Safranin and Anilin Blue with Delafield's Hematoxylin for Staining Cell Walls in Shoot Apexes

The following technic is suggested for staining cell walls in shoot apexes: After the usual preliminary steps through 50% ethyl alcohol, stain in 1 % safranin 0 for 24 hours. Rinse in tap water and place in 2% aqueous tannic acid for 2 minutes. After rinsing in tap water, stain for 2 minutes in 1 part Delafield's hematoxylin to 2 parts distilled water and rinse in tap water. Remove excess hematoxylin with acidified water (1 drop cone. HC1 in 200 ml. water), then place slides in 0.5% lithium carbonate for 5 minutes. Dehydrate through an ethyl alcohol series, then transfer from absolute alcohol to a saturated solution of anilin blue in “methyl cellosolve” for 5-10 minutes. Wash in absolute alcohol, rinse in a solution of 25% methyl salicylate, 33% xylene, 42% absolute ethyl alcohol and clear for 10 minutes in a solution of 2 parts methyl salicylate, 1 part xylene, 1 part absolute ethyl alcohol. Transfer through two changes of xylene and mount in “clarite” or suitable alternate. The resulting preparations will have clearly defined, dark-staining cell walls and will photograph well when “Super Panchro-Press, Type B” film (Eastman Kodak Co.) is used in conjunction with suitable Wratten filters.     

Asymmetric Okazaki piece synthesis during replication of simian virus 40 DNA in vivo.

We have pulse-labeled simian virus 40 (SV40)-infected monkey cells with ³H-thymidine (³H-dThd) and have hybridized the viral Okazaki pieces (rapidly labeled short DNA chains found during DNA replication, < 250 nucleotides long) and SV40 “intermediate sized” DNA (longer nascent strands, up to full replicon size) to the separated strands of two SV40 DNA restriction fragments, one lying to either side of the origin of bidirectional DNA replication. As much as 5 fold more Okazaki piece DNA hybridized to one strand than to the other strand of each restriction fragment. The excess Okazaki piece DNA was in the strands oriented 3′ → 5′ away from the replication origin (the strands which are expected to be synthesized discontinuously). Neither the duration of the labeling period nor the temperature of the cells during labeling significantly altered this hybridization asymmetry. With respect to the hybridization of “intermediate sized” DNA, a reverse asymmetry was detected (1.7 fold more radioactivity in the strands oriented 5′ → 3′ away from the origin for a 1 min pulse label at 22°C). The effects on these hybridization asymmetries of preincubating the infected cells with FdUrd prior to pulse-labeling were also determined.We also measured the size of the Okazaki pieces using gel electrophoresis under denaturing conditons after releasing the pieces from the filter-bound DNA strands. The size distribution of the Okazaki piece DNA from each strand was the same (～ 145 nucleotides, weight average; 200–250 nucleotides, maximum size), indicating that the hybridization asymmetry resulted from a difference in the number rather than the size of the pieces in each strand.The simplest interpretation of our results is that SV40 DNA is synthesized semidiscontinuously: the strand with 3′ → 5′ orientation away from the origin is synthesized in short Okazaki pieces which are subsequently joined together, while the strand with 5′ → 3′ orientation away from the origin is synthesized continuously. Some models of two-strand discontinuous synthesis, however, cannot be ruled out.     

台湾海峡晚更新世人类肱骨化石

产自台湾海峡海底的人类右肱骨石化程度高; 个体大而粗壮 ,三角肌粗隆发育 ,骨干上下两半段不在同一纵轴上 ,形成 6 5°的夹角 ,这些显示不同于新石器时代和现代人的原始性状 ,其演化水平和日本的港川人及欧洲的克罗马农人相当。与人化石一同捞出的哺乳动物化石有古菱齿象、野马、最后鬣狗、达氏四不像鹿等 ,表明其时代为晚更新世晚期。     

Staining Plant and Animal Chromosomes by the Feulgen-Acetocarmine Sequence

A technic, some fundamentals of which were first worked out on brome grass, has been considerably extended and adapted to the somatic chromosomes of salmon. Fresh salmon eggs were quickly pierced in 45% acetic acid and fixed therein for 4 minutes. The eggs were then placed in N HCl at 60°C. for 8 minutes and thereafter transferred to Feulgen stain for 30 to 45 minutes. Subsequently, each stained embryo was dissected out and divided in two, each half being placed on a slide in a drop of acetocarmine stain. The pieces were well macerated and, after covering with a cover slip, maceration was completed by tapping. Heavy pressure was gradually applied to the cover slip in order to flatten the chromosome complements. A square screw-type laboratory hose clamp was then used to maintain this pressure while a liquid gelatin seal was applied around the edges. The slide, with the clamp on, was placed in the refrigerator overnight. Before the slide was scanned, the clamp was removed permanently. After each scanning period the slide was returned to the refrigerator. Photomicrographs of well-spread chromosomes in one optical plane were enlarged and tracings made from them. These tracings together with the photomicrographs were used for chromosome analysis.     

So what is a species anyway? A primatological perspective

Since Darwin's time, the question “what a species” has provoked fierce disputes and a tremendous number of publications, from short opinion papers to thick volumes. 1 The debates covered fundamental philosophical questions, such as: Do species exist at all independently of a human observer or are they just a construct of the human mind to categorize nature's organismic diversity and serve as a semantic tool in human communication about biodiversity? 2 - 4 or: Are species natural kinds (classes) or individuals that are “born” by speciation, change in course of time, and finally “die” when they go extinct or diverge into new species? 5 - 8 Also included was the problem of species as taxa (taxonomic) versus species as products of the speciation process (evolutionary). 9 More pragmatic issues arose, such as: How can we reliably delineate and delimitate species? 10 , 11 The great interest in what a species is reflects the importance of “species” as fundamental units in most fields of biology, especially evolutionary biology, ecology, and conservation. 2 , 12 - 14     

Einwirkung des immobilisierten Präparates „Sirenin”︁ aus Bacillus mesentericus auf Milch als Substrat

Comparative investigations for the possibilities of milk coagulation by the action of soluble and immobilized (on DEAE-cellulose by adsorption) bacterial enzyme preparation “syrenin” in periods from 30–120 minutes at 5°C are carried out. The milk clotting activities of both preparations are equalized after 40 minutes. The laboratory experiments for producing a bulgarian white brine cheese by the immobilized “syrenin” show a tendency to decrease the losses of substance into the whey and to increase the curdle yields in comparison with these made by soluble preparation.     

Taking the stress out of blood collection: comparison of field blood‐sampling techniques for analysis of baseline corticosterone

Many ecological studies use stress hormones to assess the condition, health or disturbance levels of wild organisms. Common blood sampling protocols for this research involve trapping individuals and taking blood within three minutes to obtain a “baseline” for analysis of stress hormones (“conventional method”). In some situations it may be difficult to get an accurate measure of baseline values; therefore, alternative sampling techniques may be preferable. We compared corticosterone levels in samples taken via a newly developed, minimally invasive blood sampling technique with corticosterone levels in blood taken via the conventional method. We collected samples from incubating adult common terns Sterna hirundo via blood sucking bugs (Heteroptera, Triatominae) contained in “dummy eggs” (“bug method”) and compared measured corticosterone concentrations to concentrations in blood taken from the same birds using the conventional method. We found no significant differences in mean or variance of baseline corticosterone levels between samples collected via the different methods. This suggests that the bug method offers a viable alternative for hormone sampling.     

Sonication and Other Improvements on Jeffrey's Technique for Macerating Wood

Large, up to pencil-size pieces of wood, rather than slivered wood, are macerated in Jeffrey's fluid, which consists of ca. 1:1 aqueous nitric acid and aqueous chromic acid (each 10% or weaker). Staining is done in ca. 1% solution of safranin in absolute alcohol. The intact pieces of wood can be very briefly sonicated in xylene to release isolated elements and connected cell groups. Various alternative steps are noted. Discussion focuses on the practical and theoretical advantages derived from the aforementioned innovations.     

Modalités de prescription des inhibiteurs de la phospodiestérase de type 5

Phosphodiesterase type 5 inhibitors, sildenafil, tadalafil and vardenafil, are “on demand” oral treatments for erectile dysfunction. Sildenafil must be taken 1 hour before sexual activity, while tadalafil can be taken 30 minutes to 12 hours before sexual activity and vardenafil can be taken 25 to 60 minutes before sexual activity. Sexual stimulation is necessary. The main contraindications are the concomitant use of nitric oxide donors or nitrates and serious heart problems (which are a contraindication to sexual intercourse). In some cases (men over the age of 65, hepatic or renal impairment, association with certain drugs), phosphodiesterase type 5 inhibitors are not recommended or the dosage needs to be adapted.     

Dictionary of protein secondary structure: Pattern recognition of hydrogen-bonded and geometrical features

For a successful analysis of the relation between amino acid sequence and protein structure, an unambiguous and physically meaningful definition of secondary structure is essential. We have developed a set of simple and physically motivated criteria for secondary structure, programmed as a pattern-recognition process of hydrogen-bonded and geometrical features extracted from x-ray coordinates. Cooperative secondary structure is recognized as repeats of the elementary hydrogen-bonding patterns “turn” and “bridge.” Repeating turns are “helices,” repeating bridges are “ladders,” connected ladders are “sheets.” Geometric structure is defined in terms of the concepts torsion and curvature of differential geometry. Local chain “chirality” is the torsional handedness of four consecutive C^α positions and is positive for right-handed helices and negative for ideal twisted β-sheets. Curved pieces are defined as “bends.” Solvent “exposure” is given as the number of water molecules in possible contact with a residue. The end result is a compilation of the primary structure, including SS bonds, secondary structure, and solvent exposure of 62 different globular proteins. The presentation is in linear form: strip graphs for an overall view and strip tables for the details of each of 10.925 residues. The dictionary is also available in computer-readable form for protein structure prediction work.     

Establishment of Trisomic Series of Millet (Setaria italica L. Beauv.)

The young-ears of “Yugu No. 1”, a millet (Setaria italica (L.) Beauv. ) variety of good quality, high yielding and stress-resistance, were chosen to induce callus on N6 medium. After 3—5 times of subcultures, the callus was treated with colchicine (0.02%) for 48 hours and then transferred hack to subculture medium to restore growth, after which the callus was transferred to differentiation medium and subsequently, tetraploid plants were obtained. Through crossing, using tetraploid plants as female parent and diploid plant as male parent, 5 triploid plants were found among 2100 offspring plants. The triploid plants were fertilized with pollens from diploid plants. Among the plants developed from the seeds harvested on triploid plants, 9 kinds of trisomics (totally 283 plants) were identified according to chromosomal number and morphologic feature. Each kind of the trisomics has specific marker feature: (Figures in parentheses represent the number of plants obtained) Triplo-1 (52) has rolling leaves, Triplo-2 (18) is dark green, Triplo-3 (43) is bushy and has degenerated spikelets at the tip of panicles: Triplo-4 (58) has long bristle: Triplo-5 (16) has slender stalks: Triplo-6 (36) has twisted panicle neck; Triplo-7 (44) is semi-erect; Triplo-8 (8) has thick panicles; Triplo-9 (8) is pseudonormal (Similar with diploid of “Yugu No. 1”).     

不同种类无机盐对灰毡毛忍冬“渝蕾1号”悬浮培养体系中细胞生长和绿原酸含量的影响

为提高灰毡毛忍冬"渝蕾1号"悬浮培养体系中绿原酸的含量,该研究探讨了B_5培养基中不同浓度的无机盐对灰毡毛忍冬"渝蕾1号"悬浮培养细胞生物量及绿原酸含量的影响,通过在悬浮培养体系中添加不同浓度的无机盐,采用重量法测定灰毡毛忍冬"渝蕾1号"悬浮培养细胞的生物量及采用高效液相色谱法测定绿原酸的含量。结果表明:当硝态氮和铵态氮配比与B_5培养基中硝态氮和铵态氮配比一致时,即NO_3~-/NH_4~+摩尔比值为13∶1时,培养体系有利于细胞的生长和绿原酸的积累。当KNO_3浓度为3.5 g·L~(-1)时,细胞生物量达到最大,为19.26 g·L~(-1);当KNO_3在较低浓度(0.5 g·L~(-1)和1.5 g·L~(-1))时,积累较多的绿原酸。NO_3~-的两项研究结果均与对照浓度(2.5g·L~(-1))有一定的差异。另外,对(NH_4)_2SO_4来说,在高于对照浓度0.134 g·L~(-1),即浓度为0.268 g·L~(-1)时,生物量和绿原酸含量都达到了最大。P、Ca、Mg三种矿质元素的研究结果表明,当Na H_2PO_4·2H_2O浓度为0.10 g·L~(-1)、Ca Cl_2的浓度为0.20 g·L~(-1)时,细胞的生长和绿原酸的积累均可达到最大值;而对Mg~(2+)来说,低浓度促进细胞的生长,高浓度促进绿原酸的积累。兼顾细胞生物量和绿原酸含量两个指标,需选择适中的浓度。这些结果均与对照浓度有一定的差异。这说明灰毡毛忍冬"渝蕾1号"悬浮细胞所需无机盐的浓度与B_5培养基无机盐的浓度有一定的差异,选择适宜的浓度可促进其悬浮细胞的生长及次生代谢产物绿原酸的积累。该研究结果为绿原酸的工业化生产打下了基础。     

A Modified Hypotonic Treatment for Increasing the Frequency and Quality of Meiotic Metaphases from Spermatocytes of the Syrian Hamster

A modified hypotonic treatment for the spermatocytes of the Syrian hamster (Mesocricetm auratus) has been developed using a micro-osmometer to determine optimum conditions for the frequency and quality of Metaphase I and II figures. A suspension of germinal cells from the tubules is made in 3.2% sodium citrate solution and allowed to stand for 5 minutes. Water is then added until the concentration of the sodium citrate is reduced to 1.4%. This is followed after 15 minutes by fixation and air drying. The number and quality of the metaphase figures is enhanced considerably by this technique. A comparison is made of blood, plasma and testicular osmolality in several animal species and corresponding values for various solutions used for cytogenetic techniques.     

Antigen binding to lymphoid cells from unimmunized mice: IV. Shedding and reappearance of multiple antigen binding Ig receptors of T- and B-lymphocytes

Patterned antigen-binding cells (ABC) can bind two antigens and show “islands” of Ig receptor with mixed specificity. However, these cells, when unfixed, lose most of their bound fluorescent antigen within minutes upon warming above 0 °C. Residual antigen moves to one pole of the cell forming a “cap” within 5 min at room temperature. If such patterned ABC are capped with a single antigen, receptors to a second antigen can be detected on a portion of the capped cells, but only in the cap. The frequency of capped, “double” ABC approximated the frequency of patterned “double” ABC originally present.If lymphoid cells are mixed with fluorescent antigens at 0 °C and then incubated for 4 hr at 37 °, no ABC are found. When the cells are then fixed and the fluorescent antigens readded, new antigen-binding Ig receptors can be shown to have reappeared on the cell surface during the 4-hr incubation. The reappearance of antigen receptor could be inhibited by prior addition of either 10^?2M sodium azide or 50 μg/ml cycloheximide, implying that the receptors were actively synthesized by the cell. These inhibitors did not prevent shedding, but azide did inhibit the capping process. Both B-cells (bone marrow or spleen cells) and T-cells (splenic T-cells or 99.5% pure cortisone-resistant T-cells) were shown to regenerate multispecific ABC to the frequency found prior to incubation.     

Place de l’analyse du mouvement dans un bilan de fertilité: Valeurs prédictives des différents paramètres mesurés

Routine evaluation of semen characteristics-spermiogram-includes estimation of the percentage of motile sperm; however it does not provide quantitative informations about sperm movement characteristics, except under the form of qualitative appreciations (slow, sluggish, yawing, non progressive, etc.). Flagellar function is indeed directly involved in the migration of spermatozoa through the female genital tract, and in the fertilization process by itself (migration through the zona pellucida requires special motility state, generally called “hyperactivation”). Sperm flagellar movements can now be indirectly investigated by analysing movements of sperm head, which are more easily detectable under phase contrast illumination: video signals are digitalized then sperm tracks are reconstructed by the computer from coordinates of sperm centroids (these systems are called “computer-assisted semen analysis” or “CASA”). CASA systems are now so performing and rapid that sperm movement analysis (SMA) can be proposed in the same time of routine semen analysis. However SMA, together with other functional tests, offer more interest in some particular situations as unexplained infertility, or unexpected failure of IVF. Numerous studies have tried to identify the most discriminant parameters, generally by means of multiple regression analysis. The interpretation of litterature data is difficult because of several differences in the protocol design, concerning either the measurements conditions (before or after sperm washing and selection) or the nature of the functional test: migration into cervical mucus, zona-free hamster egg penetration, IVF, etc. Moreover, the most discriminant factors are generally represented by classical parameters as % of normal forms or of motile sperm. In the present study we showed that only the factor “% of motility” allowed a significant discrimination according to different classes of fertilization rate (FR) in an IVF system. FR increased with hyperactivation rate (HA), but the statistical test was not significant. However, the more numerous cases of IVF failure were found in the group corresponding to very low HA rate (0–5%). We conclude that one major interest of SMA is to reveal some flagellar dyskinesia (i.e. corresponding to low values of amplitude lateral displacement of the head, or straight line velocity). These cases could then benefit of assisted reproductive techniques well adapted to this motion dysfunction, as subzonal insemination (SUZI) or intra-cytoplasmic sperm injection (ICSI).     

Autoradiography of Water-Soluble Materials in Plant Tissues

Samples of tissue with water-soluble substances are lyophilized and doubly-infiltrated with parlodion and paraffin. Sections are mounted on an adhesive-coated cover slip with chloroform. The reverse side of the cover slip is coated successively with a thin layer of Harleco resin and a thick layer of Kodak “Opaque”. A corner of the cover slip is broken off as a marker. The cover slip is assembled with a covering glass slide on a nuclear track plate (Kodak NTB-2) with protective coating, the section being in contact with the emulsion, and held in place during radioactive exposure. Before development, the cover slip and plate are exposed briefly to light and then disassembled. Following development of the plate and removal of the layer of resin and opaque from the cover slip, staining of the section is optional. Lining up of the section with its autoradiograph can be accurate within 1-5μ.     

The Karyotypes of Angelica dahurica and Their Taxonomical Significance (Umbelliferae)

The present paper deals mainly with the karyotype analysis of five materials in Angelica dahurica collected in Yanbian of Jilin, Anguo of Hebei, Yuxian of Henan, Hangzhou of Zhejiang and Suining of Sichuan. They are under the names “Dongbeidahuo”, “Qibaizhi”, “Yubaizhi”, ”Hangbaizhi” and “Chuanbaizhi” respectively. Among then “Dongbeidahuo” is a wild plant, which occurs in northeastern China, and the others are cultivated as important crude drugs in some provinces. “Qi-Baizhi” and “Yubaizhi” have been identified as conspecific with the wild Baizhi-“Do-Ngbeidahuo” (A. dahurica) according to the external morphological features, whereas the other cultivated ones, “Hangbaizhi” and “Chuanbaizhi”, treated as a variety (A. dahurica var. formosana). The results of karyotype analysis are shown in Plate 1, 2, with the formula 2n=22 =12 m+2 m^SAT+4sm+4st. The karyotypes described here are constantly characterized by satellites attached to the fourth pair of metacentric chromosomes and differ from the published reports on the other species of the genus. It is reasonable to say that the five materials collectively named “Baizhi” are taxonomically closely related to each other and could be regarded as conspecific. Since the second chromosome pair is submetacentric in “Dongbeidahuo”, it may be justifiable to separate the wild plant from the cultivated ones and treat them as two separate varieties.     

GROWTH AND ORGANIZED DEVELOPMENT OF CULTURED CELLS. V. THE GROWTH OF COLONIES FROM FREE CELLS ON NUTRIENT AGAR

When angiosperm cells are cultured in a liquid medium they may grow in the form of free, single cells and form small to large groups of cells. This has been shown in earlier papers. This paper deals with the growth of strains of cells (Daucus carota and Haplopappus gracilis cells were used), washed and filtered free from the larger groups, on nutrient agar media in Petri dishes, thus simulating familiar microbiological technique. Each discrete member of a suspension is referred to as a “unit.” On the order of 1–10% of the separate units of a suspension may be induced to grow into viable colonies, depending on the strain and the conditions employed. Whereas at least 30% of the free single carrot cells were shown to be capable of division, only up to about 4% continued their growth to form macroscopically visible colonies when they were widely dispersed. Coconut milk promotes the growth of carrot cells into colonies. Both coconut milk and napthaleneacetic acid, which interact synergistically, arc required for the growth of Haplopappus cells. Various techniques which affect viability (the frequency with which units grow into colonies) were investigated. The viability of carrot units was found (1) to increase with their density on the plates; (2) to decrease upon washing the suspensions prior to plating; (3) to increase with increasing initial size; and (4) to decrease to a vanishingly low value in rigorously filtered suspensions which consist principally of single cells, although the single cells were found to grow with appreciable frequency when the larger units were also present; and (5) to increase dramatically (100-fold) when a rigorously filtered suspension was plated on a medium upon which pieces of growing cultured tissue were placed. Thus, the induction of growth in free cells is enhanced, even in an environment nutritionally optimal for the growth of the larger cell masses, by as yet unknown factors which are contributed by the cells themselves, or by adjacent cells or groups of cells. It is suggested that within a group of cells growing in culture, and perhaps also in the organized growing regions of intact plants, the dividing cells are nourished or stimulated by adjacent but less frequently dividing cells. The implications of these results are discussed.