Detection of cytokinins and auxin in plant tissues using histochemistry and immunocytochemistry

We report a new method for histochemical localization of cytokinins (CKs) in plant tissues based on bromophenol blue/silver nitrate staining. The method was validated by immunohistochemistry using anti-trans-zeatin riboside antibody. Indole-3-acetic acid (auxin, IAA) was localized by anti-IAA antibody in plant tissues as a proof for IAA histolocalization. We used root sections, because they are major sites of CKs synthesis, and insect galls of Piptadenia gonoacantha that accumulate IAA. Immunostaining confirmed the presence of zeatin and sites of accumulation of IAA indicated by histochemistry. The colors developed by histochemical reactions in free-hand sections of plant tissues were similar to those obtained by thin layer chromatography (TLC), which reinforced the reactive sites of zeatin. The histochemical method for detecting CKs is useful for galls and roots, whereas IAA detection is more efficient for gall tissues. Therefore, galls constitute a useful model for validating histochemical techniques due to their rapid cell cycles and relatively high accumulation of plant hormones.     

A Lectin-like Molecule is Discharged from Mucocysts of Tetrahymena pyriformis in the Presence of Insulin

ABSTRACT. By use of a monoclonal antibody directed against purified lectin from the sponge Geodia cydonium it was demonstrated that the mucocysts of Tetrahymena pyriformis contain a substance immunologically similar to that found in G. cydonium . In extracts of T. pyriformis the monoclonal antibody recognizes a 36 kDa protein; binding could be abolished by adsorption of the antibody with (i) crude extract, (ii) purified lectin from G. cydonium and (iii) a 29 aa long peptide. In addition the data show that 10^-6 M of insulin causes first the release of mucocyst material, which reacts with the lectin antibody, and second its subsequent redistribution on the surface of the somatic cilia and the oral field.     

Stability of metal ion complexes formed with methyl phosphate and hydrogen phosphate

 The acidity constants of methyl phosphoric acid, CH₃OPO(OH)₂, and orthophosphoric acid, HOPO(OH)₂, and the stability constants of the 1 : 1 complexes formed between Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, or Cd²⁺ and methyl phosphate, CH₃OPO₃ ^2–, or hydrogen phosphate, HOPO₃ ^2–, were determined by potentiometric pH titration in aqueous solution (25  °C;I = 0.1 M, NaNO₃). On the basis of previously established log K versus pK _a straight-line plots for the complexes of simple phosphate monoesters and phosphonate derivatives, R-PO₃ ^2–, where R is a noncoordinating residue, it is shown that the stability of the M(CH₃OPO₃) complexes is solely determined (as one might expect) by the basicity of the –PO₃ ^2– residue. It is emphasized that the mentioned reference lines may also be used to reveal increased complex stabilities, for example, for certain complexes formed with 8-quinolyl phosphate the occurrence of 7-membered chelates can be proven in this way; the same procedure is also applicable to complexes of nucleotides, etc. The M(HOPO₃) complexes are slightly more stable (on average by 0.08 log unit) than it is expected from the basicity of HPO₄ ^2–; this observation is attributed to a more effective solvation, including hydrogen bonding, than is possible with CH₃OPO₃ ^2– species. Received: 9 November 1995 / Accepted: 5 February 1996     

Comparison of pome fruit orchard inhabiting spider assemblages at different geographical scales

1 The composition of pome fruit orchard inhabiting spider assemblages was investigated at different geographical scales (Holarctic, European, inter- and intraregional levels within Hungary) using previous faunistic studies and data collected in Hungary between 1995 and 1997. Samples in Hungary were taken from the canopy and herb layer of apple and pear orchards in five markedly different fruit-growing regions by beating and sweep-netting methods. 2 The composition of canopy spider assemblages of apple orchards was analysed for the Holartic region and found to be determined by latitude at family level, and by the main zoogoegraphical regions at genus level. At the European scale, both the genus and species composition changed along a north–south gradient. 3 A comparison among apple and pear orchards located in different regions in Hungary, showed that both foliage- and grass-dwelling spider assemblages varied considerably in species composition and dominance order. 4 Within the same region, both the foliage- and grass-dwelling spider assemblages showed moderate differences in apple and pear orchards submitted to different treatments. Although the assemblages of spiders inhabiting the canopy and the herbaceous layer can be unambiguously distinguished, some overlap still occurs. 5 We conclude that the composition of spider assemblages is basically determined by geographical location. Although both pesticide treatments and available prey densities can influence the population of spiders, such factors are of moderate importance when compared with the effect of regionality, even when considered at smaller scale. However, most members of the family Theridiidae and the large orb-weavers (Araneidae) decreased considerably in treated plots. Scale-specific differences are thus relevant in determining the composition of prey–predator systems in orchards, and should be taken into account when designing integrated pest management (IPM) programs for apple and pear orchards.     

Self-association and protonation of adenosine 5'-monophosphate in comparison with its 2'- and 3'-analogues and tubercidin 5'-monophosphate (7-deaza-AMP)

The concentration dependence of the chemical shifts of the protons H-2, H-8 and H-1' for 2'-, 3'- and 5'-AMP2- and of the protons H-2, H-7, H-8 and H-1' for tubercidin 5'-monophosphate (= 7-deaza-AMP2-; TuMP2-) has been measured in D2O at 27 degrees C to elucidate the self-association of the nucleoside monophosphates (NMPs). The results are consistent with the isodesmic model of indefinite non-cooperative stacking; the association constants for all four NMPs are very similar: K approximately 2 M-1. These 1H-NMR measurements and those on the dependence of the chemical shifts on the pD of the solutions indicate that the NMP2- species exist predominately in the anti conformation. Comparison of the shift data for 5'-TuMP and 5'-AMP shows that no hydrogen bonding between N-7 and -PO3H- occurs; hence, the previously observed and confirmed 'wrongway' chemical shift [Martin, R. B. (1985) Acc. Chem. Res 18, 32] connected with the deprotonation of the -PO3H- group most probably results from the anisotropic properties of the phosphate group which is in the anti conformation close to N-7. From the dependence between the chemical shift and the pD of the solutions the acidity constants were calculated for the four protonated NMPs, and for adenosine and D-ribose 5'-monophosphate. The measurements also allow an estimation of the first acidity constant of H3(5'-AMP)+ (pKDD3(AMP) = 0.9 and pKHH3(AMP) = 0.4). The values for pKHH2(NMP) and pKHH(NMP) were also determined from potentiometric pH titrations in aqueous solution (I = 0.1 M, NaNO3; 25 degrees C). The agreement of the results obtained by the two methods is excellent. The position of the phosphate group at the ribose moiety and the presence of N-7 in the base moiety influence somewhat the acid-base properties of the mentioned NMPs. Measurements with 5'-AMP in 50% (v/v) aqueous dioxane show that lowering of the solvent polarity facilitates removal of the proton from the H+(N-1) site while the -PO2-3 group becomes more basic; this increases the pH range in which the monoprotonated H(5'-AMP)- species is stable and which is now also extended into the physiological pH region. Some consequences of this observation for biological systems are indicated.     

Characteristics of complement receptor-bearing cells in the spleens of tumor-bearing mice.

In the course of mammary tumor development, a population of nylon nonadherent cells with CR appears in the spleens of tumor-bearing mice although none are ever detected in normal mice. These cells apparently arise in response to immunologic stimulation. In a series of studies we have further characterized subsets of T cells (CR+ and CR-) with regard to their responses to mitogens in the lymphocyte transformation assay. Nylon column nonadherent cells from the spleens of tumor-bearing mice were rosetted in a complement receptor assay using EAC rosetting, and CR+ cells were separated from CR- by centrifugation in a discontinuous Ficoll gradient. CR+ T cells responded strongly to PHA and Con A and in addition responded to LPS, an activity not usually associated with conventional T cells. In contrast, CR- T cells from tumor-burdened mice responded to PHA but failed to respond to Con A or LPS.     

Stability and structure of binary and ternary complexes of alpha-lipoate and lipoate derivatives with Mn2+, Cu2+, and Zn2+ in solution.

The stabilities of the 1:1 complexes of Mn²⁺, Cu²⁺, and Zn²⁺ with lipoate and its chainshortened catabolites, viz., bisnorlipoate and tetranorlipoate, were studied by potentiometric titrations in water containing 50% dioxane (I = 0.1, NaClO₄; 25 °C). A comparison of the stabilities of these complexes with those of simple carboxylates reveals that the catabolite complexes formed with Cu²⁺ and Zn²⁺ are more stable than expected from only the basicity of the carboxylate groups. This is evidence that chelates involving the disulfide group are formed. The stability of all Mn²⁺ complexes is determined by the basicity of the carboxylate groups. The same pattern of stability holds for the mixed-ligand complexes formed by Cu²⁺ or Zn²⁺, 2,2′-bipyridyl, and lipoate or one of its derivatives. It is evident that the disulfide group of the 1,2-dithiolane moiety can participate in the formation of binary and ternary complexes. The somewhat less-pronounced coordinating properties of the 1,2-dithiolane moiety compared with the tetrahydrothiophene moiety are discussed. It is apparent that the electron density at S(1) and S(2) in the dithiolane moiety of lipoate is not equivalent: S(1) is favored over S(2) in electrophilic reactions; possible biological implications are indicated.