Assessing temporal dynamics of predation and effectiveness of caterpillar visual defense using sawfly larval color and resting posture as a model

Caterpillars (Lepidoptera and Symphyta larvae) employ diverse visual defensive tactics, and effectiveness of such tactics may be highly dynamic across time due to seasonal changes in the predator assemblages and their preferences. However, this has rarely been studied especially in tropical regions. Here we assessed temporal changes in the defensive value of caterpillar color and shape, using six types of plasticine dummy caterpillars: three colors (green, black, and white) × two shapes (curled and straight). These dummy caterpillars were deployed five times over different seasons in tropical forests of Xishuangbanna (China) and, as a comparison, twice in a temperate forest of Hirosaki (Japan). The colors and shapes of dummy caterpillars simulate visual traits of black sawfly larvae which take the curled resting posture in tropical rainforests of Xishuangbanna, apparently masquerading excrements commonly found on plants, while in Hirosaki there is no black-curled sawfly larvae and few excrements on plants. We found no significant effects of caterpillar colors or shapes on predation in Hirosaki. In contrast, black and curled caterpillars received significantly lower predation by birds in Xishuangbanna constantly across time. However, we were unable to provide evidence that the black-curled sawfly larvae are masquerading as excrements. Shapes of the dummy caterpillars also affected the predation by ants and parasitoid wasps at certain times. This is the first report on ecological function of the curled posture of sawfly larvae, and we demonstrated the importance to assess the temporal dynamics of predation and effectiveness of defensive tactics in tropical forests.     

Metabolism of l-β-lysine by a Pseudomonas: Purification and properties of 3-keto-6-acetamidohexanoate cleavage enzyme

Extracts of Pseudomonas B4 grown with l-β-lysine (3,6-diaminohexanoate) as the main energy source are shown to contain a 3-keto-6-acetamidohexanoate cleavage enzyme that converts 3-keto-6-acetamidohexanoate and acetyl · CoA reversibly to 4-acetamidobutyryl · CoA and acetoacetate. The enzyme catalyzes the third step in β-lysine degradation. In unfractionated extracts cleavage enzyme activity is generally assayed spectrophotometrically by coupling the forward reaction with excess 4-acetamidobutyryl · CoA thiolesterase, derived from the same organism, and measuring the rate of CoASH formation by reaction with 5,5-dithiobis(2-nitrobenzoic acid). Enzyme freed of thiolesterase is conveniently assayed by using 4-acetamidobutyryl · CoA and acetoacetate as substrates and measuring acetyl · CoA formation by means of citrate synthase reaction in the presence of 5,5-dithiobis(2-nitrobenzoic acid). The cleavage enzyme has been purified 38-fold to a specific activity of 237 mU/mg. The stoichiometry, equilibrium constant, molecular weight, and various kinetic properties of the enzymatic reaction have been determined. The substrate specificity of the Pseudomonas enzyme differs markedly from that of the analogous 3-keto-5-aminohexanoate cleavage enzyme of Clostridium subterminale strain SB4 and is broader. In the forward reaction 3-ketohexanoate can replace 3-keto-6-acetamidohexanoate, and propionyl · CoA can replace acetyl · CoA as a substrate. In the backward reaction, 4-acetamidobutyryl · CoA can be replaced by any of several CoA thiolesters including the butyryl, valeryl, 4-propionamidobutyryl, 3-acetamidopropionyl, and β-alanyl derivatives, and acetoacetate can be replaced by 2-methylacetoacetate. The products of these reactions have been characterized. Unlike the cleavage enzyme of Clostridium subterminale strain SB4, the Pseudomonas enzyme is not stimulated by Co²⁺ or Mn²⁺ and is not inhibited by EDTA, 5,5-dithiobis(2-nitrobenzoic acid), or p-chloromercuribenzoate. Tracer experiments indicate that carbon atoms 1 and 2 of acetoacetate are derived from carbon atoms 1 and 2 of 3-keto-6-acetamidohexanoate, and carbon atoms 3 and 4 of acetoacetate are derived from the acetyl group of acetyl · CoA. The cleavage enzyme is not formed in detectable amounts when Pseudomonas B4 is grown in a peptone-yeast extract medium.     

Activation of a cryptic splice site in the tax gene of HTLV-I by a single nucleotide change

We identified a T-to-C mutation 2 nucleotides (nt) upstream from the AG in a GT-AG intron between exons 2 and 3 in the human T-cell leukemia virus type I (HTLV-I) tax mRNA. This mutation resulted in the preferential usage of an alternative splice site, causing a 75-nt elongation of tax mRNA and reduced production of viral antigens. When the clone containing this T-to-C mutation was reverted to the wild-type (T) DNA sequence, normal splicing of tax mRNA ensued and viral production was restored. These results suggest that the nucleotide at the position 2nt upstream from the AG in a GT-AG intron is important for the proper splicing of the HTLV-I tax gene, although it is not considered important for splicing in eukaryotes.     

Cdc2-mediated phosphorylation of Kid controls its distribution to spindle and chromosomes

The chromokinesin Kid is important in chromosome alignment at the metaphase plate. Here, we report that Kid function is regulated by phosphorylation. We identify Ser427 and Thr463 as M phase-specific phosphorylation sites and Cdc2-cyclin B as a Thr463 kinase. Kid with a Thr463 to alanine mutation fails to be localized on chromosomes and is only detected along spindles, although it retains the ability to bind DNA or chromosomes. Localization of rigor-type mutant Kid, which shows nucleotide-independent microtubule association, is also confined to the spindle, implying that strong association of Kid with the spindle can sequester it from chromosomes. T463A substitution in DNA-binding domain-truncated Kid consistently enhances its spindle localization. At physiological ionic strength, unphosphorylated Kid shows ATP-independent microtubule association, whereas Thr463-phosphorylated Kid shows ATP dependency. Moreover, the stalk region of unphosphorylated Kid interacts with microtubules and the interaction is weakened when Thr463 is phosphorylated. Our data suggest that phosphorylation on Thr463 of Kid downregulates its affinity for microtubules to ensure reversible association with spindles, allowing Kid to bind chromosomes and exhibit its function.     

Ultrastructural distribution of growth hormone and prolactin mRNAs in normal rat pituitary cells: a comparison between preembedding and postembedding methods

In situ hybridization (ISH) at the electron microscopic level is essential for elucidating the intracellular distribution and role of mRNA in protein synthesis. We describe our electron microscopic ISH method using biotinylated oligonucleotide probes for rat growth hormone and prolactin mRNAs and compare the preembedding method with the postembedding method. Preembedding electron microscopic ISH localized rat growth hormone and prolactin mRNAs on the polysomes of the rough endoplasmic reticulum (RER). Rat growth hormone mRNA was distributed diffusely on the RER, whereas rat prolactin mRNA was scattered and distributed focally. Thus there might be a specific translational site for prolactin mRNA on the RER. Rat growth hormone mRNA signals were also recognized on the polysomes of the RER, using the postembedding method with streptavidin gold conjugate. The hybridization signal intensity using the postembedding method was lower, and non-specific signals were more frequent, in comparison with the preembedding method. The preembedding method thus appears to be easier and better than the postembedding method from the viewpoint of utility and preservation of mRNA. Electron microscopic ISH is considered to be an important tool for evaluating the intracellular localization of mRNA and the site of specific hormone synthesis on the RER.     

Plant growth retardant activity of polycycloalkanols structurally related to 4-howoisotwistanols

Various polycycloalkanols structurally related to the plant growth retardants, 4-homoisotwistanols, were prepared and their effect on the growth of cucumber seedlings in complete darkness investigated in order to obtain information on structure-activity relationships. 4-Homobrendan-2-ols, bicyclo[3.3.1]-nonan-1-ol and adamantan-1-ol showed almost the same inhibitory activity as the 4-homoisotwistanols, but 4-homobrendan-3-ol and bicyclo-[3.3.1]nonan-2-ol were only moderately active or almost inactive. No simple relationship was apparent between structure and activity.     

Metabolism of L-beta-lysine in a Pseudomonas: conversion of 6-N-acetyl-L-beta-lysine to 3-keto-6-acetamidohexanoate and of 4-aminobutyrate to succinic semialdehyde by different transaminases.

Some kinetic properties of two new species of transaminase found in extracts of a β-lysine-utilizing Pseudomonas are reported. Transaminase A catalyzes transamination between 6-N-acetyl-l-β-lysine (3-amino-6-acetamidohexanoate) and α-ketoglutarate to form 3-keto-6-acetamidohexanoate and glutamate. Transaminase B catalyzes a reaction between 4-aminobutyrate and pyruvate to form succinic semialdehyde and alanine. The formation of both transaminases is induced by growth of the bacteria on l-β-lysine, although transaminase B is also produced in the absence of this substrate. Transaminase A requires pyridoxal phosphate for activity. The β-keto acid formed from acetyl-β-lysine by transaminase A has been purified and characterized by decarboxylation, conversion to a formazan, reduction to a stable β-hydroxy acid, and conversion of the latter to its methyl ester. Transaminase B, unlike previously reported transaminases utilizing 4-aminobutyrate, cannot use α-ketoglutarate as an amino group acceptor. This enzyme is not stimulated by addition of pyridoxal phosphate, but is inhibited by hydroxylamine or cyanide. Both transaminases appear to function in the main pathway of β-lysine degradation.     

Tumor vascular targeting using a tumor-tissue endothelium-specific monoclonal antibody as an effective strategy for cancer chemotherapy.

In this study, we attempted to develop tumor vascular targeting with a tumor tissue endothelium-specific monoclonal antibody. TES-23, which strongly and selectively recognizes tumor tissue endothelial cells, was chemically conjugated with Neocarzinostatin (NCS), and the anti-tumor effect was examined. The immunoconjugate, TES-23-NCS, showed, through the use of tumor hemorrhagic necrosis, a marked anti-tumor effect on KMT-17 tumors in rats at a dosage of 17 micrograms/kg (NCS equivalent) without any side effects, probably due to specific tumor vascular injury. By contrast, TES-23 alone (107 micrograms/kg), NCS alone (17 micrograms/kg), and Mopc-NCS (Mopc, 107 micrograms/kg; NCS, 17 micrograms/kg), the immunoconjugate of control antibody, did not have any anti-tumor activities. By tissue distribution analysis, TES-23 and TES-23-NCS showed high accumulation in KMT-17 tumors 1 h after intravenous administration. Moreover TES-23 also accumulated in Sarcoma-180 tumors in mice 1 h after intravenous administration. These results suggest that TES-23 may be a candidate for a potential tumor vascular targeting agent that is applicable to a wide variety of tumor types.