H－ras在颊粘膜正常、良性、癌前及恶性变上皮中的表达：免疫组织化学定量研究

本文通过建立图象分析方法对免疫组织化学反应结果进行定量,检测观察Ｈ－ｒａｓ在口腔颊粘膜上皮在正常（Ｎ）、慢性炎症（ＩＦ）、癌旁上皮（ＥＡＣ）和鳞癌（ＳＣＣ）的变化过程中的表达并进行分析。结果显示Ｈ－ｒａｓ在ＳＣＣ组中,以中等分化的ＳＣＣ无论是Ｈ－ｒａｓ表达的量还是细胞阳性率都较高。此外,组织学观察显示,Ｈ－ｒａｓ在处于分化末期但尚未角化的正常上皮细胞中有较高的表达。本文结果显示了Ｈ－ｒａｓ的过表达与上皮细胞的会化程度密切相关。本研究还显示,所采用的阳性区域透光值、平均总透光值及阳性反应区域与阴性反应区域比值可靠并有相关性。这进一步说明了用免疫组化定量方法检测Ｈ－ｒａｓ癌基因表达的精确和可靠性。     

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Lectin-binding sites on the plasma membranes of rabbit spermatozoa: Changes in surface receptors during epididymal maturation and after ejaculation

Modifications in rabbit sperm plasma membranes during epididymal passage and after ejaculation were investigated by used of three lectins: concanavalin A (Con A); Ricinus communis I (RCA(I)); and wheat germ agglutinin (WGA). During sperm passage from caput to cauda epididymis, agglutination by WGA drastically decreased, and agglutination by RCA(I) slightly decreased, although agglutination by Con A remained approximately unchanged. After ejaculation, spermatozoa were agglutinated to a similar degree or slightly less by Con A, WGA, and RCA(I), compared to cauda epididymal spermatozoa. Ultrastructural examination of sperm lectin-binding sites with ferritin- lectin conjugates revealed differences in the densities of lectin receptors in various sperm regions, and changes in the same regions during epididymal passage and after ejaculation. Ferritin-RCA(I) showed abrupt changes in lectin site densities between acrosomal and postacrosomal regions of sperm heads. The relative amounts of ferritin-RCA(I) bound to heads of caput epididymal or ejaculated spermatozoa. Tail regions were labeled by ferritin RCA(I) almost equally on caput and cauda epididymal spermatozoa, but the middle-piece region of ejaculated spermatozoa was slightly more densely labeled than the principal-piece region, and these two regions on ejaculated spermatozoa were labeled less than on caput and cuada epididymal spermatozoa. Ferritin-WGA densely labeled the acrosomal region of caput epididymal spermatozoa, although labeling of cauda epidiymal spermatozoa was relatively sparse except in the apical area of the acrosomal region. Ejaculated spermatozoa bound only a few molecules of ferritin-WGA, even at the highest conjugate concentrations used. Caput epididymal, but not cauda epididymal or ejaculated spermatozoa, bound ferritin-WGA in the tail regions. Dramatic differences in labeling densities during epididymal passage and after ejaculation were not found with ferritin-Con A.     

Interaction of 92-kDa type IV collagenase with the tissue inhibitor of metalloproteinases prevents dimerization, complex formation with interstitial collagenase, and activation of the proenzyme with stromelysin.

Secreted metalloproteases initiating proteolytic degradation of collagens and proteoglycans play a critical role in remodeling of the connective tissue. Activation of the secreted proenzymes and interaction with their specific inhibitors TIMP and TIMP-2 are responsible for regulation of enzyme activity in extracellular space. We have previously demonstrated that 92- and 72-kDa Type IV procollagenases, in contrast to interstitial collagenase (ClI), form specific complexes with TIMP and the related inhibitor TIMP-2, respectively. The physiologic significance of the proenzyme-inhibitor complex and the mechanism of activation of Type IV collagenases remained unclear. Here, we demonstrate that in the absence of TIMP, 92-kDa Type IV procollagenase (92T4Cl) can form a covalent homodimer and a novel complex with ClI. In the presence of TIMP, the formation of a 92T4Cl proenzyme complex with TIMP prevents dimerization, formation of the complex with ClI, and activation of the 92T4Cl proenzyme by stromelysin, a related metalloprotease. The proenzyme homodimer is unable to form a complex with TIMP. All TIMP-free forms of the proenzyme can be activated by stromelysin. The 92T4Cl-ClI complex can be activated to yield a complex active against both gelatin and fibrillar Type I collagen, suggesting a mechanism for cooperative action of two enzymes in reducing collagen fibrils to small peptides under physiologic conditions.     

A mouse-adapted SARS-coronavirus causes disease and mortality in BALB/c mice

No single animal model for severe acute respiratory syndrome (SARS) reproduces all aspects of the human disease. Young inbred mice support SARS-coronavirus (SARS-CoV) replication in the respiratory tract and are available in sufficient numbers for statistical evaluation. They are relatively inexpensive and easily accessible, but their use in SARS research is limited because they do not develop illness following infection. Older (12- to 14-mo-old) BALB/c mice develop clinical illness and pneumonitis, but they can be hard to procure, and immune senescence complicates pathogenesis studies. We adapted the SARS-CoV (Urbani strain) by serial passage in the respiratory tract of young BALB/c mice. Fifteen passages resulted in a virus (MA15) that is lethal for mice following intranasal inoculation. Lethality is preceded by rapid and high titer viral replication in lungs, viremia, and dissemination of virus to extrapulmonary sites accompanied by lymphopenia, neutrophilia, and pathological changes in the lungs. Abundant viral antigen is extensively distributed in bronchial epithelial cells and alveolar pneumocytes, and necrotic cellular debris is present in airways and alveoli, with only mild and focal pneumonitis. These observations suggest that mice infected with MA15 die from an overwhelming viral infection with extensive, virally mediated destruction of pneumocytes and ciliated epithelial cells. The MA15 virus has six coding mutations associated with adaptation and increased virulence; when introduced into a recombinant SARS-CoV, these mutations result in a highly virulent and lethal virus (rMA15), duplicating the phenotype of the biologically derived MA15 virus. Intranasal inoculation with MA15 reproduces many aspects of disease seen in severe human cases of SARS. The availability of the MA15 virus will enhance the use of the mouse model for SARS because infection with MA15 causes morbidity, mortality, and pulmonary pathology. This virus will be of value as a stringent challenge in evaluation of the efficacy of vaccines and antivirals.     

Phylogenetic utility of the nuclear gene arginine decarboxylase: an example from Brassicaceae

Arginine decarboxylase (ADC) is an important enzyme in the production of putrescine and polyamines in plants. It is encoded by a single or low-copy nuclear gene that lacks introns in sequences studied to date. The rate of Adc amino acid sequence evolution is similar to that of ndhF for the angiosperm family studied. Highly conserved regions provide several target sites for PCR priming and sequencing and aid in nucleotide and amino acid sequence alignment across a range of taxonomic levels, while a variable region provides an increased number of potentially informative characters relative to ndhF for the taxa surveyed. The utility of the Adc gene in plant molecular systematic studies is demonstrated by analysis of its partial nucleotide sequences obtained from 13 representatives of Brassicaceae and 3 outgroup taxa, 2 from the mustard oil clade (order Capparales) and 1 from the related order Malvales. Two copies of the Adc gene, Adc1 and Adc2, are found in all members of the Brassicaceae studied to data except the basal genus Aethionema. The resulting Adc gene tree provides robust phylogenetic data regarding relationships within the complex mustard family, as well as independent support for proposed tribal realignments based on other molecular data sets such as those from chloroplast DNA.      

Effects of aerobic exercise therapy and cognitive behavioural therapy on functioning and quality of life in amyotrophic lateral sclerosis: protocol of the FACTS-2-ALS trial

Background

Neuropathic pain must be correctly diagnosed for optimal treatment. The questionnaire named Neuropathic Pain Symptom Inventory (NPSI) was developed in its original French version to evaluate the different symptoms of neuropathic pain. We hypothesized that the NPSI might also be used to differentiate neuropathic from non-neuropathic pain.

Methods

We translated the NPSI into German using a standard forward-backward translation and administered it in a case-control design to patients with neuropathic (n = 68) and non-neuropathic pain (headache and osteoarthritis, n = 169) to validate it and to analyze its discriminant properties, its sensitivity to change, and to detect neuropathic pain subgroups with distinct profiles.

Results

Using a sum score (the NPSI-G score), we found sensitivity to change (r between 0.37 and 0.5 for pain items of the graded chronic pain scale) and could distinguish between neuropathic and other pain on a group basis, but not for individual patients. Post hoc development of a discriminant score with optimized diagnostic properties to distinguish neuropathic pain from non-neuropathic pain resulted in an instrument with high sensitivity (91%) and acceptable specificity (70%). We detected six different pain profiles in the patient group with neuropathic pain; three profiles were found to be distinct.

Conclusions

The NPSI-G potentially combines the properties of a diagnostic tool and an instrument to identify subtypes of neuropathic pain.     

Duality of interaction outcomes in a plant–frugivore multilayer network

In plant–animal interactions, species are commonly labeled as either mutualists or antagonists, based on the most common, most studied, or most easily observed outcome. Nevertheless, evidence from simple systems comprising 2–4 species suggests that those labels are an oversimplification: individual species often function in both roles, either simultaneously or at different places or times. We include both mutualistic and antagonistic interactions between mammals and seeds in a multilayer network, to explore for the first time the community‐level consequences of the dual roles played by some species. We tested whether negative and positive interactions within a plant–frugivore network are separated into different modules, or whether they overlap due to the presence of frugivores that both kill and disperse seeds. The frugivorous diets of nonvolant small mammals were studied at one dry tropical forest site in southeastern Brazil by analyzing fecal samples from individuals captured in live traps. Seed viability was assessed with a tetrazolium test to determine the outcome of those interactions, as estimated by whether or not seeds survived gut passage. Interactions were analyzed as a weighted multilayer network, subdivided into one potentially mutualistic (live seeds deposited) and one antagonistic (dead seeds deposited) layer. The two layers had similar structure with high overlap between them. Some mammal species exhibited highly central, dual roles, acting both as antagonists and mutualists, in many cases of the same plant species. Dispersal service by most of these small mammals is accompanied by seed destruction, suggesting that the selective pressures exerted by those animals on the plants is much more complex than often assumed. Our results demonstrate that the complexity of plant–frugivore networks can not be fully understood without proper incorporating measures of seed fate following gut passage.