Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding α-crystallin

Unlike many pathogens that are overtly toxic to their hosts, the primary virulence determinant of Mycobacterium tuberculosis appears to be its ability to persist for years or decades within humans in a clinically latent state. Since early in the 20th century latency has been linked to hypoxic conditions within the host, but the response of M. tuberculosis to a hypoxic signal remains poorly characterized. The M. tuberculosis α-crystallin (acr) gene is powerfully and rapidly induced at reduced oxygen tensions, providing us with a means to identify regulators of the hypoxic response. Using a whole genome microarray, we identified >100 genes whose expression is rapidly altered by defined hypoxic conditions. Numerous genes involved in biosynthesis and aerobic metabolism are repressed, whereas a high proportion of the induced genes have no known function. Among the induced genes is an apparent operon that includes the putative two-component response regulator pair Rv3133c/Rv3132c. When we interrupted expression of this operon by targeted disruption of the upstream gene Rv3134c, the hypoxic regulation of acr was eliminated. These results suggest a possible role for Rv3132c/3133c/3134c in mycobacterial latency.

Ceramide formation during heat shock: a potential mediator of alpha B-crystallin transcription.

Ceramide has been identified as a potential second messenger that may mediate cell differentiation and apoptosis after exposure to hormonal agonists such as 1 alpha, 25-dihydroxyvitamin D3, tumor necrosis factor alpha, or gamma-interferon. The secondary cellular events that follow ceramide generation remain undefined. We report that in NIH WT-3T3 cells, ceramide induces an enhancement of gene transcription of alpha B-crystallin, a small heat shock protein. The levels of alpha B-crystallin, as measured by Northern blot and immunoblot analyses, were increased by the addition of an exogenous short-chain ceramide, N-acetylsphingosine, or by increasing endogenous intracellular ceramide by inhibition of glucosylceramide synthase. Similar effects were not seen in the expression of the closely related gene, Hsp25. To ascertain whether ceramide-mediated gene transcription was a feature of the heat shock response, cell ceramide was measured in heat shocked cells and observed to be elevated 2-fold immediately upon the return of cells to 37 degrees C. Thus ceramide formed after heat shock treatment of 3T3 cells may mediate the transcription events associated with the cell stress response.

Disruption of the gene for hsp30, an alpha-crystallin-related heat shock protein of Neurospora crassa, causes defects in thermotolerance.

The alpha-crystallin-related heat shock proteins are produced by all eukaryotes, but the role of these proteins in thermoprotection remains unclear. To investigate the function of one of these proteins, we disrupted expression of the single-copy hsp30 gene of Neurospora crassa, using repeat-induced point mutagenesis, and we generated and characterized mutant strains that were deficient in hsp30 synthesis. These strains could grow at high temperature and they acquired thermotolerance from a heat shock. However, the hsp30-defective strains proved to be extremely sensitive to the combined stresses of high temperature and carbohydrate limitation, enforced by the addition of a nonmetabolizable glucose analogue. Under these conditions, their survival was reduced by 90% compared with wild-type cells. This sensitive phenotype was reversed by reintroduction of a functional hsp30 gene into the mutant strains. The mutant cells contained mitochondria from which a 22-kDa protein was readily extracted with detergents, in contrast to its retention by the mitochondria of wild-type cells. Antibodies against hsp30 coimmunoprecipitated a protein also of approximately 22 kDa from wild-type cells. Results of this study suggest that hsp30 may be important for efficient carbohydrate utilization during high temperature stress and that it may interact with other mitochondrial membrane proteins and function as a protein chaperone.

Pax-6 is essential for lens-specific expression of zeta-crystallin.

Pax-6 is essential for normal eye development and has been implicated as a "master gene" for lens formation in embryogenesis. Guinea pig zeta-crystallin, a taxon-specific enzyme crystallin, achieves high expression specifically in lens through use of an alternative promoter. Here we show that Pax-6 binds a site in this promoter, which is essential for lens-specific expression. Lens and lens-derived cells exhibit a tissue-specific pattern of alternative splicing of Pax-6 transcripts and Pax-6 is expressed in adult lenses and cells that support zeta-crystallin expression. These results suggest that zeta-crystallin is a natural target gene for Pax-6 and that this Pax family member has a direct role in the continuing expression of tissue-specific genes.

Pax-6 and lens-specific transcription of the chicken delta 1-crystallin gene.

The abundance of delta-crystallin in the chicken eye lens provides an advantageous marker for tissue-specific gene expression during cellular differentiation. The lens-specific expression of the delta 1-crystallin gene is governed by an enhancer in the third intron, which binds a positive (delta EF2) and negative (delta EF1) factor in its core region. Here we show by DNase I footprinting, electrophoretic mobility-shift assays, and cotransfection experiments with the delta 1-promoter/enhancer fused to the chloramphenicol acetyltransferase reporter gene that the delta 1-crystallin enhancer has two adjacent functional Pax-6 binding sites. We also demonstrate by DNase I footprinting that the delta EF1 site can bind the transcription factor USF, raising the possibility that USF may cooperate with Pax-6 in activation of the chicken delta 1- and alpha A-crystallin genes. These data, coupled with our recent demonstration that Pax-6 activates the alpha A-crystallin gene, suggest that Pax-6 may have been used extensively throughout evolution to recruit and express crystallin genes in the lens.

Alpha B-crystallin, a new independent marker for poor prognosis in head and neck cancer

Objectives. Gene expression profiling has provided many insights into tumor progression but translation to clinical practice has been limited. We have previously identified a list of potential markers by the differences of expression profiling of seven matched head and neck cancer (HNSCC) tumors with autologous normal oral mucosa (NOM). Alpha B-crystallin (CRYAB) was in the top 5% of genes identified with statistically significant differences in expression between tumor and NOM at the mRNA level. The objective was to confirm this in routine paraffin sections at the protein level. Study Design: The level of alpha B-crystallin was determined in tumors of 62 HNSCC patients whose prognosis was known for 5 years. Methods. Immunohistochemical detection of alpha B-crystallin expression was performed on HNSCC paraffin sections. Results. Univariate survival analysis identified lack of alpha B-crystallin staining as an independent prognostic marker for disease-free interval (P < 0.001) and overall survival (P < 0.002) of HNSCC patients over the 5-year observation period. Notably, all 13 patients (100%), including 5 patients with nodal disease whose tumors lacked alpha B-crystallin had no recurrences (P < 0.001). Nineteen of 27 node-negative patients stained positive for alpha B-crystallin and seven of the 19 (36.8%) had recurrences. Conclusion: Presence or absence of expression of alpha B-crystallin was a powerful marker for prognosis in this series of patients.

Age-related development of a refractive index plateau in the human lens : evidence for a distinct nucleus

The human lens comprises two distinct regions in which the refractive index changes at different rates. The periphery contains a rapidly increasing refractive index gradient, which becomes steeper with age. The inner region contains a shallow gradient, which flattens with age, due to formation of a central plateau, of RI = 1.418, which reaches a maximum size of 7.0 × 3.05 mm around age 60 years. Formation of the plateau can be attributed to compression of fibre cells generated in prenatal life. Present in prenatal but not in postnatal fibre cells, γ-crystallin may play a role in limiting nuclear cell compression.

Quantitative and qualitative changes in gene expression patterns characterize the activity of plaques in multiple sclerosis

Multiple sclerosis (MS) is a complex autoimmune disorder of the CNS with both genetic and environmental contributing factors. Clinical symptoms are broadly characterized by initial onset, and progressive debilitating neurological impairment. In this study, RNA from MS chronic active and MS acute lesions was extracted, and compared with patient matched normal white matter by fluorescent cDNA microarray hybridization analysis. This resulted in the identification of 139 genes that were differentially regulated in MS plaque tissue compared to normal tissue. Of these, 69 genes showed a common pattern of expression in the chronic active and acute plaque tissues investigated (Pvalue<0.0001, ρ=0.73, by Spearman's ρ analysis); while 70 transcripts were uniquely differentially expressed (≥1.5-fold) in either acute or chronic active tissues. These results included known markers of MS such as the myelin basic protein (MBP) and glutathione S-transferase (GST) M1, nerve growth factors, such as nerve injury-induced protein 1 (NINJ1), X-ray and excision DNA repair factors (XRCC9 and ERCC5) and X-linked genes such as the ribosomal protein, RPS4X. Primers were then designed for seven array-selected genes, including transferrin (TF), superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1), GSTP1, crystallin, alpha-B (CRYAB), phosphomannomutase 1 (PMM1) and tubulin β-5 (TBB5), and real time quantitative (Q)-PCR analysis was performed. The results of comparative Q-PCR analysis correlated significantly with those obtained by array analysis (r=0.75, Pvalue<0.01, by Pearson's bivariate correlation). Both chronic active and acute plaques shared the majority of factors identified suggesting that quantitative, rather than gross qualitative differences in gene expression pattern may define the progression from acute to chronic active plaques in MS.

Fatty acid uptake and incorporation into phospholipids in the rat lens

PURPOSE. Phospholipids are a major component of lens fiber cells and influence the activity of membrane proteins. Previous investigations of fatty acid uptake by the lens are limited. The purpose of the present study was thus to determine whether exogenous fatty acids could be taken up by the rat lens and incorporated into molecular phospholipids. METHODS. Lenses were incubated with fluorescently labeled palmitic acid and then analyzed by confocal microscopy. Concurrently, lenses incubated with either fluorescently labeled palmitic acid or the more physiologically relevant (13)C(18)-oleic acid were sectioned into nuclear and cortical regions and analyzed by highly sensitive and structurally selective electrospray ionization tandem mass spectrometry techniques. RESULTS. The detection of fluorescently labeled palmitic acid, even after 16 hours of incubation, was limited to approximately the outer 25% to 30% of the rat lens. Mass spectrometry also revealed the presence of free (13)C(18)-oleic acid in the cortex but not the nucleus. No evidence could be found for incorporation of fluorescently labeled palmitic acid into phospholipids; however, a low level of (13)C(18)-oleic acid incorporation into phosphatidylethanolamine (PE), specifically PE (PE 16:0/(13)C(18) 18:1) was detected in the lens cortex after 16 hours. CONCLUSIONS. These data demonstrate that uptake of exogenous (e.g., dietary fatty acids) by the lens and their incorporation into phospholipids is minimal, most likely occurring only during de novo synthesis in the outermost region of the lens. This finding adds support to the hypothesis that once synthesized there is no active remodeling or turnover of fiber cell phospholipids.

Annealing-free high-mobility diketopyrrolopyrrole-quaterthiophene copolymer for solution-processed organic thin film transistors

A donor-acceptor polymer semiconductor, PDQT, comprising diketopyrrolopyrrole (DPP) and β-unsubstituted quaterthiophene (QT) for organic thin film transistors (OTFTs) is reported. This polymer forms ordered layer-by-layer lamellar packing with an edge-on orientation in thin films even without thermal annealing. The strong intermolecular interactions arising from the fused aromatic DPP moiety and the DPP-QT donor-acceptor interaction facilitate the spontaneous self-assembly of the polymer chains into close proximity and form a large π-π overlap, which are favorable for intermolecular charge hopping. The well-interconnected crystalline grains form efficient intergranular charge transport pathways. The desirable chemical, electronic, and morphological structures of PDQT bring about high hole mobility of up to 0.97 cm2/(V·s) in OTFTs with polymer thin films annealed at a mild temperature of 100 °C and similarly high mobility of 0.89 cm2/(V·s) for polymer thin films even without thermal annealing.

非晶状体βγ晶状体蛋白与三叶因子蛋白复合物诱导兔胸主动脉环内皮依赖的持续收缩效应

脊椎动物中,非晶状体βγ-晶状体蛋白广泛分布于各种组织,但是功能知之甚少.三叶因子在创伤修复与肿瘤发生中具有重要作用,其分子作用机制尚不清楚.非晶状体βγ晶状体蛋白与三叶因子蛋白复合物(βγ-CAT)是一个从大蹼铃蟾皮肤分泌物中分离的一类全新的蛋白复合物.研究表明,βγ-CAT能够诱导离体的兔胸主动脉产生快速而持续的收缩,结合药理学抑制剂,细胞培养,激光共聚焦显微镜和免疫荧光原位组化,从细胞和分子水平对其作用机制进行研究.结果表明:.βγ-CAT诱导兔胸主动脉产生的收缩效应为剂量依赖(2-35 nmol/L)和内皮依赖(P＜0.01).在βγ-CAT(25nmol/L)处理的主动脉环的内皮细胞层检测到肿瘤坏死因子-α的释放.同时,βγ-CAT能够诱导原代培养的兔胸主动脉内皮细胞(RAEC)快速释放肿瘤坏死因子-α,βγ-CAT(25nmol/L)分别处理5和30min,RAEC释放的肿瘤坏死因子-α的浓度分别为(34.17±5.10)pg/mL和(98.01±4.67)pg/mL(P＜0.01).表明肿瘤坏死因子-α在βγ-CAT诱导兔胸丰动脉产生的收缩效应中发挥重要作用.为进一步深入研究非晶状体βγ晶状体蛋白与三叶因子的生理功能提供了新的思路和线索.

电镜分析非晶状体βγ晶状体蛋白与三叶因子蛋白复合物在人红细胞膜上的孔道形成效应

非晶状体βγ晶状体蛋白与三叶因子蛋白复合物(non-lens βγ-crystallin and trefoil factor complex,βγ-CAT)是一个从大蹼铃蟾(Bombina maxima)皮肤分泌物中分离的大然分子量为72kDa的全新的蛋白复合物.本研究测定了βγ-CAT处理红细胞后引起细胞内钾离子外流与溶血效应的时效曲线,结合扫描电子显微镜和透射电子显微镜分析βγ-CAT处理红细胞引起的早期形态学变化.结果表明:βγ-CAT(3 nmol/L)37℃处理红细胞5 min,(93.31±5.89)%的细胞内钾离子迅速外流(P＜0.01),相心溶解率为(13.12±1.92)%(P＜0.05).电子显微镜观察发现红细胞形态发生明显变化,细胞体枳增加,肿胀.少数红细胞表面向外形成棘状异常突起,部分肿胀细胞内的血红蛋白通过棘状突起缺口向细胞外喷射血红蛋白.表明βγ-CAT通过在红细胞膜上形成孔道使细胞内钾离子迅速外流导致红细胞内渗透压改变而溶血.其结果为理解βγ-CAT的溶血机制提供了直接的形态学证据.

Independent Transport and Sorting of Functionally Distinct Protein Families in Tetrahymena thermophila Dense Core Secretory Granules

Dense core granules (DCGs) in Tetrahymena thermophila contain two protein classes. Proteins in the first class, called granule lattice (Grl), coassemble to form a crystalline lattice within the granule lumen. Lattice expansion acts as a propulsive mechanism during DCG release, and Grl proteins are essential for efficient exocytosis. The second protein class, defined by a C-terminal beta/gamma-crystallin domain, is poorly understood. Here, we have analyzed the function and sorting of Grt1p (granule tip), which was previously identified as an abundant protein in this family. Cells lacking all copies of GRT1, together with the closely related GRT2, accumulate wild-type levels of docked DCGs. Unlike cells disrupted in any of the major GRL genes, Delta GRT1 Delta GRT2 cells show no defect in secretion, indicating that neither exocytic fusion nor core expansion depends on GRT1. These results suggest that Grl protein sorting to DCGs is independent of Grt proteins. Consistent with this, the granule core lattice in Delta GRT1 Delta GRT2 cells appears identical to that in wild-type cells by electron microscopy, and the only biochemical component visibly absent is Grt1p itself. Moreover, gel filtration showed that Grl and Grt proteins in cell homogenates exist in nonoverlapping complexes, and affinity-isolated Grt1p complexes do not contain Grl proteins. These data demonstrate that two major classes of proteins in Tetrahymena DCGs are likely to be independently transported during DCG biosynthesis and play distinct roles in granule function. The role of Grt1p may primarily be postexocytic; consistent with this idea, DCG contents from Delta GRT1 Delta GRT2 cells appear less adhesive than those from the wild type.

EPITAXIAL LATERAL OVERGROWTH OF GaN ON SILICON-ON-INSULATOR

From a single process, GaN layers were laterally overgrown on maskless stripe-patterned (111) silicon-on-insulator (SOI) substrates by metalorganic chemical vapor deposition. The influence of stress on the behavior of dislocations at the coalescence during growth was observed using transmission electron microscopy (TEM). Improvement of the crystallin equality of the GaN layer was demonstrated by TEM and micro-Raman spectroscopy. Furthermore, the benefits of SOI substrates for GaN growth are also discussed.

大蹼铃蟾（Bombina maxima）皮肤分泌物非晶状体-晶状体蛋白与三叶因子复合物细胞生物学活性和分子作用机制

非晶状体-晶状体蛋白质(non-lens -crystallins)在脊椎动物中以一簇的基 因家族的形式存在，在各种上皮细胞中广泛表达，但对其在体内承担的功能，人 们几乎一无所知。三叶因子蛋白(trefoil factors, TFFs)主要分布在胃肠道上皮和两 栖动物皮肤表面，许多研究表明该家族的蛋白质，在粘膜保护，损伤修复和肿瘤 抑制中具有重要的作用；但是自发现以来，TFFs 一直作为孤儿配基存在，对于 其作用的机制了解得很少。人们从来没有把两个家族的蛋白质联系在一起来考虑 过。 本实验室从中国特有的两栖动物大蹼铃蟾(Bombina maxima)皮肤分泌物中， 分离到这两个家族的蛋白质的天然结合在一起的复合物，并命名为：非晶状体- 晶状体蛋白和三叶因子蛋白复合物（non-lens -crystallin and treifol factor complex, -CAT）。尽管在低剂量下，-CAT 已对小鼠，大鼠和兔有致死活性， 但其结构与人源的non-lens -crystallins 和TFFs 的同源性，提示了它可能在正 常的生理功能中起到重要作用，也为揭示两类重要的蛋白质的功能和作用机制提 供了可能性。本研究工作，在多种细胞株中，对-CAT 的生物学活性作了详细 的研究，并对其作用机制进行了进一步深入的探讨。 我们原代培养了人脐静脉血管内皮细胞(HUVEC) ，兔主动脉内皮细胞 (RAEC)，兔心内皮细胞(REEC)；培养了多种肿瘤细胞株。-CAT 能够引起多种 贴壁细胞的脱落。-CAT 在高剂量下能够引起这些细胞的凋亡；但是在不同的 细胞株中，发生凋亡的通路可能是不同的。在低剂量下，-CAT 能够促进细胞 的迁移，对HUVEC 具有诱导伤口修复的活性。 以HUVEC 细胞为模型，我们探讨了-CAT 的作用机制。在激光共聚焦显 微镜下，观察到-CAT 诱导HUVEC 发生囊泡化，这个效应是剂量依赖的；囊 泡化的发生不依赖于NH4Cl 的存在，但是NH4Cl 能够增大囊泡化的效应。在荧 光染料Cy3 直接标记-CAT 时，观察到-CAT 被定向运输到细胞核上。荧光染 料FITC 分别标记-CAT 的轻链和重链的多克隆抗体，免疫荧光染色发现，在较 短的时间(5 min)内, -CAT 已经进入细胞，并有部分分子被运输到细胞核上。随 着时间增加到30 min，轻链和重链在核上的比例也渐增加；到2 小时，-CAT的重链全部集中在细胞核上，而-CAT 的轻链则退出核外，主要聚集在细胞核 周围。核定位显示-CAT 分子进入HUVEC 细胞核，可能在基因的转录调节中 发挥作用。我们运用基因芯片检测了加药处理前后细胞蛋白质表达水平的变化。 在四组重复实验中均显示，加入-CAT 处理后，有121 个基因发生上调，这些 基因在调节细胞的生存和死亡中具有复杂的功能。其中核受体蛋白质家族 （NR4A1 等）的变化最为明显。而其他的基因包括调节细胞早期生长，凋亡， 炎症反应的相关基因和金属蛋白酶。同时，加入-CAT 处理后，仅有2 个基因 发生下调，包括胶原I，这为解释细胞发生脱落和调亡提供了分子基础。 本研究工作揭示了非晶状体-晶状体蛋白和三叶因子蛋白的相互作用，共 同定位到细胞核上，并调节基因的转录，首次提示非晶状体-晶状体蛋白可能 参与一条全新的细胞信号调节途径，在组织平衡，肿瘤发生和胚胎发育中起到重 要的作用；同时也为三叶因子蛋白的分子作用机制的解析提供了一种新的可能 性。