Functional analysis of natural mutations in two TWIST protein motifs

Autosomal-dominant hypercholesterolemia (ADH) has been identified as a major risk factor for coronary vascular disease (CVD) and is associated with mutations in the low-density lipoprotein receptor (LDLR) and the apolipoprotein B (APOB) gene. Since 1991 DNA samples from clinically diagnosed ADH patients have been routinely analyzed for the presence of LDLR and APOB gene mutations. As of 2001, 1,641 index patients (164 index patients per year) had been identified, while from 2001 onward a more sensitive, high-throughput system was used, resulting in the identification of 1,177 new index patients (average=294 index patients per year). Of these 1,177 index cases, 131 different causative genetic variants in the LDLR gene and six different causative mutations in the APOB gene were new for the Dutch population. Of these 131 mutations, 83 LDLR and four APOB gene mutations had not been reported before. The inclusion of all 2,818 index cases into the national screening program for familial hypercholesterolemia (FH) resulted in the identification of 7,079 relatives who carried a mutation that causes ADH. Screening of the LDLR and APOB genes in clinically diagnosed FH patients resulted in approximately 77% of the patients being identified as carriers of a causative mutation. The population of patients with ADH was divided into three genetically distinct groups: carriers of an LDLR mutation (FH), carriers of an APOB mutation (FDB), and non-LDLR/non-APOB patients (FH3). No differences were found with regard to untreated cholesterol levels, response to therapy, and onset of CVD. However, all groups were at an increased risk for CVD. Therefore, to ultimately identify all individuals with ADH, the identification of new genes and mutations in the genes that cause ADH is of crucial importance for the ongoing national program to identify patients with ADH by genetic cascade screening.     

Analysis of the subcellular localization of the proteins Rep, Rep' and Cap of porcine circovirus type 1

Porcine circovirus type 1 (PCV1) encodes two major ORFs. The cap gene comprises the major structural protein of PCV, the rep gene specifies Rep and Rep', which are both essential for initiating the replication of the viral DNA. Rep corresponds to the full-length protein, whereas Rep' is a truncated splice product that is frame-shifted in its C-terminal sequence. In this study, the cellular localization of PCV1-encoded proteins was investigated by immune fluorescence techniques using antibodies against Rep, Rep' and Cap and by expression of viral proteins fused to green and red fluorescence proteins. Rep and Rep' protein co-localized in the nucleus of infected cells as well as in cells transfected with plasmids expressing Rep and Rep' fused to fluorescence proteins, but no signal was seen in the nucleoli. Rep and Rep' carry three potential nuclear localization signals in their identical N-termini, and the contribution of these motifs to nuclear import was experimentally dissected. In contrast to the rep gene products, the localization of the Cap protein varied. While the Cap protein was restricted to the nucleoli in plasmid-transfected cells and was also localized in the nucleoli at an early stage of PCV1 infection, it was seen in the nucleoplasm and the cytoplasm later in infection, suggesting that a shuttling between distinct cellular compartments occurs.     

医疗数据交换规格MML(Medical Markup Language)3.0汉化版的制作

MML(Medical Markup Language)是一套不同医疗设施间的数据交换规格。于1995年在日本被开发。MML从版本2.21开始使用XML(eXtensible Markup Language)作为标记语言。而最新版本3.0又遵循HL7Clincal Document Architecture(CDA),包含14模块和36个数据定义表格。目前在中国,还没有一个使用XML来结构整个病历内容的规格。鉴于MML的柔韧性,我们制作了一个基于3.0版本的汉化版。日本与中国虽然诊疗流程、病历记录的内容等都很相似,但是也有一些,比如民族的表现、中医诊断分类,医师资格分类等都是日本不存在的或者分类不同的信息。另外,因为国情不同,医疗保险制度也完全不同。为了使MML能在中国的医院适用,我们追加和更改了12个数据定义表格,并重新制作了医疗保险信息模块。MML汉化版不止是一个对原规格的翻译和说明,它还考虑了本地的需要。因此,使用MML汉化版在中国的医疗设施间进行医疗数据交换已经成为可能。     

眼球固定器在眼内肿瘤X-线放射外科治疗中的应用

目的:眼内肿瘤X-线放射外科定位受眼球旋转的影响,常规定位方法误差极大,眼肌缝扎痛苦较大.本文探讨X-线放射外科治疗眼内肿瘤眼球无创固定方法、CT定位方法、精度及扫描方法.材料与方法:应用研制的微真空角膜接触眼球固定器固定眼球进行眼内肿瘤CT定位,并进行验证扫描,研究CT定位的精度及扫描方法.结果:利用微真空角膜接触眼球固定器进行眼内肿瘤CT定位精度为0.81 mm,最大误差为1.18mm.重复扫描筛板在三维方向上误差均小于1mm.利用此方法对14例眼内肿瘤进行CT定位,肿瘤边界显示清晰,固定方法无创,局部反应轻微,效果满意.结论:利用微真空角膜接触眼球固定器进行眼内肿瘤X-线放射外科定位精度高,无创,解决了眼球旋转的问题,效果满意.     

Characterization of Atlantic halibut (Hippoglossus hippoglossus) Mx protein expression

Mx proteins are antiviral GTPases that are induced by type I interferons in vertebrates. An Atlantic halibut Mx cDNA (HHMx) was recently cloned. In this work, a polyclonal antiserum against HHMx protein was generated that detected a 71 kDa protein in the nuclei of Chinook salmon embryo cells transfected with the HHMx cDNA. Mx protein expression in organs of halibut was studied by immunoblot analysis after injection with the double-stranded RNA poly I:C or infectious pancreatic necrosis virus. Poly I:C stimulated increased Mx protein expression in liver, kidney, heart, spleen, gills and intestine. The Mx protein level in liver reached a maximum after 3 days and remained elevated for 14 days after treatment. IPNV infection resulted in increased Mx protein in liver from 4 to at least 35 days. Immunocytochemical detection of Mx proteins in blood smears from poly I:C treated halibut indicated that a cytoplasmic Mx form might exist in this species. Detection of Mx proteins in blood leukocytes could thus work as an early non-lethal test for viral infections.     

A spatial filtering technique to detect and localize multiple sources in the brain

Summary An algorithm for localization of electromagnetic activity in the central nervous system is explored. This algorithm generates a neural activity index map within the brain by passing surface recordings through a set of spatial filters. The covariance matrix of the surface recordings is used to optimize the spatial filters' responses. This approach is studied in simulated situations and in real data. The simulations show the method's capability to detect areas of activity without prior knowledge of the number of sources. The resolving power of the method increases with number of electrodes and signal-to-noise ratio, and it decreases with depth. The analysis of the electrophysiological data indicates that the method can distinguish simultaneously active areas in a realistic fashion. The analyzed recordings are bilateral median SEP responses, an epoch of spike activity showing several active regions and a recording with eye movement superimposed on spike activity. The method and the results are discussed in relation to current localization techniques.The authors wish to thank Dr. J. M. Guerit, Dr. R.E. Lasky and G. Rook for their valuable suggestions. We thank Dr. K. Hecox for his support and R. Birrenkot for preparing the figures.     

Human chromosome 3: high-resolution fluorescencein situ hybridization mapping of 40 uniqueNotl linking clones homologous to genes and cDNAs

Forty newNotl linking clones representing sequence tagged sites (STSs) were mapped by fluorescencein situ hybridization (FISH) to different regions of human chromosome 3 (HSA3). Clone NL1-245, containing human aminoacylase 1, was localized to 3p21.2–p21.1. Our previous localization of the CLC-2 chloride channel protein gene was refined to 3q27. Clone NL2-316 most likely contains a translocon-associated protein -subunit gene and was mapped to 3q23–q24. To our knowledge, this is the first time this gene has been mapped. OneNoti linking clone (NL1-229) probably contains a new protein phosphatase gene. This clone was mapped to 3p25. FiveNoti linking clones probably contain human expressed sequence tags (ESTs), as they possess sequences with a high level of identity (>90%) to cDNA clones. Other clones show 56–85% homology to known mammalian and human genes with various functions, including oncogenes and tumour-suppressor genes. These clones might represent new genes.accepted for publication by M. Schmid     

Contribution of the forebrain archistriatal gaze fields to auditory orienting behavior in the barn owl

A region in the barn owl forebrain, referred to as the archistriatal gaze fields (AGF), is shown to be involved in auditory orienting behavior. In a previous study, electrical microstimulation of the AGF was shown to produce saccadic movements of the eyes and head, and anatomical data revealed that neurons in the AGF region of the archistriatum project directly to brainstem tegmental nuclei that mediate gaze changes. In this study, we investigated the effects of AGF inactivation on the auditory orienting responses of trained barn owls. The AGF and/or the optic tectum (OT) were inactivated pharmacologically using the GABA_A agonist muscimol. Inactivation of the AGF alone had no effect on the probability or accuracy of orienting responses to contralateral acoustic stimuli. Inactivation of the OT alone decreased the probability of responses to contralateral stimuli, but the animals were still capable of orienting accurately toward stimuli on about 60% of the trials. Inactivation of both the AGF and the OT drastically decreased the probability of responses to 16–21% and, on the few trials that the animals did respond, there was no relationship between the final direction of gaze and the location of the stimulus. Thus, with the AGF and OT both inactivated, the animals were no longer capable of orienting accurately toward acoustic stimuli located on the contralateral side. These data confirm that the AGF is involved in gaze control and that the AGF and the OT have parallel access to gaze control circuitry in the brainstem tegmentum. In these respects, the AGF in barn owls is functionally equivalent to the frontal eye fields in primates.     

Expression and localization of mutant p16 proteins in melanocytic lesions from familial melanoma patients

Little is known about the correlation between the loss of p16 expression and tumor progression in familial melanoma; no systematic study has been conducted on p16 expression in melanocytic tumors from patients carrying germline CDKN2A mutations. We analyzed 98 early primary lesions from familial patients, previously tested for germline CDKN2A status, by quantitative immunohistochemistry using 3 p16 antibodies. We found that p16 expression was inversely correlated with tumor progression and was significantly lower in melanomas, including in situ lesions, than in nevi. Of other features analyzed, tumor thickness showed the most significant correlation with p16 levels. Lesions from mutation-negative patients displayed combined nuclear and cytoplasmic staining. However, some mutation-positive lesions (ie, G101W, 113insR, M53I, R24P, and 33ins24), including benign nevi, showed nuclear mislocalization, confirming previous studies suggesting that subcellular distribution indicates functional impairment of p16.     

The effect of eye position on auditory lateralization

The present study examines whether the direction of gaze can influence sound lateralization. For this purpose, dichotic stimuli with variable interaural level difference (ILD) were presented under different conditions of visual fixation. In experiment 1, subjects with their head fixed directed their gaze to a given target, simultaneously adjusting the ILD of continuous pure tone or noise stimuli so that their location was perceived in the median plane of the head. The auditory adjustments were significantly correlated with gaze direction. During eccentric fixation, the psychophysical adjustments to the median plane shifted slightly toward the direction of gaze. The magnitude of the shift was about 1–3 dB, over a range of fixation angles of 45° to either side. The eye position effect, measured as a function of pure-tone frequency, was most pronounced at 2 kHz and showed a tendency to decrease at lower and higher frequencies. The effect still occurred, although weaker, even when the eyes were directed to eccentric positions in darkness and without a fixation target. In experiment 2, the adjustment method was replaced by a two-alternative forced-choice method. Subjects judged whether sound bursts, presented with variable ILDs, were perceived on the left or right of the median plane during fixation of targets in various directions. Corresponding to experiment 1, the psychometric functions shifted significantly with gaze direction. However, the shift was only about half as large as that found in experiment 1. The shift of the subjective auditory median plane in the direction of eccentric gaze, observed in both experiments, indicates that dichotic sound is localized slightly to the opposite side, i.e., to the left when the gaze is directed to the right and vice versa. The effect may be related to auditory neurons which exhibit spatially selective receptive fields that shift with eye position.