Epidermal growth factor receptor in human brain tumors.

The expression of epidermal growth factor receptor (EGF-R) was examined in 27 primary human brain tumors (7 glioblastomas, 10 astrocytomas, 5 oligodendrogliomas, 1 schwannoma, 1 ganglioneuroma, 1 medulloblastoma, 1 ependymoma, 1 histiocytic lymphoma), in 6 brain metastases from lung carcinomas and in 20 meningiomas. Peritumoral tissues histologically normal excised surgically along with a large tumor were used as control. All plasma membranes from brain tissues tested showed specific EGF binding. The EGF receptor is expressed at low levels in the control human brain and at very high levels in 60% of the total intracranial tumors studied. When the various histological types of tumors were analyzed, the higher percentage of positive tumors was found with the meningiomas (85%) and the glioblastomas (71%), while the lower percentage of positivity was found with the oligodendrogliomas (40%) and the astrocytomas (30%). A good correlation between binding and total amount of EGF-R protein detected by Western Blot was also observed.     

Small potassium ion channel proteins encoded by chlorella viruses

Kcv, a 94-aa protein encoded by Paramecium bursaria chlorella virus 1, is the smallest known protein to form a functional potassium ion channel and basically corresponds to the "pore module" of potassium channels. Both viral replication and channel activity are inhibited by the ion channel blockers barium and amantadine but not by cesium. Genes encoding Kcv-like proteins were isolated from 40 additional chlorella viruses. Differences in 16 of the 94 amino acids were detected, producing six Kcv-like proteins with amino acid substitutions occurring in most of the functional domains of the protein (N terminus, transmembrane 1, pore helix, selectivity filter, and transmembrane 2). The six proteins form functional potassium selective channels in Xenopus oocytes with different properties including altered current kinetics and inhibition by cesium. The amino acid changes together with the different properties observed in the six Kcv-like channels will be used to guide site-directed mutations, either singularly or in combination, to identify key amino acids that confer specific properties to Kcv.     

Soluble interleukin 2 receptor in lung cancer. An indirect marker of tumor activity?

Circulating levels of the soluble interleukin 2 receptor (sIL-2R) could provide an in vivo measure of the immunologic response to human tumors. We performed a total of 326 sIL-2R serum assays in 126 patients with lung cancer (67 at diagnosis, 59 during and after treatment), 112 patients with pulmonary benign diseases, and 63 voluntary healthy subjects. Patients with lung cancer had a median value of sIL-2R of 791 U/ml, which was superior to that of both controls (398 U/ml, p less than 0.001) and patients with noninflammatory benign diseases (583 U/ml, p less than 0.02). However, infectious pulmonary disorders, such as tuberculosis and pneumonia, were associated with the highest values of the substance (median, 1150 U/ml; p less than 0.001). At the diagnosis of lung cancer, sIL-2R correlated neither with the stage of disease nor with the cell type. On the contrary, posttreatment levels of the receptor were significantly related to disease status (RO = .41, p less than 0.002), particularly in the subgroup of nonsurgical patients (RO = .48, p less than 0.001). Patients with abnormal sIL-2R levels had a nearly significant reduction in survival as compared with patients with normal values (p less than 0.1). Measurements of sIL-2R could be useful in monitoring patients under treatment for bronchogenic carcinoma, as well as in prognostication. In this setting, sIL-2R might open a new class of biologic markers, providing information that is complementary to those of the more classic tumor-derived markers.     

From the Cover: Structural basis for the mutual antagonism of cAMP and TRIP8b in regulating HCN channel function

cAMP signaling in the brain mediates several higher order neural processes. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels directly bind cAMP through their cytoplasmic cyclic nucleotide binding domain (CNBD), thus playing a unique role in brain function. Neuronal HCN channels are also regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit that antagonizes the effects of cAMP by interacting with the channel CNBD. To unravel the molecular mechanisms underlying the dual regulation of HCN channel activity by cAMP/TRIP8b, we determined the NMR solution structure of the HCN2 channel CNBD in the cAMP-free form and mapped on it the TRIP8b interaction site. We reconstruct here the full conformational changes induced by cAMP binding to the HCN channel CNBD. Our results show that TRIP8b does not compete with cAMP for the same binding region; rather, it exerts its inhibitory action through an allosteric mechanism, preventing the cAMP-induced conformational changes in the HCN channel CNBD.Hyperpolarization-activated cyclic nucleotide-gated (HCN1–4) channels are the molecular determinants of the h-current (I_h), which regulates critical neuronal properties, including membrane resting potential, dendritic excitability, and intrinsic rhythmicity (1). HCN channels are dually regulated by voltage and by binding of cAMP to the cyclic nucleotide binding domain (CNBD), which is found on the cytoplasmic C-terminal tail of the channel. The CNBD exerts a tonic inhibition on the channel pore, with the opening transition of the channel being allosterically coupled to the conformational changes in the CNBD induced by cAMP binding (2). Thus, the closed-to-open transition of the channel is thought to reflect the transition from the cAMP-free conformation to the cAMP-bound conformation of the CNBD, which stabilize, respectively, the closed and open states of the channel (2, 3). The C-linker, an α-helical folded domain that connects the CNBD to the pore region, conveys the regulation of channel gating from the CNBD to the pore (4–6). As a result of this allosteric mechanism, the binding of cAMP shifts the voltage dependence of the HCN channel opening to more positive potentials and increases maximal I_h at extreme negative voltages, where voltage gating is complete.In addition to cAMP, HCN channels in the brain are regulated by auxiliary proteins, such as TRIP8b, a cytosolic β-subunit of neuronal HCN channels, which inhibits channel activation by antagonizing the effects of cAMP (7–9). We have previously shown that TRIP8b_core, an 80-aa sequence located in the TRIP8b protein core that directly interacts with the C-linker/CNBD region of HCN channels, is necessary and sufficient to prevent all of the effects of cAMP on the channel (10, 11). TRIP8b_core decreases both the sensitivity of the channel to cAMP [half maximal concentration (k_1/2)] and the efficacy of cAMP in inducing channel opening [half activation voltage (V_1/2)]; conversely, cAMP binding inhibits these actions of TRIP8b. These mutually antagonistic effects are well described by a cyclic allosteric model in which TRIP8b binding reduces the affinity of the channel for cAMP, with the affinity of the open state for cAMP being reduced to a greater extent than the cAMP affinity of the closed state (11).A second important action of TRIP8b is to reduce maximal current through HCN channels in the absence of cAMP (11). As a consequence, application of cAMP produces a larger increase in maximal I_h in the presence of TRIP8b than in its absence. The observation that TRIP8b exerts opposing influences on the two major actions of cAMP on HCN channel function, namely, reduces the effect of cAMP to shift the voltage dependence of channel gating but enhances the effect of cAMP to increase maximal current, has important implications for the ability of cAMP to modulate neuronal excitability in vivo. Thus, the relative extent by which neuromodulatory transmitters alter maximal I_h or shift the voltage dependence of HCN channel gating can vary widely among distinct classes of neurons (12–14). The differential expression of TRIP8b may provide a mechanistic explanation for this finding, because in neurons with high levels of TRIP8b expression, cAMP will exert a larger action to enhance maximal current, and a smaller action to alter the voltage dependence of channel gating, compared with neurons in which TRIP8b expression is low. Such fine-tuning broadens the range of physiological actions that cAMP can exert to modulate neuronal firing.In the present study, we address the structural basis for the mutually antagonistic effects of cAMP and TRIP8b on HCN channel function. Although our previous biochemical and electrophysiological data strongly support the hypothesis that TRIP8b and cAMP binding sites do not overlap, direct structural information on the TRIP8b–CNBD complex is required to validate the allosteric antagonism model of interaction between the two ligands. A plausible hypothesis for the antagonistic effect of TRIP8b and cAMP is that each of the two ligands stabilizes the CNBD in a conformation that decreases the affinity for the other. To test this hypothesis, we first generated the 3D structure of the cAMP-free HCN2 channel CNBD using solution NMR spectroscopy and then characterized its interaction with the TRIP8b_core fragment. By comparing the cAMP-free with the available cAMP-bound HCN2 channel CNBD structure (15, 16), we reconstruct the full conformational changes induced by cAMP binding, revealing critical transitions occurring in the P- and C-helices of the CNBD, and further highlighting the role of the N-terminal helical bundle in transducing the movements of the CNBD to the channel pore. We next identify, through NMR titration, site-directed mutagenesis, and biochemical interaction assays, the binding site of TRIP8b_core on the cAMP-free form of the HCN2 channel CNBD. Our results demonstrate that cAMP and TRIP8b do not directly compete for the same binding region and support a model of mutual allosteric inhibition between cAMP and TRIP8b. Finally, our results clarify the mechanism by which TRIP8b antagonizes the effect of cAMP on channel gating: TRIP8b directly interacts with two mobile elements that drive the ligand-induced conformational changes in the CNBD. TRIP8b binding to the CNBD therefore prevents the cAMP-induced transition and stabilizes the channel in the cAMP-free conformation.     

Transitioning outcome measures: relationship between the CMTPedS and CMTNSv2 in children,adolescents, and young adults with Charcot‐Marie‐Tooth disease

Long‐term studies of Charcot‐Marie‐Tooth (CMT) disease across the entire lifespan require stable endpoints that measure the same underlying construct (e.g., disability). The aim of this study was to assess the relationship between the CMT Pediatric Scale (CMTPedS) and the adult CMT Neuropathy Score (CMTNSv2) in 203 children, adolescents, and young adults with CMT. There was a moderate curvilinear correlation between the CMTPedS and the CMTNSv2 (Spearman's rho ρ = 0.716, p < 0.0001), although there appears to be a floor effect of the CMTNSv2 in patients with a milder CMT phenotype. Univariate analyses indicate that the relationship between the CMTPedS and CMTNSv2 scores improves with worsening disease severity and advancing age. Although one universal scale throughout life would be ideal, our data supports the transition from the CMTPedS in childhood to the CMTNSv2 in adulthood as a continuum of measuring lifelong disability in patients with CMT.     

Auditory temporal envelope processing in a patient with left-hemisphere damage

Abstract

Auditory temporal envelope processing was investigated in a patient showing a mild speech identification impairment following left-hemisphere damage. Three tasks evaluated the patient's ability to: (1) detect a sinusoidal amplitude modulation (SAM) applied to a white noise, as a function of modulation rate (i.e. her ‘temporal modulation transfer function’ or TMTF); (2) discriminate between two white noises amplitude modulated by time-reversed temporally asymmetric envelopes; and (3) identify white noises amplitude modulated by the temporal envelope of speech stimuli. Measurements of intensity discrimination thresholds were performed as a control task. Compared to normal data, the results obtained with the brain-damaged patient showed: (1) increased thresholds for the detection of SAM; (2) increased thresholds for the discrimination of temporal asymmetry; and (3) a deficit in the identification of speechenvelope noise stimuli. In contrast, intensity discrimination thresholds were within the normal range. Taken together, the results indicate a general impairment in auditory temporal acuity, which is now specified as a deficit in the coding of envelope rate and shape, and a deficit in the ability to use temporal envelope cues in speech processing. These results support the hypothesis that left-hemisphere damage is associated with an impairment in time analysis, which may cause, in turn, speech intelligibility disorders.     

Validity of the Italian Version of the Pittsburgh Sleep Quality Index (PSQI)

The aim of this study is to validate the Italian version of the Pittsburgh Sleep Quality Index (PSQI), comparing five different groups of individuals (healthy young and elderly, sleep apnoea syndrome patients, depressed patients, individuals with dementia) by both questionnaire scores and polysomnographic measures. Fifty individuals (10 for each group) participated in the study. Each of them filled in the PSQI and slept for two consecutive nights in the sleep laboratory. The PSQI showed an overall reliability coefficient (Cronbach’s α) of 0.835, indicating a high degree of internal consistency. The mean PSQI global score showed significant differences between groups, with an impaired overall quality of sleep in patients’ groups with respect to both the healthy groups. Results also indicated that the best cut-off score (differentiating “good” from “bad” sleepers) is 5. Pittsburgh Sleep Quality Index is a useful, valid and reliable tool for the assessment of sleep quality, with an overall efficiency comparable to the mother language version and differentiate “good” from “bad” sleepers. The Italian version of the questionnaire provides a good and reliable differentiation between normal and pathological groups, with higher scores reported by people characterized by impaired objectively evaluated sleep quality.