Progress in the Standardization of Stains the Standardization of Eosin and Related Dyes

Decolorizers which are distinctly acidic or basic in their chemical nature give abnormally high decolorization in the Gram stain for bacteria. Acidic substances yield more regular results. Ideally an “inert” decolorizer should be used, but ordinarily such substances will not dissolve the dye or dye-mordant precipitate from the smear. The most practical substances seem to be those so very slightly acidic in character as to be practically inert, such as acetone or alcohol, or a mixture of such substances.     

Variations in endogenous gibberellins in developing bean seeds I. Occurrence of neutral and acidic substances

Activities of separated and chromatographed substances in the nonacidic, acidic ethyl acetate and acidic butanol fractions from bean seeds, Phaseolus vulgaris L., cv. Bountiful and Kentucky Wonder, were measured in the Progress No. 9 dwarf pea bioassay grown under red light. Activity in the nonacidic fraction was shown to be attributable only to neutral substances and was free of acidic gibberellin-like substances. As the seed matures, neutral substances and one of the acidic butanol-soluble substances (B-I) increase in activity. The acidic ethyl acetate substances and butanol-soluble substance (B-II) initially increase and then almost disappear.     

Is alcoholic acidic silver nitrate reagent really specific for the histochemical localization of ascorbic acid.

The histochemical localization of ascorbic acid in plant tissues with the alcoholic acidic silver nitrate reagent is shown here to be not specific for ascorbic acid, since some of the polyphenolic substances, including flavonoids, which are known to be widely distributed in plant tissues, are also able to reduce the acidic alcoholic silver nitrate reagent at low temperature (0-4 degrees C) and at pH 2 to 2.5 in dark. This method may perhaps be used for animal tissues where flavonoid pigments do not occur in such large quantities as they do in plants. I therefore, come to the inevitable conclusion that the use of alcoholic acidic silver nitrate reagent in localizing ascorbic acid in plant tissues may be highly misleading.     

Cutaneous antipredatory secretions and pheromones in anurans and urodeles

The article analyses the main chemical signals used by anurans and urodeles for social interactions such as defence and reproduction. Some emblematic examples have been selected from the most significative reports. The antipredatory arsenal of many frogs and toads includes secretions of cutaneous glands, randomly distributed on the body or localised in “critical” skin regions. These substances act as repellent, alarm or venom, with specific toxicity and pharmacological actions. Other chemical cues facilitate social interactions. These “pheromones” allow animals to recognize conspecifics and to identify their sex, reproductive condition and social status. In many cases courtship pheromones play a crucial role in increasing male success. Evidence such as that suggests that selective pressures from environmental and social constraints produced the high incidence of chemical signalling typical of the amphibia, a view confirmed by the similarity of chemical cues across different taxa.     

Histochemical demonstration of acidic glycosaminoglycans in the cell nuclei of the iris and other tissues.

Using histochemical methods, the presence of acidic glycosaminoglycans in the cell nuclei of 51 human irides and a series of monkey organs was demonstrated. In general, these substances are sensitive to testicular hyaluronidase and chondroitinase ABC and also to Streptomyces hyaluronidase, when using special staining methods. The specificity of testicular hyaluronidase was tested by inhibition with heparin. By simultaneously staining with alcian blue and Feulgen, acidic glycosaminoglycans can be distinguished from the nucleic acids. Sporadically, hyaluronidase-resistant substances with a specific acidic glycosaminoglycan stainability occur. We assume the existence of various acidic glycosaminoglycans in the cell nuclei. Aging changes were not traceable with constancy.     

Electron microscopic examination of nuclear inclusions induced by Vinca alkaloids

Nuclear inclusions (filament bundles, crystalloid structures, “nuclear bodies”, tubular structures) in Ehrlich ascites cells induced by Vinca alkaloids were studied with a transmission electron microscope. The appearance of nuclear inclusions caused by VLB or DA-VCR may be attributed to the precipitation of tubulin, actin or some other acidic proteins.     

Variations in Endogenous Gibberellins in Developing Bean Seeds II. Changes Induced in Acidic and Neutral Fractions by GA(1)

Immature (8-mm), medium mature (11-mm), and mature green (16- and 17-mm) bean seeds (Phaseolus vulgaris L. cv. Kentucky Wonder and Bountiful) were incubated in gibberellin A₁ solutions for 24 hours at 20°. Extracts from the seeds were separated into nonacidic, acidic ethyl acetate, and acidic butanol fractions. These were chromatographed. The eluates of the chromatograms were tested on Progress No. 9 dwarf peas grown under red light. The level of neutral gibberellin-like substances remained unchanged in immature seed, but they increased markedly in mature green seeds. Coincident with increased levels of the neutral substances, there were significant decreases in acidic ethyl acetate-soluble gibberellin-like substances, including applied GA₁, and in 1 acidic butanol-soluble gibberellin-like substance. Seed incubation in GA₁ brought about increased activity of substance B-II in immature and medium mature seeds. The level of butanol-soluble gibberellin-like substance B-I in seeds of any size was not affected by incubation in GA₁. Considering the marked increases in activity induced in the neutral fraction and the decreases in activity of certain eluates from the chromatograms of the acidic fractions, it was concluded that the neutral fraction may serve as a reserve form of gibberellins in the dry seed. The acidic ethyl acetate substances and substance B-II may be required for normal development of the bean seed.     

Stimulatory effect of bleomycin on the synthesis of acidic glycosaminoglycans in cultured fibroblasts derived from rat carrageenin granuloma.

Cultured fibroblasts derived from rat carrageenin granuloma were treated with bleomycin and the synthesis of hexosamine-containing substances was compared with that in control cells. Four day treatment with o.1 mug bleomycin/ml resulted in a significant increase of the production of these macromolecules by the cells, though DNA synthesis was remarkably inhibited at this dose of bleomycin. The stimulatory effect could be seen as early as the second day of bleomycin treatment, and was enhanced with increasing treatment time. Further fractionation of the hexosamine-containing substances revealed that synthesis of acidic glycosaminoglycans was more sensitive to bleomycin than that of glycoproteins, i.e., acidic glycosaminoglycans increased by 80% and glycoproteins by 53% after four day treatment with 0.1 mug bleomycin/ml. The increased components of acidic glycosaminoglycans included not only hyaluronic acid but also sulphated glycosaminoglycans. Collagen synthesis was increased by 23% by the same dose of bleomycin. N-Acetyl-beta-glucosaminidase, one of the degradation enzymes for acidic glycosaminoglycans released into the cultured medium, was decreased significantly by bleomycin.     

结合代谢组学和转录组学分析恶臭假单胞菌Y-9主动稳定胞内外pH的机制

【目的】揭示恶臭假单胞菌(Pseudomonas putida) Y-9在氨氧化过程中主动调节胞外和胞内pH稳态机制。【方法】在初始pH为7.19和9.40的硝化培养基中培养Y-9生长48 h，利用代谢组学对比分析Y-9氨氧化过程中的显著差异代谢产物并预测解离常数(pKa)；结合转录组学对比分析Y-9氨氧化过程中的显著差异调控基因。【结果】Y-9在初始pH为7.19的相对酸性条件下，产生麦芽糖醇提高胞外pH；通过上调脱氨酶、脱亚胺酶和阳离子转运相关基因在相对酸性环境中的表达来维持细胞内pH稳定性。在初始pH为9.40的碱性条件下，产5-氨基戊酸3和草氨酸等有机酸及酸性物质降低胞外pH；通过调控NADH脱氢酶、细胞色素、ATP合酶和氨基酸转运相关基因的表达来维持细胞内酸度，应对碱性环境。【结论】本研究结果首先发现了Y-9具有稳定胞外pH的能力，探讨了其胞内pH稳态机制，拓展了对微生物与环境相互作用的认知，为进一步认识微生物脱氮过程中系统pH稳定机理提供了理论依据。     

Lability of equol to acidic hydrolysis procedures.

If equol is subjected to acidic hydrolysis by refluxing with 1.5 n HCl, a large proportion of the equol is altered to other substances. Subjection to enzymic hydrolysis by aryl sulfatase does not bring about any such alterations.     

Ultracentrifugation studies of the location of the site involved in the interaction of pig heart lactate dehydrogenase with acidic phospholipids at low pH. A comparison with the muscle form of the enzyme

Lactate dehydrogenase (LDH) from the pig heart interacts with liposomes made of acidic phospholipids most effectively at low pH, close to the isoelectric point of the protein (pH = 5.5). This binding is not observed at neutral pH or high ionic strength. LDH-liposome complex formation requires an absence of nicotinamide adenine dinucleotides and adenine nucleotides in the interaction environment. Their presence limits the interaction of LDH with liposomes in a concentration-dependent manner. This phenomenon is not observed for pig skeletal muscle LDH. The heart LDH-liposome complexes formed in the absence of nicotinamide adenine dinucleotides and adenine nucleotides are stable after the addition of these substances even in millimolar concentrations. The LDH substrates and studied nucleotides that inhibit the interaction of pig heart LDH with acidic liposomes can be ordered according to their effectiveness as follows: NADH > NAD > ATP = ADP > AMP > pyruvate. The phosphorylated form of NAD (NADP), nonadenine nucleotides (GTP, CTP, UTP) and lactate are ineffective. Chemically cross-linked pig heart LDH, with a tetrameric structure stable at low pH, behaves analogously to the unmodified enzyme, which excludes the participation of the interfacing parts of subunits in the interaction with acidic phospholipids. The presented results indicate that in lowered pH conditions, the NADH-cofactor binding site of pig heart LDH is strongly involved in the interaction of the enzyme with acidic phospholipids. The contribution of the ATP/ADP binding site to this process can also be considered. In the case of pig skeletal muscle LDH, neither the cofactor binding site nor the subunit interfacing areas seem to be involved in the interaction.     

Is alcoholic acidic silver nitrate reagent really specific for the histochemical localization of ascorbic acid?

Summary The histochemical localization of ascorbic acid in plant tissues with the alcoholic acidic silver nitrate reagent is shown here to be not specific for ascorbic acid, since some of the polyphenolic substances, including flavonoids, which are known to be widely distributed in plant tissues, are also able to reduce the acidic alcoholic silver nitrate reagent at low temperature (0–4°C) and at pH 2 to 2.5 in dark. This method may perhaps be used for animal tissues where flavonoid pigments do not occur in such large quantities as they do in plants. I therefore, come to the inevitable conclusion that the use of alcoholic acidic silver nitrate reagent in localizing ascorbic acid in plant tissues may be highly misleading.     

The Growth Substances separated from Plant Extracts by Chromatography. I

Methods for the chromatographic separation on paper of indolecompounds and for the direct biological assay of the chrornatograinsusing the Avena coleoptile straight-growth method are described.Reagents for the detection of the indole-3-carboxylic acids,indole-3-acetonitrile, and gramirte as coloured spots on chromatogramsare compared and the areas of such spots are shown to be proportionalto the logarithms of the quantities of substance present. The procedure of chromatography described is shown not to involvea loss of indole-3-acetic acid activity if chromatography isdone in darkness and chrornatograms are not stored in lightand air. Methods are described for the extraction of growth aubstancesfrom plant materials, the purification and chromatography, onpaper, of the extracts and the bioassay of the chromatogramsusing Avena coleoptile sections. The ether extracts, containing acidic substances, of etiolatedbroad bean and pea shoots and roots, etiolated sunflower shoots,maize roots, and potato etiolated shoots and tuber have beenchromatographed and the chromatograms bioassayed. On all chromatogramsthree areas active in Avena coleoptile section growth are found.One area of growth promotion is shown due to indole-3-aceticacid [IAA]. Another area of growth promotion and, one of growthinhibition are due to unknown substances, which are named accelerator () and inhibitor ß (ß) respectively. On chromatograms of potato tuber a fourth growth-promoting area,in addition to those described above, is detected and is shownto be probably due to indole-3-acetonitrile [IAN]. IAN or indole-3-pyruvicacid may occpr together with IAA on chrormatograms of extractsof immature maize kernels and cauliflower head respectively. On cabbage extract chromatograms the growth-promoting activitycorresponding in position with IAA is shown to be due to IAAand to IAA alone. In etiolated broad bean shoots IAA is the predominating growthsubstance in the stem and ß predominates in the firstlateral bud. The latter is suggested as an explanation of apicaldominance, and the predominance of ß in potato tuberskin is suggested as an explanation of dormancy in tubers. In the broad bean root the acidic growth-substance patterns,for the whole root and for the sections 0–2 cm. and 2–4cm. from the tip, are the same. The acidic growth substances extractable from broad bean shootsare the same whether the plant material is boiled or frozenbefore extraction.     

Retinal fate and ganglion cell differentiation are potentiated by acidic FGF in an in vitro assay of early retinal development.

One of the earliest events in vertebrate eye development is the establishment of the pigmented epithelium and neural retina. These fundamentally different tissues derive from the invaginated optic vesicle, or optic cup. Even after achieving a fairly advanced state of differentiation, the pigmented epithelium exhibits the same potential as the optic cup in that it can "transdifferentiate" into neural retina. C. M. Park and M. J. Hollenberg (Dev. Biol. 134, 201-205, 1989) discovered that administration of basic fibroblast growth factor, coupled with retinal removal, could trigger this transformation in vivo. We have developed a quantitative in vitro assay to study the role(s) of the fibroblast growth factor (FGF) family in this phenomenon and more generally in early retinal development. We found that several aspects of the process, including inhibition of pigmented epithelium differentiation, proliferation, and conversion to a retinal fate, were not strictly correlated. Both acidic and basic FGFs were found to potentiate all aspects of the process, with acidic FGF being 4 to 20 times more potent than basic FGF for inhibition of pigmentation and induction of retinal antigens. Depending upon its concentration, acidic FGF induced from 40% to 80% of the cells in the explants to produce antigens normally expressed by retinal ganglion cells, the first cell type to be generated in retinal development. Expression of such a ganglion cell marker could be directly stimulated in non-dividing cells as well as in dividing cells, indicating that conversion from the pigmented epithelial to retinal fate did not require cell division. These data suggest that acidic FGF, or a related molecule, may function in establishment of retinal fate from the optic cup. This effect may be directly or indirectly mediated by induction of retinal ganglion cell fate among multipotent progenitor cells.     

Kinetoplast DNA in the insect trypanosomes Crithidia luciliae and Crithidia fasciculata: III. Heteroduplex analysis of the C. luciliae minicircles

We have investigated the nature of the sequence heterogeneity of the minicircles of Crithidia luciliae kinetoplast DNA by EM heteroduplex analysis of minicircles cleaved with endonuclease HindIII. Approximately 40% of the minicircles showed—after denaturation and reannealing—structures indicative of sequence rearrangements: the majority contained heteroduplex “eyes” interpreted as due to inversions; about 10% of the heteroduplexes yielded structures interpreted as due to translocations and a similar fraction showed insertions and deletions. The category of “eyed” molecules was analyzed in detail: four minicircle segments were found that displayed a high incidence of such eyes indicating that the rearrangements were not scattered at random over the minicircles. Moreover, since also “eyes” were found overlapping two or three of the four regions, we postulate that these segments are capable of recombining with each other. We conclude that specific segmental rearrangements form the main basis of the minicircle sequence heterogeneity in Crithidia.     

Zirconyl Hematoxylin: a New Stain for Acidic Mucins

Most stains for acidic mucins are time-consuming to prepare and have poor stability. Zirconyl hematoxylin is easily prepared and works for a year or more. It is made by adding 5 ml freshly-made 0.1% aqueous sodium iodate, 400 mg zirconyl chloride oc-tahydrate, and 40 ml 25% aqueous glycerol, in that order, to 100 mg of hematoxylin in 5 ml of absolute ethanol and stirring for 5 min. Stain 10 min and do not “blue” the stain. Chlorazole black or kernechtrot and fast green are good counterstains. Zirconyl hematoxylin stains acidic mucins violet or red violet, regardless of how they are fixed. It stains the same mucins as alcian blue in mouse and sheep salivary glands. It shows goblet cells in mouse rectum as well as alcian blue. It stains the same stomach regions in a lizard as alcian blue. Like alcian blue and colloidal iron, zirconyl hematoxylin stains the mucin of cancerous prostate, but not normal prostate.     

The Mechanism of Staining Explained on A Chemical Basis II. General Presentation

At present the division of the factors influencing staining into those supporting a chemical mechanism or those supporting what is known as “adsorption” mechanism is questioned because the latter term cannot be definitely defined from general usage. In this paper the nature of adsorption is discussed. Data indicating definitely a chemical mechanism are not directly considered; but in many cases often cited by other writers as necessitating a non-chemical mechanism, the validity of such mechanism is questioned.     

Characterization and action of meiotic maturation inhibitors in starfish ovary

Mechanical release of oocytes from the ovary of the starfish Asterias amurensis into sea water results in “spontaneous” meiotic maturation of the oocytes. The substances blocking the maturation of Asterias oocytes have been purified from the ovary and shown to be steroid glycosides named asterosaponins A and B. The extract prepared from isolated oocytes was incapable of inhibiting oocyte maturation. The ovarian extract inhibited the production of 1-methyladenine (1-MA) in follicle cells surrounding the oocyte. The ovarian extract failed to influence 1-MA-induced maturation of the oocyte with or without follicle cells. It can be concluded from the present results that the role of the ovarian extract containing steroid glycosides is to arrest “spontaneous” production of 1-MA in follicle cells. The suppression can be overcome by the action of a gonadotropic peptide hormone released from the nerve tissue.     

A Novel Myosin-like Protein (Myocilin) Expressed in the Connecting Cilium of the Photoreceptor: Molecular Cloning, Tissue Expression, and Chromosomal Mapping

We have isolated a human cDNA clone encoding a novel acidic protein of MW 55,000 that we designated “myocilin” since it has homology to myosin and is localized preferentially in the ciliary rootlet and basal body of the connecting cilium of photoreceptor cells. The deduced amino acid sequence of human myocilin showed significant homologies with nonmuscle myosin ofDictyostelium discoideumin the N-terminal region and also with olfactomedin of bullfrog in the C-terminal region. Myocilin contained a leucine zipper-like motif similar to that seen in kinectin and other cytoskeletal proteins. These findings suggest that myocilin is a novel cytoskeletal protein involved in the morphogenesis of ciliated neuroepithelium such as photoreceptor cells. The myocilin gene (MYOC) was mapped to human chromosome 1q23–q24 by fluorescencein situhybridization.     

The Preparation of Hardened Embedding Paraffins Having Low Melting Points

Technical “stearic acid” hardens paraffins melting at 52° C. and above, and at the same time lowers the melting point. Spermaceti wax further lowers the melting point of such a mixture without much effect on the hardness. With these two substances, and one of the anti-crystallizing adjuvants already found satisfactory, embedding media yielding thin sections at room temperature and having melting point below 52° C. can easily be prepared. A general method is given, specific formulas are stated, and the behavior at various temperatures of typical embedding media of this kind is described.