Chromosomal rearrangement segregating with adrenoleukodystrophy: a molecular analysis.

The relationship between X chromosome-linked adrenoleukodystrophy and the red/green color pigment gene cluster on Xq28 was investigated in a large kindred. The DNA in a hemizygous male showed altered restriction fragment sizes compatible with at least a deletion extending from the 5' end of the color pigment genes. Segregation analysis using a DNA probe within the color pigment gene cluster showed significant linkage with adrenoleukodystrophy (logarithm of odds score of 3.19 at theta = 0.0). These data demonstrate linkage, rather than association, between a unique molecular rearrangement in the color pigment gene cluster and adrenoleukodystrophy. The DNA changes in this region are thus likely to be helpful for determining the location and identity of the responsible gene.     

From the Cover: Femtosecond quantum control of molecular bond formation

Ultrafast lasers are versatile tools used in many scientific areas, from welding to eye surgery. They are also used to coherently manipulate light–matter interactions such as chemical reactions, but so far control experiments have concentrated on cleavage or rearrangement of existing molecular bonds. Here we demonstrate the synthesis of several molecular species starting from small reactant molecules in laser-induced catalytic surface reactions, and even the increase of the relative reaction efficiency by feedback-optimized laser pulses. We show that the control mechanism is nontrivial and sensitive to the relative proportion of the reactants. The control experiments open up a pathway towards photocatalysis and are relevant for research in physics, chemistry, and biology where light-induced bond formation is important.     

Visualizing electron rearrangement in space and time during the transition from a molecule to atoms

Imaging and controlling reactions in molecules and materials at the level of electrons is a grand challenge in science, relevant to our understanding of charge transfer processes in chemistry, physics, and biology, as well as material dynamics. Direct access to the dynamic electron density as electrons are shared or transferred between atoms in a chemical bond would greatly improve our understanding of molecular bonding and structure. Using reaction microscope techniques, we show that we can capture how the entire valence shell electron density in a molecule rearranges, from molecular-like to atomic-like, as a bond breaks. An intense ultrashort laser pulse is used to ionize a bromine molecule at different times during dissociation, and we measure the total ionization signal and the angular distribution of the ionization yield. Using this technique, we can observe density changes over a surprisingly long time and distance, allowing us to see that the electrons do not localize onto the individual Br atoms until the fragments are far apart (∼5.5 Å), in a region where the potential energy curves for the dissociation are nearly degenerate. Our observations agree well with calculations of the strong-field ionization rates of the bromine molecule.     

Femtosecond direct observation of charge transfer between bases in DNA

Charge transfer in supramolecular assemblies of DNA is unique because of the notion that the pi-stacked bases within the duplex may mediate the transport, possibly leading to damage and/or repair. The phenomenon of transport through pi-stacked arrays over a long distance has an analogy to conduction in molecular electronics, but the mechanism still needs to be determined. To decipher the elementary steps and the mechanism, one has to directly measure the dynamics in real time and in suitably designed, structurally well characterized DNA assemblies. Here, we report our first observation of the femtosecond dynamics of charge transport processes occurring between bases within duplex DNA. By monitoring the population of an initially excited 2-aminopurine, an isomer of adenine, we can follow the charge transfer process and measure its rate. We then study the effect of different bases next to the donor (acceptor), the base sequence, and the distance dependence between the donor and acceptor. We find that the charge injection to a nearest neighbor base is crucial and the time scale is vastly different: 10 ps for guanine and up to 512 ps for inosine. Depending on the base sequence the transfer can be slowed down or inhibited, and the distance dependence is dramatic over the range of 14 A. These observations provide the time scale, and the range and efficiency of the transfer. The results suggest the invalidity of an efficient wire-type behavior and indicate that long-range transport is a slow process of a different mechanism.     

On the mechanism of genetic recombination: electron microscopic observation of recombination intermediates.

This paper deals with the nature of recombination intermediates. Using the electron microscope to study the DNA of the plasmid colicin E1, we have observed more than 800 molecules that appear to represent intermediates in the process of recombination. Specifically, after isolating colicin DNA and linearizing it with the restriction enzyme EcoRI, we find crossed molecules with twice the normal colicin DNA content. These forms consist of two genome-length elements held together at a region of DNA homology. The molecules can be recovered from wild type and Rec B-C host cells but are not present among the colicin DNA forms isolated from recombination-deficient Rec A cells. We have termed the experimentally observed molecules "chi forms" and believe that they represent the recombination intermediate of the Holliday model.     

Direct observation of multiple misfolding pathways in a single prion protein molecule

Protein misfolding is a ubiquitous phenomenon associated with a wide range of diseases. Single-molecule approaches offer a powerful tool for deciphering the mechanisms of misfolding by measuring the conformational fluctuations of a protein with high sensitivity. We applied single-molecule force spectroscopy to observe directly the misfolding of the prion protein PrP, a protein notable for having an infectious misfolded state that is able to propagate by recruiting natively folded PrP. By measuring folding trajectories of single PrP molecules held under tension in a high-resolution optical trap, we found that the native folding pathway involves only two states, without evidence for partially folded intermediates that have been proposed to mediate misfolding. Instead, frequent but fleeting transitions were observed into off-pathway intermediates. Three different misfolding pathways were detected, all starting from the unfolded state. Remarkably, the misfolding rate was even higher than the rate for native folding. A mutant PrP with higher aggregation propensity showed increased occupancy of some of the misfolded states, suggesting these states may act as intermediates during aggregation. These measurements of individual misfolding trajectories demonstrate the power of single-molecule approaches for characterizing misfolding directly by mapping out nonnative folding pathways.     

Direct observation of reaction intermediates for a well defined heterogeneous alkene metathesis catalyst

Grafting of [W(NAr)(=CHtBu)(2,5-Me₂NC₄H₂)₂] on a silica partially dehydroxylated at 700°C (SiO_{2- (700)}) generates the corresponding monosiloxy complex [(SiO)W(NAr)(=CHtBu)(2,5-Me₂NC₄H₂)] as the major species (≈90%) along with [(SiO)W(NAr)(CH₂tBu)(2,5-Me₂NC₄H₂)₂], according to mass balance analysis, IR, and NMR studies. This heterogeneous catalyst displays good activity and stability in the metathesis of propene. Very importantly, solid state NMR spectroscopy allows observation of the propagating alkylidene as well as stable metallacyclobutane intermediates. These species have the same reactivity as the initial surface complex [(SiO)W(NAr)(=CHtBu)(2,5-Me₂NC₄H₂)], which shows that they are the key intermediates of alkene metathesis.     

Unusual sequence of VDJ rearrangement revealed by molecular analysis in a patient with indolent lymphoma

We report a unique case of indolent lymphoma with an unusual VDJ rearrangement. Polymerase chain reaction (PCR) analysis of bone marrow at the time of diagnosis was positive for both BCL-2/JH and CDRIII rearrangements. After treatment, the patient achieved complete remission (CR) with slow disappearance of both rearrangements (CDRIII and then BCL-2/JH). Subsequently, two new CDRIII rearrangements were detected in bone marrow, peripheral blood, and lymph node tissue. After this conversion, fluorescent activated cell sorting (FACS) analysis demonstrated monoclonal disease, suggesting that both CDRIII rearrangements originated from one cell. Histological evidence of a B-cell small lymphocytic lymphoma (B-SLL) infiltrate in the bone marrow became evident approximately 1 year after the two CDRIII rearrangements appeared. Direct sequencing revealed that one of the CDRIII sequences consisted of a VDVDJ rearrangement. This is the first report of such a rearrangement in a case of indolent lymphoma. This type of rearrangement has been described to result from a secondary VDJ recombination in childhood acute lymphoblastic leukemia (ALL) leading towards oligoclonality and poorer prognosis. Our observations suggest that such a finding in an indolent lymphoma patient may precede transformation into an aggressive disease. Early detection by PCR could have substantial impact on the prognosis of such patients.     

Macrophage migration inhibitory factor: molecular, cellular and genetic aspects of a key neuroendocrine molecule

The immunological and neuroendocrine properties of macrophage migration inhibitory factor (MIF) are diverse. In this article we review the known cellular, molecular and genetic properties of MIF that place it as a key regulatory cytokine, acting within both the innate and adaptive immune responses.The unexpected and paradoxical induction of MIF secretion by low concentrations of glucocorticoids is explored. The role of MIF as a locally acting modulator of glucocorticoid sensitivity within foci of inflammation is also discussed. MIF has no homology with any other pro-inflammatory cytokine and until recently lacked a recognised transmembrane receptor. MIF has also been shown to be directly taken up into target cells and to interact with intracellular signalling molecules, including the Jun activation domain-binding protein Jab-1.Comprehensive analysis of the MIF gene has identified important functional polymorphisms and a series of genetic studies has revealed both association and linkage of MIF with inflammatory diseases. Altered MIF regulation may therefore be pivotal to acquiring chronic inflammation following an innate immune response.     

Reactive oxygen intermediates reduce inactivation of serotonin in isolated, perfused rat lungs

Reactive oxygen intermediates such as free radicals have been proposed to mediate lung injury. The present work examined whether or not enzymatically generated oxygen metabolites altered serotonin clearance. Isolated, plasma-perfused rat lungs were exposed to xanthine oxidase (XO) and hypoxanthine (HX). Pulmonary arterial pressure (Ppa) and lung weight were recorded. Fulminant edema was defined as a spontaneous weight increase exceeding 500 mg. Inactivation of serotonin was determined by superfusion bioassay. XO and HX reduced serotonin inactivation from 74 +/- 3% (mean +/- SEM) to 62 +/- 2%. This reduction was inhibited by the scavenger enzymes superoxide dismutase (SOD) and catalase and by allopurinol, an inhibitor of XO. Hydrostatic edema and perfusion per se did not decrease the pulmonary clearance of serotonin. XO and HX did not significantly alter Ppa. Fulminant edema developed in four of six lungs after exposure to XO and HX compared with none in the other groups. It was concluded that reactive oxygen intermediates inhibited serotonin inactivation in isolated rat lungs.     

Direct structural observation of a molecular junction by high-energy x-ray reflectometry

We report a direct angstrom resolution measurement of the structure of a molecular-size electronic junction comprising a single (or a double) layer of alkyl-thiol and alkyl-silane molecules at the buried interface between solid silicon and liquid mercury. The high-energy synchrotron x-ray measurements reveal densely packed layers comprising roughly interface-normal molecules. The monolayer's thickness is found to be 3-4 A larger than that of similar layers at the free surfaces of both mercury and silicon. The origins of this and the other unusual features detected are discussed in this article. Measurements of the bilayer junction with an applied potential did not show visible changes in the surface normal structure.     

细胞间黏附分子-1在红霉素抗炎机制中的作用

目的 从分子水平上探讨红霉素(EM)的抗炎作用机制.方法 体外培养人支气管上皮细胞(16HBE),将细胞随机分为8组,先加入EM干预,后加入肿瘤坏死因子α(TNF-α)刺激.分组如下:①空白对照组;②TNF-α(20 ng/ml,16 h)组;③EM(0.3 μg/ml,24 h)+TNF-α(20 ng/ml,16 h)组;④EM(3 μg/ml,24 h)+TNF-α(20 ng/ml,16 h)组;⑤EM(30 μg/ml,24 h)+TNF-α(20 ng/ml,16 h)组;⑥EM(0.3 μg/ml,48 h)+TNF-α(20 ng/ml,16 h)组;⑦EM(3 μg/ml,48 h)+TNF-α(20 ng/ml,16 h)组;⑧EM(30 μg/ml,48 h)+TNF-α(20 ng/ml,16 h).然后收集各组细胞分别提取RNA,应用逆转录聚合酶链反应(RT-PCR)方法 检测细胞间黏附分子-1(ICAM-1) mRNA的表达.因ICAM-1 RT-PCR产物分子大小约为700 bp,与原设计产物分子大小490 bp不相符,进一步作基因克隆测序了解基因之间是否具有同源性.结果 ICAM-1的RT-PCR产物即为目的 基因:ICAM-1基因.TNF-α刺激人支气管上皮细胞(16HBE)后,细胞ICAM-1mRNA表达增高.先加入不同浓度及作用时间的EM,后加入TNF-α刺激人支气管上皮细胞(16HBE),各组细胞ICAM-1mRNA表达均降低,且提示有浓度与时间依赖性.结论 TNF-α能激活人支气管上皮细胞使其ICAM-1基因表达增高,促进炎症的发生发展.EM能抑制人支气管上皮细胞ICAM-1基因表达,可能是EM的抗炎作用机制之一.     

Purification and characterization of factor VII 304-Gln: a variant molecule with reduced activity isolated from a clinically unaffected male

Factor VII (FVII) is the plasma serine protease zymogen which, on binding to its cellular receptor tissue factor (TF), initiates blood coagulation. A 47-year-old man with no clinical bleeding tendency was found to have undetectable plasma FVII activity when tested in a one- stage assay using rabbit brain TF, but 0.3 U/mL using recombinant human TF and 1.04 U/mL FVII antigen. Variant FVII purified from his plasma showed an identical migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis to wild-type zymogen. By enzyme kinetic analysis the Km of the variant using FX as a substrate was 12-fold higher than that of normal FVII. Also, the variant FVII was unable to compete with wild-type FVII for limited rabbit TF binding sites. A ligand blot procedure was used to directly demonstrate reduced binding of recombinant human TF to the variant FVII compared with normal FVII. Genetic analysis of leukocyte DNA showed a G to A mutation in the propositus' gene at codon 304 that results in the substitution of a glutamine for an arginine residue in the catalytic domain of the protease. We conclude that this region of the FVII molecule is important for its function.     

Familial isolated hypoparathyroidism: a molecular genetic analysis of 8 families with 23 affected persons

Abnormalities in the parathyroid hormone (PTH) gene as a cause of hypoparathyroidism were evaluated by linkage analysis with DNA polymorphisms adjacent to the PTH gene in 8 families in which members were affected with familial isolated hypoparathyroidism (FIH). We found that in none of the 23 affected individuals was there absence of the parathyroid hormone gene or abnormal restriction patterns to suggest recognizable deletions, insertions, or rearrangements. To determine if subtle mutations within the PTH gene were associated with hypoparathyroidism in these families, we used the Pst I and Taq I restriction-site polymorphisms in linkage analysis as markers to differentiate between PTH alleles. In 4 families, affected sibs inherited different PTH gene alleles, implying that hypoparathyroidism was not due to an abnormality in the PTH gene. In 2 other families, linkage analysis was uninformative because of inability to differentiate between PTH alleles. In 2 families, concordance was found between the inheritance of hypoparathyroidism and specific PTH alleles in affected members, suggesting that in these families, hypoparathyroidism may be due to an alteration in or near the PTH structural gene. We conclude that FIH is a diverse group of disorders and is characterized by genetic and molecular heterogeneity. In some forms of FIH the mutation that leads to PTH deficiency does not lie within the region of the structural gene for PTH. Linkage analysis using DNA polymorphisms within the PTH gene is of benefit in identifying individuals with disorders of PTH secretion or synthesis in whom DNA sequencing and expression studies of the PTH gene might succeed in establishing the molecular basis of the disease.