Regulation of the myeloperoxidase enhancer binding proteins Pu1, C-EBP alpha, -beta, and -delta during granulocyte-lineage specification.

We have compared the molecular architecture and function of the myeloperoxidase upstream enhancer in multipotential versus granulocyte-committed hematopoietic progenitor cells. We show that the enhancer is accessible in multipotential cell chromatin but functionally incompetent before granulocyte commitment. Multipotential cells contain both Pu1 and C-EBP alpha as enhancer-binding activities. Pu1 is unphosphorylated in both multipotential and granulocyte-committed cells but is phosphorylated in B lymphocytes, raising the possibility that differential phosphorylation may play a role in specifying its lymphoid versus myeloid functions. C-EBP alpha exists as multiple phosphorylated forms in the nucleus of both multipotential and granulocyte-committed cells. C-EBP beta is unphosphorylated and cytoplasmically localized in multipotential cells but exists as a phosphorylated nuclear enhancer-binding activity in granulocyte-committed cells. Granulocyte colony-stimulating factor-induced granulocytic differentiation of multipotential progenitor cells results in activation of C-EBP delta expression and functional recruitment of C-EBP delta and C-EBP beta to the nucleus. Our results implicate Pu1 and the C-EBP family as critical regulators of myeloperoxidase gene expression and are consistent with a model in which a temporal exchange of C-EBP isoforms at the myeloperoxidase enhancer mediates the transition from a primed state in multipotential cells to a transcriptionally active configuration in promyelocytes.

Reconstitution of the hippocampal mossy fiber and associational-commissural pathways in a novel dissociated cell culture system.

Synapses of the hippocampal mossy fiber pathway exhibit several characteristic features, including a unique form of long-term potentiation that does not require activation of the N-methyl-D-aspartate receptor by glutamate, a complex postsynaptic architecture, and sprouting in response to seizures. However, these connections have proven difficult to study in hippocampal slices because of their relative paucity (<0.4%) compared to commissural-collateral synapses. To overcome this problem, we have developed a novel dissociated cell culture system in which we have enriched mossy fiber synapses by increasing the ratio of granule-to-pyramidal cells. As in vivo, mossy fiber connections are composed of large dynorphin A-positive varicosities contacting complex spines (but without a restricted localization). The elementary synaptic connections are glutamatergic, inhibited by dynorphin A, and exhibit N-methyl-D-aspartate-independent long-term potentiation. Thus, the simplicity and experimental accessibility of this enriched in vitro mossy fiber pathway provides a new perspective for studying nonassociative plasticity in the mammalian central nervous system.

Conscious recollection and the human hippocampal formation: evidence from positron emission tomography.

We used positron emission tomography (PET) to examine the role of the hippocampal formation in implicit and explicit memory. Human volunteers studied a list of familiar words, and then they either provided the first word that came to mind in response to three-letter cues (implicit memory) or tried to recall studied words in response to the same cues (explicit memory). There was no evidence of hippocampal activation in association with implicit memory. However, priming effects on the implicit memory test were associated with decreased activity in extrastriate visual cortex. On the explicit memory test, subjects recalled many target words in one condition and recalled few words in a second condition, despite trying to remember them. Comparisons between the two conditions showed that blood-flow increases in the hippocampal formation are specifically associated with the conscious recollection of studied words, whereas blood-flow increases in frontal regions are associated with efforts to retrieve target words. Our results help to clarify some puzzles concerning the role of the hippocampal formation in human memory.

Learning about categories in the absence of memory.

A fundamental question about memory and cognition concerns how information is acquired about categories and concepts as the result of encounters with specific instances. We describe a profoundly amnesic patient (E.P.) who cannot learn and remember specific instances--i.e., he has no detectable declarative memory. Yet after inspecting a series of 40 training stimuli, he was normal at classifying novel stimuli according to whether they did or did not belong to the same category as the training stimuli. In contrast, he was unable to recognize a single stimulus after it was presented 40 times in succession. These findings demonstrate that the ability to classify novel items, after experience with other items in the same category, is a separate and parallel memory function of the brain, independent of the limbic and diencephalic structures essential for remembering individual stimulus items (declarative memory). Category-level knowledge can be acquired implicitly by cumulating information from multiple training examples in the absence of detectable conscious memory for the examples themselves.

Adenovirus-mediated urokinase gene transfer induces liver regeneration and allows for efficient retrovirus transduction of hepatocytes in vivo.

Retrovirus-mediated gene transfer into hepatocytes in vivo results in long-term gene expression. Limitations include the need to remove two-thirds of the liver and the relatively low frequency of gene transfer. To increase gene transfer without surgical hepatectomy, mouse hepatocytes were transduced in vivo with a recombinant adenovirus that transiently expressed urokinase, resulting in high rates of asynchronous liver regeneration. During the regenerative phase, in vivo retroviral-mediated gene transfer in hepatocytes resulted in 5- to 10-fold greater transduction efficiencies than that obtained by conventional partial hepatectomy. In 3-4 weeks, the architecture and microscopic structure of the recipient livers were normal. The two-viral system of achieving permanent transgene expression from hepatocytes in vivo offers an alternative approach to current ex vivo and in vivo gene-transfer models.