A detailed study of the periodate oxidation of sialic acids in glycoproteins

Periodate oxidation of terminalN-acetyl- andN-glycoloylneuraminic acid residues in the mucins from edible bird nest substance and pig submandibular gland, respectively, can be carried out under conditions which exclusively give rise to the formation of the C-7 analogues of these sialic acids. In contrast, the C-8 compounds can be obtained in a maximum yield of about 40%. Under identical conditions,N-glycoloylneuraminic acid is oxidized about 1.5 times faster than theN-acetylated derivative. After release of the sialic acids by acid hydrolysis, the characterization of the oxidation products was carried out by TLC, by GLC and GLC-MS of the corresponding pertrimethylsilyl derivatives, and by 500-MHz¹H-NMR spectroscopy. In addition, molar response factors for GLC analysis and extinction coefficients in the orcinol/Fe³⁺/HCl assay were determined.     

Patterns of divergence during evolution of alpha 1-proteinase inhibitors in mammals

alpha 1-Proteinase inhibitor (alpha 1-PI), a member of the serine proteinase inhibitor superfamily, has a primary role in controlling neutrophil elastase activity within the mammalian circulation. Several studies have indicated that the reactive center region of alpha 1-PI, the amino acid sequence of which is critical to recognition of and binding to target proteinases, is highly divergent within and among species. This appears to be a consequence of accelerated rates of evolution that may have been driven by positive Darwinian selection. In order to examine this and other features of alpha 1-PI evolution in more detail, we have isolated and sequenced cDNAs representing alpha 1- PI mRNAs of the mouse species Mus saxicola and Mus minutoides and have compared these with a number of other mammalian alpha 1-PI mRNAs. Relative to other mammalian mRNAs, the extent of nonsynonymous substitution is generally high throughout the alpha 1-PI mRNA molecule, indicating greater overall rates of amino acid substitution. Within and among mouse species, the 5'-half of the mRNA, but not the 3'-half, has been homogenized by concerted evolution. Finally, the reactive center is under diversifying or positive Darwinian selection in murid rodents (rats, mice) and guinea pigs yet is under purifying selection in primates and artiodactyls. The significance of these findings to alpha 1-PI function and the possible selective forces driving evolution of serpins in general are discussed.      

Temporal and spatial variability in insect grazing of the canopies of five Australian rainforest tree species

Herbivory was measured monthly for 2 years on leaves of permanently marked replicate branches in the canopies of five Australian rainforest tree species. Variability in insect grazing activities was evident with respect to several factors:

• 1 leafage — young leaves were preferred over older leaf tissue:
• 2 height — leaves closer to ground level were more heavily grazed:
• 3 light — shade leaves were preferred to sun leaves:
• 4 time — grazing was intense during spring and summer months, and almost negligible during autumn and winter, but was cumulatively similar between the 2 years:
• 5 space — grazing was extremely variable on small spatial scales such as between individual leaves and branches, but similar where hundreds of leaves were pooled on larger scales between individual canopies and among geographically different sites:
• 6 tree abundance — grazing was heavier at sites where a tree species was common than where it was rare:
• 7 host tree species.

Long term observations resulted in higher but more accurate estimates of herbivory since it was possible to quantify losses of leaves totally eaten, an event not accounted for infield methods of discrete sampling whereby leaves are merely harvested and measured for area missing. Variability in herbivory is discussed in terms of plant-insect phenologies, plant defences, successional status of tree species, and insect behaviour.     

Tyrosine phosphorylation of Munc18‐1 inhibits synaptic transmission by preventing SNARE assembly

Tyrosine kinases are important regulators of synaptic strength. Here, we describe a key component of the synaptic vesicle release machinery, Munc18‐1, as a phosphorylation target for neuronal Src family kinases (SFKs). Phosphomimetic Y473D mutation of a SFK phosphorylation site previously identified by brain phospho‐proteomics abolished the stimulatory effect of Munc18‐1 on SNARE complex formation (“SNARE‐templating”) and membrane fusion in vitro. Furthermore, priming but not docking of synaptic vesicles was disrupted in hippocampal munc18‐1‐null neurons expressing Munc18‐1_Y473D. Synaptic transmission was temporarily restored by high‐frequency stimulation, as well as by a Munc18‐1 mutation that results in helix 12 extension, a critical conformational step in vesicle priming. On the other hand, expression of non‐phosphorylatable Munc18‐1 supported normal synaptic transmission. We propose that SFK‐dependent Munc18‐1 phosphorylation may constitute a potent, previously unknown mechanism to shut down synaptic transmission, via direct occlusion of a Synaptobrevin/VAMP2 binding groove and subsequent hindrance of conformational changes in domain 3a responsible for vesicle priming. This would strongly interfere with the essential post‐docking SNARE‐templating role of Munc18‐1, resulting in a largely abolished pool of releasable synaptic vesicles.     

Dynamic subcellular localization of isoforms of the folate pathway enzyme serine hydroxymethyltransferase (SHMT) through the erythrocytic cycle of Plasmodium falciparum

Background

The folate pathway enzyme serine hydroxymethyltransferase (SHMT) converts serine to glycine and 5,10-methylenetetrahydrofolate and is essential for the acquisition of one-carbon units for subsequent transfer reactions. 5,10-methylenetetrahydrofolate is used by thymidylate synthase to convert dUMP to dTMP for DNA synthesis. In Plasmodium falciparum an enzymatically functional SHMT (PfSHMTc) and a related, apparently inactive isoform (PfSHMTm) are found, encoded by different genes. Here, patterns of localization of the two isoforms during the parasite erythrocytic cycle are investigated.

Methods

Polyclonal antibodies were raised to PfSHMTc and PfSHMTm, and, together with specific markers for the mitochondrion and apicoplast, were employed in quantitative confocal fluorescence microscopy of blood-stage parasites.

Results

As well as the expected cytoplasmic occupancy of PfSHMTc during all stages, localization into the mitochondrion and apicoplast occurred in a stage-specific manner. Although early trophozoites lacked visible organellar PfSHMTc, a significant percentage of parasites showed such fluorescence during the mid-to-late trophozoite and schizont stages. In the case of the mitochondrion, the majority of parasites in these stages at any given time showed no marked PfSHMTc fluorescence, suggesting that its occupancy of this organelle is of limited duration. PfSHMTm showed a distinctly more pronounced mitochondrial location through most of the erythrocytic cycle and GFP-tagging of its N-terminal region confirmed the predicted presence of a mitochondrial signal sequence. Within the apicoplast, a majority of mitotic schizonts showed a marked concentration of PfSHMTc, whose localization in this organelle was less restricted than for the mitochondrion and persisted from the late trophozoite to the post-mitotic stages. PfSHMTm showed a broadly similar distribution across the cycle, but with a distinctive punctate accumulation towards the ends of elongating apicoplasts. In very late post-mitotic schizonts, both PfSHMTc and PfSHMTm were concentrated in the central region of the parasite that becomes the residual body on erythrocyte lysis and merozoite release.

Conclusions

Both PfSHMTc and PfSHMTm show dynamic, stage-dependent localization among the different compartments of the parasite and sequence analysis suggests they may also reversibly associate with each other, a factor that may be critical to folate cofactor function, given the apparent lack of enzymic activity of PfSHMTm.     

Electron-microscope studies of Drosophila salivary-gland chromosomes

Summary The submicroscopic structure of the chromosomes of the larval salivary gland cells inDrosophila melanogaster has been studied by means of electron microscopy of thin sections.The three fixatives used were buffered 1% osmium tetroxide, unbuffered Zenker's solution, and buffered formaldehyde in concentrations of 0·1% and 1·0%. It was found that formalin preserves the fine structure of the chromosomes better than the other fixatives.Nucleoprotein is present in the chromosome in the form of granules 200 Å to 300 Å in diameter. The granules are connected together by strands of protein approximately 100 Å in diameter. The length of the strands between successive granules in the banded areas is 100 Å to 300 Å and in the interband regions it is 700 Å to 900 Å.Heterochromatin differs from euchromatin in the absence or reduction in the amount of strand material.The nucleolus is composed of an aggregate of granules 300 Å to 600 Å in diameter contained in an amorphous matrix. Within the nucleolus are a series of canals containing material similar in appearance to that of the euchromatic band material.Submicroscopic granules are also present in the karyoplasm and in the cytoplasm surrounding the nuclear membrane. The cytoplasmic granules are more densely distributed near the nuclear membrane.A model for chromosome structure is proposed.The Applied Fisheries Laboratory is operated by the University of Washington under Contract No. AT(45-1)540 with the United States Atomic Energy Commission.     

Seasonal variation in insect abundance among three Australian rain forests,with particular reference to phytophagous types

Monthly sweep net and light trap samples were used to examine seasonal changes in the abundance of insects in subtropical, warm and cool temperate rain forest in New South Wales. Maximum insect abundance, especially of phytophages, coincided with leaf flushing in the canopy trees. Cool temperate insect numbers were highest during the month just following the beech leaf flush, a rapid and synchronous growth event. Conversely, numbers of subtropical insects fluctuated over a longer period, in a pattern similar to the continuous growth of leaves that occurred throughout spring and summer there. The warm temperate was intermediate in its vegetation growth phenology and insect patterns. Rainforest insect abundance varied both temporally and spatially.     

The impact of defoliating insects on the growth of eucalypt saplings

The effect of spraying insecticide on eucalypt saplings was experimentally tested in a New England woodland. Replicate branches of five species were sprayed fortnightly with Thiodan over a period of 4 months. Their leaf damage, leaf flushes, stem growth, and bud production were compared with those of control branches. Loss of leaf area to insect grazers was extremely variable, ranging from very low levels (e.g. 1.8% for sprayed new leaves of Angophora floribunda) to nearly total defoliation (97.1% for unsprayed new leaves of Eucalyptus viminalis). The sprayed branches consistently suffered less herbivory and grew more than the controls.     

Anatomy of flower and fruit of <i>Vassobia breviflora</i> (Solanaceae) in the south of the southern Yungas (Argentina)

Solanaceae is a family with nearly 2400 species of cosmopolitan distribution. Vassobia breviflora is the only species of the genus present in Argentina. The goal of this work was to review and characterize the anatomy of the flower and fruit of V. breviflora from samples collected in populations of Yungas in the argentine Northwest. Conventional anatomical techniques were applied. The results showed that most flower, fruit and seed structures did not differ from those previously reported regarding the structural organization described for other species of the Solanaceae family. However, for the first time, we described the androecium, fruit, seed, floral and fruit pedicels, five types of tricomes and five types of stomata in the perianth. We found some differences in the shape of the transmission tissue and in the type of ovule with respect to that previously reported. Also, we located the parenchyma and the epidermic secretory cells of the nectary. In the context of the family Solanaceae, we discussed the function and diagnostic value of the described structures.