Manipulation of the Endocochlear Potential Reveals Two Distinct Types of Cochlear Nonlinearity

The mammalian hearing organ, the cochlea, contains an active amplifier to boost the vibrational response to low level sounds. Hallmarks of this active process are sharp location-dependent frequency tuning and compressive nonlinearity over a wide stimulus range. The amplifier relies on outer hair cell (OHC)-generated forces driven in part by the endocochlear potential, the ∼+80 mV potential maintained in scala media, generated by the stria vascularis. We transiently eliminated the endocochlear potential in vivo by an intravenous injection of furosemide and measured the vibrations of different layers in the cochlea’s organ of Corti using optical coherence tomography. Distortion product otoacoustic emissions were also monitored. After furosemide injection, the vibrations of the basilar membrane lost the best frequency (BF) peak and showed broad tuning similar to a passive cochlea. The intra-organ of Corti vibrations measured in the region of the OHCs lost the BF peak and showed low-pass responses but retained nonlinearity. This strongly suggests that OHC electromotility was operating and being driven by nonlinear OHC current. Thus, although electromotility is presumably necessary to produce a healthy BF peak, the mere presence of electromotility is not sufficient. The BF peak recovered nearly fully within 2 h, along with the recovery of odd-order distortion product otoacoustic emissions. The recovery pattern suggests that physical shifts in operating condition are a critical step in the recovery process.     

Engineering a Hyperstable Enzyme by Manipulation of Early Steps in the Unfolding Process

Protein engineering experiments have recently yielded hyperstable variants of the thermolysin-like protease from Bacillus stearothermophilus (TLP-ste). These variants contain mutations suggested by comparison of TLP-ste with its more thermostable counterpart thermolysin, as well as rationally designed mutations. The key to the successful stabilization strategy was the identification of a “weak” region that is involved in early unfolding events (“unfolding region”). Mutations in this region had large effects on stability, whereas mutations in other parts of the protein generally had minor effects. The mutational strategies that were used as well as characteristics of the engineered hyperstable biocatalysts are reviewed below.     

Two Types of Alpha Satellite DNA in Distinct Chromosomal Locations in Azara's Owl Monkey

Alpha satellite DNA is a repetitive sequence known to be a major DNA component of centromeres in primates (order Primates). New World monkeys form one major taxon (parvorder Platyrrhini) of primates, and their alpha satellite DNA is known to comprise repeat units of around 340 bp. In one species (Azara''s owl monkey Aotus azarae) of this taxon, we identified two types of alpha satellite DNA consisting of 185- and 344-bp repeat units that we designated as OwlAlp1 and OwlAlp2, respectively. OwlAlp2 exhibits similarity throughout its entire sequence to the alpha satellite DNA of other New World monkeys. The chromosomal locations of the two types of sequence are markedly distinct: OwlAlp1 was observed at the centromeric constrictions, whereas OwlAlp2 was found in the pericentric regions. From these results, we inferred that OwlAlp1 was derived from OwlAlp2 and rapidly replaced OwlAlp2 as the principal alpha satellite DNA on a short time scale at the speciation level. A less likely alternative explanation is also discussed.     

Evaluation of Two Types of Infectious Mononucleosis Antigen Slides by the Indirect Fluorescent-Antibody Technique

The efficiency of two types of antigen slides was compared by using the indirect fluorescent-antibody technique. Fifty sera from infectious mononucleosis patients were tested concurrently on the two sets of slides for antibody to Epstein-Barr virus. The indirect fluorescent-antibody serum titer readings from the epoxy slides were either equal to or twofold higher than those from the cover slip slides.     

An Assessment of Routine Chromosomal Staining Procedures in Radioautography

Unlabeled human chromosome preparations were treated with commonly employed chromosome stains as follows: (I) they were stained, destained, coated with liquid emulsion, developed, fixed, and restained; (II) stained and coated directly; or (III) coated and then stained. Of the stains tested, the methylene blue-eosin type (Giemsa, MacNeal's, Wright's) was useful for application after coating, although a similar stain (eosin-Stevenel's blue) caused formation of a heavy precipitate in the emulsion when so used. None of these stains could be employed before coating, however, even though they were removed with acid alcohol prior to dipping, because they caused chemographic grain formation in the emulsion. Aceto-orcein and Feulgen could not be employed after coating because the procedures removed the emulsion from the slides. Safranin was also found to be ineffective for staining coated preparations due to chemical changes caused by the photographic processing. The only stain which did not cause chemography, and hence can be used before coating slides, is aceto-orcein. Since this stain fades during radioautographic processing and cannot be employed after coating, we recommend secondary use of one of the methylene blue-eosin type stains for revisualization of the chromosome spread.     

Staining of Ultraviolet Irradiated Chromosomes by the Gomori Alkaline Phosphatase Technique

When a chromosome segment is selectively irradiated with an ultraviolet micro-beam, the chromosome(s), which normally appear black by medium-dark phase-contrast microscopy, become “pale” in the irradiated region (decrease in refractive index). Previous ultraviolet absorption and Feulgen staining studies indicated that all or most of the deoxyribonucleic acid is lost in this region. After fixation, the irradiated area appears pale with most of the usual staining methods. The residual material in the paled spot, however, can be stained with the Comori alkaline phosphatase technique and is seen to be directly continuous with the nonirradiated segments. With the bright field microscope, there appears to be no decrease or increase in chromosome width. It is concluded that staining by the Gomori technique is independent of the presence or absence of deoxyribonucleic acid. Positive staining of chromosomes by the nonenzymatic peroxide method of Danielli indicated that staining was due to nonspecific precipitation of calcium phosphate rather than to enzymatic activity.     

Selective Staining of Neurosecretory Material in Semithin Epoxy Sections by Gomori's Aldehyde Fuchsin

The epoxy resin was removed from semithin (1 μm) sections by immersing them for 30 sec in sodium methoxide (Mayor et al., J. Biophys. Biochem. Cytol., 9: 909-10, 1961) and then processed as follows: (1) left for 1-3 hr at 60 C in a mixture of formalin, 25 ml; glacial acetic acid, 5 ml; CrO₃, 3 gm; and distilled water, 75 ml: (2) oxidized 10 min in a 1:1:6 v/v mixture of 2.5% KMnO₄, 5% H₂SO₄ and distilled water: (3) bleached in 1% oxalic acid, and (4) stained for 15 min in aldehyde fuchsin, 0.125% in 70% alcohol, or in a 1% aqueous solution of toluidine blue. The neurosecretory material is selectively stained.     

Specific Staining of Egg-Shell Material in Trematodes and Cestodes

The material which forms the egg-shell in trematodes is an orthodihydroxyphenol-protein complex (Stephenson, 1947) which originates in the cells of the so-called vitellaria (Dawes, 1946). It has been found that both malachite and methyl green in aqueous solution have a marked and specific affinity for this material after formolsaline fixation. The same was found to be true for the egg-shell material in pseudophyllidean cestodes. These stains thus provide the helminthologist with a simple method by means of which the process of egg-shell formation in these groups may be worked out.     

Aceto-Iron-Haematoxylin for Staining Chromosomes in Squashes of Plant Material

Haematoxylin can be used successfully in the acetic squash technic if adequate mordanting is provided, (a) in the stain—composed of 4% haematoxylin and 1% iron alum in 45% acetic acid—and (b) in a step that combines additional fixation, mordanting and maceration in a 1:1 HCl-alcohol mixture, to which is added chrome alum, iron alum and iodic acid: 0.1 gm of each to 6 ml of HCl-alcohol. The material is usually given a preliminary fixation in 1:3 acetic alcohol, then macerated, fixed and mordanted in the acidified alum-HIO₃ step for 10 min, transferred to Carney's fluid (6:3:1) for 10-20 min, squashed in a drop of stain and gently heated. In some species, the preliminary fixation may be omitted. The method yields intensely and selectively stained chromatin. To secure consistently good results, the stain can be diluted with 45% acetic acid, and the iodic acid omitted for some plant materials.     

分子印迹技术用于生物大分子的识别

分子印迹技术是一种人工合成具有分子识别功能的介质的一种新技术,近年来在许多领域都得到很大的发展。本文介绍了分子印迹技术的发展现状,尤其对生物大分子的分子印迹技术进行了详细论述,对生物大分子印迹采用的功能单体、印迹分子的种类、印迹的方法、印迹的机理、存在的问题和应用的前景等分别进行了讨论。     

窝雏数处理对两种雀形目幼鸟生长的影响

基于 1 997～ 1 999年野外实验 ,对高寒草甸小云雀和黄嘴朱顶雀两种雀形目鸟的窝雏数进行增减处理。结果表明 ,对照组的幼鸟生长率和离巢体重都大于增加组 ,说明窝雏数增加后 ,幼鸟质量下降。随着窝雏数增加 ,这两种幼鸟生长率显著下降 (小云雀 :r =-0 965 ,P =0 0 3 5 <0 0 5 ;朱顶雀 :r =-0 82 8,P =0 0 2 2 <0 0 5 )。窝雏数改变对小云雀幼鸟出飞重影响不显著 (r =-0 41 8,P =0 5 2 8>0 0 5 ) ,而对黄嘴朱顶雀有显著的影响 (r=-0 90 1 ,P =0 0 1 4<0 0 5 )。     

Two Steps of Gas Exchange in Leaf Photosynthesis

Photosynthesis and transpiration of excised leaves of Taraxacum officinale L. and a few other species of plants were measured, using an open gas analysis system. The rates of CO₂ uptake and transpiration increased in two steps upon illumination of stomata-bearing epidermis of these leaves at a light intensity of 50 mW × cm⁻². Abscisic acid inhibited only the second step of gas exchange. Illumination of the astomatous epidermis of hypostomatous leaves caused only the first step of gas exchange. These data indicate that the first and second steps arise from cuticular and stomatal gas exchange, respectively. The rate of the cuticular photosynthesis in a Taraxacum leaf reached saturation at a light intensity of 5 mW × cm⁻², and the rates of the stomatal photosynthesis and transpiration reached saturation at a higher intensity of 35 mW × cm⁻². The cuticular photosynthesis of a Taraxacum leaf was 18% of the stomatal photosynthesis at 50 mW × cm⁻² and 270% at 5 mW × cm⁻². The other species of leaves showed the same trend. The importance of cuticular CO₂ uptake in leaf photosynthesis, especially under low light intensity was stressed from these data.     

Lipoprotein FtsB in Streptococcus pyogenes Binds Ferrichrome in Two Steps with Residues Tyr137 and Trp204 as Critical Ligands

Lipoprotein FtsB is a component of the FtsABCD transporter that is responsible for ferrichrome binding and uptake in the Gram-positive pathogen Streptococcus pyogenes. In the present study, FtsB was cloned and purified from the bacteria and its Fch binding characteristics were investigated in detail by using various biophysical and biochemical methods. Based on the crystal structures of homogeneous proteins, FtsB was simulated to have bi-lobal structure forming a deep cleft with four residues in the cleft as potential ligands for Fch binding. With the assistance of site-directed mutagenesis, residue Trp204 was confirmed as a key ligand and Tyr137 was identified to be another essential residue for Fch binding. Kinetics experiments demonstrated that Fch binding in FtsB occurred in two steps, corresponding to the bindings to Tyr137 at N-lobe and Trp204 from C-lobe, respectively, and so that closing the protein conformation. Without either residue Tyr137 or Trp204, Fch binding in the protein as mutants Fch-Y137A and Fch-W204A may have a loose conformation, resembling the apo-proteins in proteolysis resistance and migration behaviors in native gel. This study revealed the inconsistence in the key amino acids among Fch-binding proteins from Gram-positive and –negative bacteria, providing interesting findings for understanding the differences between Gram-positive and –negative bacteria in the mechanism of iron uptake via siderophore (Fch) binding and transport.