The use of a neutral peptide aptamer scaffold to anchor BH3 peptides constitutes a viable approach to studying their function.

The B-cell CLL/lymphoma-2 (Bcl-2) family of proteins are important regulators of the intrinsic pathway of apoptosis, and their interactions, driven by Bcl-2 homology (BH) domains, are of great interest in cancer research. Particularly, the BH3 domain is of clinical relevance, as it promotes apoptosis through activation of Bcl-2-associated x protein (Bax) and Bcl-2 antagonist killer (Bak), as well as by antagonising the anti-apoptotic Bcl-2 family members. Although investigated extensively in vitro, the study of the BH3 domain alone inside cells is more problematic because of diminished secondary structure of the unconstrained peptide and a lack of stability. In this study, we report the successful use of a novel peptide aptamer scaffold - Stefin A quadruple mutant - to anchor and present the BH3 domains from Bcl-2-interacting mediator of cell death (Bim), p53 upregulated modulator of apoptosis (Puma), Bcl-2-associated death promoter (Bad) and Noxa, and demonstrate its usefulness in the study of the BH3 domains in vivo. When expressed intracellularly, anchored BH3 peptides exhibit much the same binding specificities previously established in vitro, however, we find that, at endogenous expression levels, Bcl-2 does not bind to any of the anchored BH3 domains tested. Nonetheless, when expressed inside cells the anchored PUMA and Bim BH3 α-helices powerfully induce cell death in the absence of efficient targeting to the mitochondrial membrane, whereas the Noxa helix requires a membrane insertion domain in order to kill Mcl-1-dependent myeloma cells. Finally, the binding of the Bim BH3 peptide to Bax was the only interaction with a pro-apoptotic effector protein observed in this study.

Development of peptide aptamer microarrays for detection of HPV16 oncoproteins in cell extracts.

Protein microarrays represent an emerging technology that promises to facilitate high-throughput proteomics. The major goal of this technology is to employ peptides, full-length proteins, antibodies, and small molecules to simultaneously screen thousands of targets for potential protein-protein interactions or modifications of the proteome. This article describes the performance of a set of peptide aptamers specific for the human papillomavirus (HPV) type 16 oncoproteins E6 and E7 in a microarray format. E6 and E7 peptide aptamer microarrays were probed with fluorescence-labeled lysates generated from HPV-infected cervical keratinocytes expressing both E6 and E7 oncoproteins. Peptide aptamer microarrays are shown to detect low levels of E6 and E7 proteins. Peptide aptamers specific for cellular proteins included on these microarrays suggested that expression of CDK2, CDK4, and BCL-6 may be affected by HPV infection and genome integration. We conclude that peptide aptamer microarrays represent a promising tool for proteomics and may be of value in biological and clinical investigations of cervical carcinogenesis.

A peptide aptamer to antagonize BCL-6 function.

BCL-6 is a transcription factor essential for germinal centre B-cell development. The BCL-6 gene is involved in diffuse large-cell lymphoma and overexpressed in other types of non-Hodgkin's lymphoma and in high-grade breast cancer. BCL-6 is a transcriptional repressor whose N-terminal POZ domain mediates protein-protein interactions to exert its effects. Reasoning that disruption of POZ domain-mediated interactions may be an effective route to antagonizing the effects of BCL-6 in lymphoma, we screened a library for peptide aptamers that specifically bind to BCL-6 POZ and not the POZ domains of related proteins and describe here the first of these reagents, Apt48. Apt48 binds BCL-6 POZ in a manner distinct from the transcriptional corepressor SMRT, yet was found to prevent BCL-6-mediated repression of a luciferase reporter gene. Apt48 also reproduced several previously validated effects of BCL-6 inhibition. Notably, expression of the differentiation markers CD69, Blimp-1 and cyclin D2 was increased in B-cell lines when Apt48 was expressed. We also show that expression of Apt48 restores cytokine-mediated growth arrest to BCL-6 overexpressing cells. Thus, we have identified a peptide aptamer that affects a function of BCL-6 that is required to prevent differentiation of proliferating B cells.

Creutzfeldt-Jakob disease associated with high titer of antithyroid autoantibodies: case report and literature review.

Hashimoto's encephalopathy and Creutzfeldt-Jakob disease (CJD) often have similar clinical features and may be confused, especially at onset. A 61-year-old woman developed rapidly progressive ataxia, myoclonus and dementia, with abnormalities seen on electroencephalography (EEG). Serum analysis disclosed high titers of antithyroid autoantibodies. Both clinical course and autopsy led to a definitive diagnosis of CJD. This case and a literature review of previous cases confirm that CJD may be confused with Hashimoto's encephalopathy. EEG, clinical and laboratory findings (including the positivity of 14.3.3 protein in the cerebrospinal fluid) are not conclusive for a differential diagnosis, especially at early stages. Only the results of genetic exams can allow a definitive diagnosis in a small percentage of cases while patients are still alive. In patients with unclear symptomatology and rapid onset of myoclonus, dementia and ataxia, the presence of antithyroid antibodies should be examined. If their levels are abnormal, corticosteroid therapy remains mandatory.

Localization of yeast telomeres to the nuclear periphery is separable from transcriptional repression and telomere stability functions.

The left telomere of Saccharomyces chromosome VII was often localized near the nuclear periphery, even in cells lacking the silencing proteins Sir3 or Hdf1. This association was lost in late mitotic cells and when transcription was induced through the telomeric tract. Although in silencing competent cells there was no correlation between the fraction of cells in which a telomeric gene was repressed and the fraction of cells in which it was localized to the periphery, no condition was found where the telomere was both silenced and away from the periphery. We conclude that localization of a telomere to the nuclear periphery is not sufficient for transcriptional repression nor does it affect the stability function of yeast telomeres.

SH3GLB, a new endophilin-related protein family featuring an SH3 domain.

A new cDNA encoding a protein of 362 amino acids designated SH3GLB1, for SH3 domain GRB2-like endophilin B1, was identified in a yeast two-hybrid screen devoted to the identification of new partners interacting with the apoptosis inducer Bax. SH3GLB1 shows strong similarities to the SH3 domain-containing proteins of the endophilin family and presumably represents the human homologue of the potential Caenorhabditis elegans SH3 containing-protein identified by systematic translation of the C. elegans genome (GenBank Accession No. U46675). Reversing prey to bait in the yeast screen, a second protein, SH3GLB2, of 395 amino acids showing 65% identity to SH3GLB1 was identified as an interacting partner of SH3GLB1. The discovery of SH3GLB1 itself in the screening with SH3GLB1 as a bait and further mapping experiments demonstrated that a core coiled-coil-type region is required for the formation of SH3GLB homo- and/or heterodimers, whereas the SH3 domain is not involved in these interactions. Interestingly, the similarities with the endophilin proteins cover the entire sequence of the SH3GLB family, suggesting a common fold and presumably a common mode of action. Furthermore, SH3GLB members colocalize to the cytoplasmic compartment of the cell together with Bax and are excluded from the nucleus. SH3GLB1 and SH3GLB2 do not significantly influence the onset and time course of Bax-mediated apoptosis in HeLa or 293T cells.

Seizure disorder mimicking an acute confusional state as clinical presentation of neurocysticercosis: neuroimaging, EEG findings and clinical correlations.

Epilepsy is the main clinical manifestation of neurocysticercosis (NC). We studied an adult subject who presented a seizure disorder mimicking an acute confusional state as clinical expression of NC. Diagnosis was made with neuroimaging and western blot determination of specific antibodies on serum. Computed tomography and magnetic resonance imaging displayed multiple calcifications and a few transitional cysts in the cerebral parenchyma. Electroencephalography showed a pattern of periodic lateralized epileptiform discharges (PLEDs) which could be related topographically to a cystic lesion located in the left parietal lobe. In our view there was a clear pathogenic correlation between the seizure disorder and the parasitic cyst located in the left parietal lobe. Neither antiepileptic drugs nor steroids were prescribed. Follow-up to one year ruled out other clinical manifestations of the disease. This case is an example of acute symptomatic seizure related to a transitional cystic lesion of NC.

SAC3 may link nuclear protein export to cell cycle progression.

Selective movement of proteins between the nucleus and the cytoplasm is a regulatory mechanism exploited extensively by the eukaryotic cell. We have identified the evolutionarily conserved Sac3 protein, which was implicated previously in the regulation of mitosis [Bauer, A. & Kölling, R. (1996) J. Cell Sci. 109, 1575-1583] as a novel mediator of nuclear protein export. We show that Sac3p is localized to the nuclear pore, where it interacts with nucleoporins. Loss of SAC3 function results in a block in nuclear export of a nuclear export signal-containing reporter protein. Our results also demonstrate that SAC3 interacts genetically with the nuclear protein export factors Crm1p/Xpo1p and Yrb2p. Taken together, these data indicate a link between nuclear protein export and transition through the cell cycle.

Targeted modification and transportation of cellular proteins.

Peptide aptamers are proteins selected from combinatorial libraries that display conformationally constrained variable regions. Peptide aptamers can disrupt specific protein interactions and thus represent a useful method for manipulating protein function in vivo. Here, we describe aptamer derivatives that extend the range of functional manipulations. We isolated an aptamer with increased affinity for its Cdk2 target by mutagenizing an existing aptamer and identifying tighter binding mutants with calibrated two-hybrid reporter genes. We used this and other anti-Cdk2 aptamers as recognition domains in chimeric proteins that contained other functional moieties. Aptamers fused to the catalytic domain of a ubiquitin ligase specifically decorated LexA-Cdk2 with ubiquitin moieties in vivo. Aptamers against Cdk2 and another protein, Ste5, that carried a nuclear localization sequence transported their targets into the nucleus. These experiments indicate that fusion proteins containing aptameric recognition moieties will be useful for specific modification of protein function in vivo.

Regulated nuclear localization of stress-responsive factors: how the nuclear trafficking of protein kinases and transcription factors contributes to cell survival.

The details of nuclear transport mechanisms are emerging rapidly, largely through work with model organisms. Here, we briefly describe these advances, with an emphasis on the remaining challenges. We then address the nuclear transport of some high profile cellular regulators, including p53 and the proto-oncogene PKB/Akt. We discuss the mechanisms that contribute to the differential subcellular localization of these proteins. Finally, we analyse the provocative patterns that emerge from our overview.

Species-specific, postentry barriers to primate immunodeficiency virus infection.

By using replication-defective vectors derived from human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV(mac)), and murine leukemia virus (MuLV), all of which were pseudotyped with the vesicular stomatitis virus (VSV) G glycoprotein, the efficiency of postentry, early infection events was examined in target cells of several mammalian species. Titers of HIV-1 vectors were significantly lower than those of SIV(mac) and MuLV vectors in most cell lines and primary cells from Old World monkeys. By contrast, most New World monkey cells exhibited much lower titers for the SIV(mac) vector compared with those of the HIV-1 vector. Prosimian cells were resistant to both HIV-1 and SIV(mac) vectors, although the MuLV vector was able to infect these cells. Cells from other mammalian species were roughly equivalent in susceptibility to the three vectors, with the exception of rabbit cells, which were specifically resistant to the HIV-1 vector. The level of HIV-1 vector expression was very low in transduced cells of rodent, rabbit, cow, and pig origin. Early postentry restriction of primate immunodeficiency virus infection exhibits patterns largely coincident with species borders and applies to diverse cell types within an individual host, suggesting the involvement of species-specific, widely expressed cellular factors.

Elimination of replication block protein Fob1 extends the life span of yeast mother cells.

A cause of aging in yeast is the accumulation of circular species of ribosomal DNA (rDNA) arising from the 100-200 tandemly repeated copies in the genome. We show here that mutation of the FOB1 gene slows the generation of these circles and thus extends life span. Fob1p is known to create a unidirectional block to replication forks in the rDNA. We show that Fob1p is a nucleolar protein, suggesting a direct involvement in the replication fork block. We propose that this block can trigger aging by causing chromosomal breaks, the repair of which results in the generation of rDNA circles. These findings may provide a novel link between metabolic rate and aging in yeast and, perhaps, higher organisms.

Anatomic correlates of painful tonic spasms in multiple sclerosis.

Painful tonic spasms (PTS) are now regarded as a typical symptom of multiple sclerosis but pathologic or radiologic findings rarely have been described. We report clinical and magnetic resonance imaging records of five original cases. In all of them, lesions likely responsible for unilateral PTS involved the motor pathway at the level of the posterior limb of the internal capsule or the cerebral peduncle on the opposite side. Closeness of motor fibers seems to be the most important underlying anatomic factor because it enables involvement of a higher proportion of axons by a single demyelinating lesion and radial spread of ephaptic activation. In turn, preservation of the underlying pyramidal-spinal tract could make it easier for the pathologic discharge to reach the peripheral effectors and generate PTS.

Temporal differences in the appearance of NEP-B78 and an LBR-like protein during Xenopus nuclear envelope reassembly reflect the ordered recruitment of functionally discrete vesicle types.

In this work, we have used novel mAbs against two proteins of the endoplasmic reticulum and outer nuclear membrane, termed NEP-B78 and p65, in addition to a polyclonal antibody against the inner nuclear membrane protein LBR (lamin B receptor), to study the order and dynamics of NE reassembly in the Xenopus cell-free system. Using these reagents, we demonstrate differences in the timing of recruitment of their cognate membrane proteins to the surface of decondensing chromatin in both the cell-free system and XLK-2 cells. We show unequivocally that, in the cell-free system, two functionally and biochemically distinct vesicle types are necessary for NE assembly. We find that the process of distinct vesicle recruitment to chromatin is an ordered one and that NEP-B78 defines a vesicle population involved in the earliest events of reassembly in this system. Finally, we present evidence that NEP-B78 may be required for the targeting of these vesicles to the surface of decondensing chromatin in this system. The results have important implications for the understanding of the mechanisms of nuclear envelope disassembly and reassembly during mitosis and for the development of systems to identify novel molecules that control these processes.

Regulated nucleo/cytoplasmic exchange of HOG1 MAPK requires the importin beta homologs NMD5 and XPO1.

MAP kinase signaling modules serve to transduce extracellular signals to the nucleus of eukaryotic cells, but little is known about how signals cross the nuclear envelope. Exposure of yeast cells to increases in extracellular osmolarity activates the HOG1 MAP kinase cascade, which is composed of three tiers of protein kinases, namely the SSK2, SSK22 and STE11 MAPKKKs, the PBS2 MAPKK, and the HOG1 MAPK. Using green fluorescent protein (GFP) fusions of these kinases, we found that HOG1, PBS2 and STE11 localize to the cytoplasm of unstressed cells. Following osmotic stress, HOG1, but neither PBS2 nor STE11, translocates into the nucleus. HOG1 translocation occurs very rapidly, is transient, and correlates with the phosphorylation and activation of the MAP kinase by its MAPKK. HOG1 phosphorylation is necessary and sufficient for nuclear translocation, because a catalytically inactive kinase when phosphorylated is translocated to the nucleus as efficiently as the wild-type. Nuclear import of the MAPK under stress conditions requires the activity of the small GTP binding protein Ran-GSP1, but not the NLS-binding importin alpha/beta heterodimer. Rather, HOG1 import requires the activity of a gene, NMD5, that encodes a novel importin beta homolog. Similarly, export of dephosphorylated HOG1 from the nucleus requires the activity of the NES receptor XPO1/CRM1. Our findings define the requirements for the regulated nuclear transport of a stress-activated MAP kinase.

Sclerosteosis: report of a case in a black African man.

Sclerosteosis is a rare genetic disorder of bone modelling, similar to, but distinct from, van Buchem disease; it has been described almost exclusively in Afrikaners of South Africa, a white population of Dutch ancestry. Isolated cases have been reported in a girl in Japan, a boy in Spain, and in multiracial families in Brazil and USA. Here we report a case of sclerosteosis in a black man born in Senegal. He presented with the full features of the disease: tall stature; syndactyly: nail dysplasia; massive sclerosis of the long tubular bones, the ribs, the pelvis and the skull; multiple cranial nerve involvement: optic atrophy, facial palsy and trigeminal neuralgia. Radiologic examination, visual and brainstem auditory evoked potentials, computerized tomography and magnetic resonance imaging of the skull were performed. This seems to be the first case of the disease in a black African individual, with no known relationship with Dutch ancestry.

Visual evoked potentials in hypothyroidism: a long-term evaluation.

Visual Evoked Potentials (VEP) were measured in 9 new-diagnosed hypothyroid female patients--mean age 46 +/- 12 ys--before treatment, during (with monthly evaluations) thyroid hormone replacement therapy and after long-term therapy, at the achievement as well as one year after having achieved and maintained euthyroidism. Three of the hypothyroids had abnormally prolonged latencies (m.v. 131.7 +/- 7.9 ms), while 7 had lower than normal amplitude (m.v. 2.3 +/- 2.8 microV). No remarkable change of amplitude was observed after the achievement of euthyroidism, after a mean time of 5.9 +/- 4.9 months (range 2-14 months). A significant shortening of latency (m 128.3 +/- 7.6 ms), even still higher than the control value (m 122.7 +/- 3.7 ms) was found. Significant correlation between P100 latency and thyroid hormone levels was found for TT4 (r = 0.3353; p = 0.005), TT3 (r = 0.2568; p = 0.032) and FT4 (r = 0.3572; p = 0.002). No further improvement in P100 latency (m 129.5 +/- 7.2 ms; p = 0.037) was found one year after the achievement of euthyroidism, while a remarkable amplitude increase (m 9.2 +/- 3.4 micro; p = 0.001) was observed. Our findings indicate that, as well as other studied parameters, VEP are reversibly alterated in hypothyroidism, probably in relation to metabolic rather than to structural alterations. Moreover, VEP can represent a useful neurophysiologic parameter for quantitation of SNC involvement in hypothyroidism.

Contribution of central neuropathy to postural instability in IDDM patients with peripheral neuropathy.

OBJECTIVE: To evaluate the contribution of central neuropathy on postural impairment observed in diabetic patients with peripheral neuropathy. RESEARCH DESIGN AND METHODS: Central sensory and motor nervous propagation, nerve conduction velocity, and static posturography were assessed in the following age-matched subjects: 7 IDDM patients with peripheral neuropathy (group DN), 18 IDDM patients without peripheral neuropathy (group D), and 31 control subjects (group C). Somatosensory-evoked potentials (SEPs) during tibial nerve stimulation were recorded, and the spine-to-scalp sensory central conduction time (SCCT) was evaluated. Motor-evoked potentials (MEPs) were recorded from leg muscles during magnetic transcranial brain stimulation, and the scalp-to-spine motor central conduction time (MCCT) was evaluated. The following posturographic parameters were calculated from the statokinesigram: trace length, trace surface, velocity of body sway with its standard deviation, and VFY (a parameter derived from the velocity variance and the anteroposterior mean position of the body). RESULTS: SCCT was significantly higher in the DN group than in the C and D groups (P < 0.001). MCCT was similar in all groups. Posturographic parameters were all significantly impaired in the DN group (P < 0.01). While posturographic parameters showed a direct relationship with some parameters of peripheral nerve conduction, no correlations were observed with SEP and MEP central conduction time. These results were also confirmed by logistic regression, which indicates peripheral neuropathy as the only implicating factor in postural instability (odds ratio 0.22, 95% CI 0.07-0.75) after data reduction by means of factor analysis. CONCLUSIONS: Although diabetic patients with peripheral neuropathy show a delay in central sensory conduction, postural instability may be fully explained by the presence of peripheral neuropathy.