurse cells, "late S" patterns of bromodeoxyuridine incorporation are observed similar to those in mitotic cells. CycE protein still cycles in cycE01672 germ-line cysts but at reduced levels, and it is found throughout a longer fraction of the cell cycle. Our experiments support the view that oscillating levels of CycE control the polyploid S phase. Moreover, they indicate that a checkpoint linking the presence of unreplicated DNA to the CycE oscillator is lacking, leading to incomplete replication of late-replicating sequences such as satellite DNAs. Unexpectedly, two to three of the 16 cells in cycE01672 cysts frequently differentiate as oocytes, implicating cell-cycle programming in oocyte determination.

Semantic memory following pediatric head injury: relationship to age, severity of injury, and MRI.

The effects of closed head injury (CHI) severity (mild vs. severe) and age at injury were analyzed in a longitudinal study (3. 12 months postinjury) of semantic memory which used magnetic resonance imaging (MRI) to characterize focal brain lesions. Semantic memory was evaluated by word and category fluency, semantic verification, semantic clustering in word list recall, and vocabulary. Episodic memory was assessed by word list recall. Comparison of normal control (n = 104) data with the patients' data (n = 77) at 3 months postinjury disclosed semantic and episodic memory deficits in the severe CHI patients. Analysis of the longitudinal data revealed significant effects of age at injury for all of the semantic memory measures. The effects of injury severity were confined to the latency of verifying correct statements. Volume of left frontal and extrafrontal lesions was predictive of performance on several semantic memory measures, but less robust for right hemisphere lesions.

Behavioral outcomes after pediatric closed head injury: relationships with age, severity, and lesion size.

This study investigated the behavioral outcomes and adaptive functioning of 138 children with mild to severe closed head injury in the 6- to 16-year age range. Each child was evaluated with the Personality Inventory for Children-Revised. A subset of this sample (n = 77) received the Vineland Adaptive Behavior Scales. Results revealed little evidence for group differences based on severity of closed head injury on scales associated with psychopathology on the Personality Inventory for Children-Revised. However, children with severe closed head injury were viewed as experiencing more difficulties than children with mild-moderate closed head injury on those components of the Personality Inventory for Children-Revised most closely associated with cognitive functions. In addition, on the Vineland Adaptive Behavior Scales, severely injured children had lower scores on the Communication and Socialization scales than children with mild-moderate injury. Relationships between the size of frontal and extrafrontal lesions from concurrent magnetic resonance imaging and behavioral outcomes were not apparent. This study suggests that outcome measures assessing adaptive behavior and cognitive functions are more sensitive to severity of closed head injury than parent-based scales of internalizing and externalizing psychopathology.

Frontal lobe changes after severe diffuse closed head injury in children: a volumetric study of magnetic resonance imaging.

In view of the pathophysiology and biomechanics of severe closed head injury (CHI) in children, we postulated that the frontal lobes sustain diffuse injury, even in the absence of focal brain lesions detected by magnetic resonance imaging (MRI). This study quantitated the morphological effects of CHI on the frontal lobes in children who sustained head trauma of varying severity. The MRI findings of 14 children who had sustained severe CHIs (Glasgow Coma Scale score of < or = 8) were compared with the findings in a matched group of 14 children having sustained mild head injuries (Glasgow Coma Scale score of 13-15). The patients ranged in age from 5 to 15 years at the time of their MRIs, which were acquired at least 3 months postinjury. MRI findings revealed no focal areas of abnormal signal in the frontal lobes. Volumetric analysis disclosed that the total prefrontal cerebrospinal fluid increased and the gray matter volume decreased in the patients with severe CHI, relative to the mildly injured comparison group. Gray matter volume was also reduced in the orbitofrontal and dorsolateral regions of the brains of children with severe CHI, relative to the children who sustained mild head trauma. These volumetric findings indicate that prefrontal tissue loss occurs after severe CHI in children, even in the absence of focal brain lesions in this area. Nearly two-thirds of the children who sustained severe CHIs were moderately disabled after an average postinjury interval of 3 years or more, whereas 12 of the 14 patients with mild CHIs attained a good recovery (2 were moderately disabled) by the time of study.(ABSTRACT TRUNCATED AT 250 WORDS)

Dissociation between delayed alternation and memory after pediatric head injury: relationship to MRI findings.

This study investigated the usefulness of a delayed alternation task in characterizing the cognitive sequelae of closed head injury in children and adolescents. Verbal learning and memory (California Verbal Learning Test) were also studied for comparison. Sixty-two closed head injury patients (mean age, 9.6 years), who were studied after an average postinjury interval of 20 months, were divided according to both their lowest postresuscitation Glasgow Coma Scale score (3 to 8 versus 9 to 15) and age range (5 to 7 years versus 8 to 16 years) at the time of testing. Magnetic resonance imaging was performed to evaluate the relationship of focal brain lesions to cognitive and memory performance. Fifty-six neurologically normal children (mean age, 9.9 years) were tested on the same measures. The results disclosed no relationship between delayed alternation performance and severity of injury. In contrast, verbal memory was impaired in the severely-injured patients, relative to both controls and less severely-injured patients. Frontal lobe (but not extrafrontal) lesion size incremented the Glasgow Coma Scale score in predicting verbal memory, but there was no relationship between focal brain lesions and delayed alternation performance. In contrast to the tendency for more efficient delayed alternation performance in the 5- to 7-year-old subjects than in the 8- to 16-year-old subjects, verbal memory significantly improved with age in the closed head injury and control groups. Notwithstanding our essentially negative findings for delayed alternation, it is possible that this task may be useful for assessing frontal lobe injury in younger children or infants.(ABSTRACT TRUNCATED AT 250 WORDS)

Cognition in relation to magnetic resonance imaging in head-injured children and adolescents.

To investigate the relationship between cognitive sequelae and magnetic resonance imaging (MRI) findings following closed head injury of varying severity in the pediatric age range, 76 head-injured children and adolescents were studied at least 3 months after trauma and compared with 57 normal controls. Problem solving, planning, verbal and design fluency, memory, and response modulation were assessed. Significant effects of injury were obtained on all of the cognitive measures. Cognitive impairment was more consistently present on the various outcome measures in children who were 6 to 10 years old at the time of the study than in the older children and adolescents. Magnetic resonance imaging disclosed areas of abnormal signal in the frontal lobes of 42 patients, whereas focal lesions restricted to the extrafrontal region were found in 15 children. Regression analyses disclosed that taking into account the size of frontal lobe lesion enhanced the relationship between cognitive performance and the severity of injury.

Effects of transcallosal surgery on interhemispheric transfer of information.

The effects of transcallosal surgery on interhemispheric transfer of information were investigated in five patients who underwent partial section of the corpus callosum for evacuation of a brain tumor. In comparison with normal controls, postoperative findings indicated subtle motor and/or sensory manifestations of hemispheric disconnection which tended to resolve overtime. However, in no case were the disconnection effects disabling in the performance of daily activities.