Relationship between type and age of the inoculum cultures and betalains biosynthesis by Beta vulgaris hairy root culture

From a study of the relationship between the type and age of the inocula, and the growth and biosynthesis of betalains in a Beta vulgaris hairy root culture, the best results were achieved with a 14 d inoculum grown in submerged culture giving 42 mg betalains (16 mg betacyanins and 26 betaxanthins) and 1.5 g dry biomass in 40 ml medium.     

A continuous fluorimetric assay for glycosidase activity: Human N-acetyl-β-d-hexosaminidase

The fluorescence intensity of 4-methylumbelliferone (λ_excitation = 320 nm, λ_emission = 450 nm) is approximately 120-fold greater than those of 4-methylumbelliferyl glycosides over a pH range from 3 to 7. Therefore, continuous fluorimetric monitoring of the enzymatic release of 4-methylumbelliferone from its corresponding glycoside can be performed. The technique we developed is suitable for kinetic studies of various glycosidases operating within the indicated pH range. This procedure was found to be accurate, sensitive, and rapid, as shown using N-acetyl-β-d-hexosaminidases A and B isolated from human placenta.     

The protein concentration of crude cell and tissue extracts as estimated by the method of dye binding: comparison with the Lowry method.

This paper describes a new, inexpensive, and highly sensitive voltammetric assay for aromatic l-amino acid decarboxylase (AADC) activity in rat and human brains by highperformance liquid chromatography (hplc). l-Dopa was used as substrate and d-dopa for the blank. After isolating dopamine formed enzymatically from l-dopa on a small Amberlite CG-50 column, the dopamine in the column eluate was assayed by hplc with a voltammetric detector. Dihydroxybenzylamine was added to each incubation mixture as an internal standard and therefore this assay was highly reproducible. The peak height in hplc was linear from 100 fmol to 100 pmol of dopamine. The values obtained by this method agreed with those by radioassay using [1-¹⁴C]dopa. The enzyme activity in rat cerebral cortex and in Parkinsonian caudate nucleus, in which the activity was too low to be measured even with the radioassay, could be measured accurately.     

Further studies on the growth and differentiation of single,isolated cells of tobacco in vitro

Summary Single cells of hybrid tobacco callus, isolated from germinating seeds, were grown individually in microchambers in the absence of any other cells, in fresh, liquid, coconut milk-medium. These produced a conlony of 50–75 cells in 10–15 days. In all instances the plane of the first cell division, and in most cases also that of the second division, were at right angles to the long axis of the cell. There was more variation in the size, shape and pattern of the second and subsequent divisions in single cells isolated from fresh stem pith and seed callus than those from old stem callus. Upon transfer from the microchambers to agar medium the colony of cells gave rise to a huge mass of callus tissue in about 3 months. In no instance did the cell colonies or any stages preceding them resemble or simulate any stages of normal embryogeny of tabacco. Single-cell clones obtained by this method from seed callus or fresh pith callus produced shoots with leaves and roots in synthetic medium with various indoleacetic acid-kinetin combinations. Normal plants established from the above cultures in the greenhouse flowered in due course of time. This method offers the possibility of producing a very large number of clones or identical plants in species where vegetative propagation is not otherwise possible, apart from its use in studies on the genetics and morphogenesis in higher plants.     

Molecular phylogeny of the genus Alticola (Cricetidae, Rodentia) as inferred from the sequence of the cytochrome b gene

Central Asian mountain voles Alticola is one of the least known groups of voles both in evolution and life history. This genus includes three subgenera Alticola s.str., Aschizomys and Platycranius, and belongs to the tribe Clethrionomyini comprising also red‐backed voles Clethrionomys and oriental voles Eothenomys. In order to elucidate the phylogenetic relationships within Alticola and to examine its position within the tribe, mitochondrial cytochrome b (cyt b) gene variation was estimated, and the results were compared with morphological and palaeontological data. Maximum likelihood (ML), neighbor‐joining (NJ), maximum parsimony (MP) and Bayesian phylogenetic analyses show that the genus Alticola does not appear to be a monophyletic group since the representatives of Aschizomys branch within Clethrionomys, whereas two other subgenera (Alticola and Platycranius) form a separate monophyletic clade. Flat‐headed vole Alticola (Platycranius) strelzowi is nested within the nominative subgenus showing close association with A. (Alticola) semicanus. Surprisingly, the two species of Aschizomys do not form a monophyletic group. The results of the relaxed‐clock analysis suggest that the Alticola clade splits from the Clethrionomys stem in early Middle Pliocene while basal cladogenetic events within Alticola s.str. dates back to the late Middle to early Late Pliocene. A scenario of evolution in Clethrionomyini is put forward implying rapid parallel morphological changes in different lineages leading to the formation of Alticola‐like biomorphs adapted to mountain and arid petrophilous habitats. Corresponding author: Vladimir S. Lebedev, Zoological Museum, Moscow State University, B. Nikitskaya 6, 125009 Moscow, Russia. E‐mail: wslebedev@hotmail.com Anna A. Bannikova, Lomonosov Moscow State University, Vorobievy Gory, 119992 Moscow, Russia. E‐mail: hylomys@mail.ru Alexey S. Tesakov, Geological Institute RAS, Pyzhevsky 7, 119017 Moscow, Russia. E‐mail: tesak@ginras.ru Natalia I. Abramson, Zoological Institute RAS, Universitetskaya nab. 1, 199034 St Petersburg, Russia. E‐mail: lemmus@zin.ru     

A Genome Scale Screen for Mutants with Delayed Exit from Mitosis: Ire1-Independent Induction of Autophagy Integrates ER Homeostasis into Mitotic Lifespan

Proliferating eukaryotic cells undergo a finite number of cell divisions before irreversibly exiting mitosis. Yet pathways that normally limit the number of cell divisions remain poorly characterized. Here we describe a screen of a collection of 3762 single gene mutants in the yeast Saccharomyces cerevisiae, accounting for 2/3 of annotated yeast ORFs, to search for mutants that undergo an atypically high number of cell divisions. Many of the potential longevity genes map to cellular processes not previously implicated in mitotic senescence, suggesting that regulatory mechanisms governing mitotic exit may be broader than currently anticipated. We focused on an ER-Golgi gene cluster isolated in this screen to determine how these ubiquitous organelles integrate into mitotic longevity. We report that a chronic increase in ER protein load signals an expansion in the assembly of autophagosomes in an Ire1-independent manner, accelerates trafficking of high molecular weight protein aggregates from the cytoplasm to the vacuoles, and leads to a profound enhancement of daughter cell production. We demonstrate that this catabolic network is evolutionarily conserved, as it also extends reproductive lifespan in the nematode Caenorhabditis elegans. Our data provide evidence that catabolism of protein aggregates, a natural byproduct of high protein synthesis and turn over in dividing cells, is among the drivers of mitotic longevity in eukaryotes.     

Aggregation-Prone Motifs in Human Immunoglobulin G

Therapeutic antibodies of many different IgG subclasses (IgG1, IgG2 and IgG4) are used in the treatment of various cancers, rheumatoid arthritis and other inflammatory and infectious diseases. These antibodies are stored for long durations under high concentrations as required in the disease treatment. Unfortunately, these antibodies aggregate under these storage conditions, leading to a decrease in antibody activity and raising concerns about causing an immunological response. Thus, there is a tremendous need to identify the aggregation-prone regions in different classes of antibodies. We use the SAP (spatial-aggregation-propensity) technology based on molecular simulations to determine the aggregation-prone motifs in the constant regions of IgG1 classes of antibodies. Mutations engineered on these aggregation-prone motif regions led to antibodies of enhanced stability. Fourteen aggregation-prone motifs are identified, with each motif containing one to seven residues. While some of these motifs contain residues that are neighbors in primary sequence, others contain residues that are far apart in primary sequence but are close together in the tertiary structure. Comparison of the IgG1 sequence with those of other subclasses (IgG2, IgG3 and IgG4) showed that these aggregation-prone motifs are largely preserved among all IgG subclasses. Other broader classes of antibodies (IgA1, IgD, IgE and IgM), however, differed in these motif regions. The aggregation-prone motifs identified were therefore common to all IgG subclasses, but differ from those of non-IgG classes. Moreover, since the motifs identified are in the constant regions, they are applicable for all antibodies within the IgG class irrespective of the variable region. Thus, the motif regions identified could be modified on all IgGs to yield antibodies of enhanced stability.     

Intracellular organelle transport: few motors, many signals

Bidirectional microtubule-dependent organelle transport in melanophores is regulated by cAMP through organelle-bound protein kinase A (PKA); however, the mechanisms responsible for this regulation are unknown. A recent study by Gelfand and colleagues demonstrates that, in addition to PKA, transport is regulated by the organelle-bound mitogen-activated protein kinase (MAPK) signaling components ERK and MEK, whose activity is required for bidirectional transport along microtubules. This pathway apparently acts downstream of PKA, suggesting that bidirectional organelle transport is regulated by a hierarchical cascade of signaling pathways.